References 14 website: JMR, http://fluoroquinolonethyroid.com

References 14

Links, Abstracts, Articles, etc.

These links should work as of 2016; sometimes you have to click on them several times; if they don’t work, then Google/search the titles

V-ATPase, Topo2A/B ATPase activity,

Topo2B, Topos for transcription, miRNA

V-ATPase List of the 23 genes: http://www.genenames.org/cgi-bin/genefamilies/set/415 VATPase link

https://www.hindawi.com/journals/njos/2014/675430/ Vacuolar H+-ATPase: An Essential

Multitasking Enzyme in Physiology and Pathophysiology

http://jeb.biologists.org/content/212/3/341.long Inhibitors of V-ATPases: old and new players

https://www.ncbi.nlm.nih.gov/pubmed/18511251 The V-type H+-ATPase in vesicular trafficking:

targeting, regulation and function

http://www.cancertreatmentreviews.com/article/S0305-7372(09)00119-4/abstract?cc=y= V-ATPase

inhibitors and implication in cancer treatment.

https://en.wikipedia.org/wiki/Bafilomycin

http://www.sciencedirect.com/science/article/pii/S1097276503003976 Revised Nomenclature for

Mammalian Vacuolar-Type H-ATPase Subunit Genes

https://www.ncbi.nlm.nih.gov/pubmed/26442671 Mapping the H(+) (V)-ATPase interactome:

identification of proteins involved in trafficking, folding, assembly and phosphorylation. “Within any

given cell, the V-ATPase holoenzyme subunit profile differs among organelles and plasma membrane

domains3,10. For example, the 56-kDa B subunit has two isoforms, one ubiquitous (B2) and one that is

expressed in specialized proton-secreting cells (B1). In general, the subunit isoforms responsible for

References 14 website: JMR, http://fluoroquinolonethyroid.com

activities such as acidification of endosomes and lysosomes are “ubiquitous” and their dysfunction is

incompatible with life. V-ATPase activity is essential for post-implantation development11, and no

disease-causing mutations in these isoforms have been identified. However, loss-of-function mutations of

tissue- or cell type-specific12 isoforms, can lead to specific, but non-fatal, disease conditions such as

osteopetrosis, distal renal tubular acidosis, deafness, osteoporosis13, Parkinsonism14 and impairment of

insulin secretion15. Conversely, V-ATPase activity exacerbates metastasis in some cancers and its

inhibition is an emerging strategy to block cancer progression16. “

http://www.nature.com/nrm/journal/v3/n2/full/nrm729.html The vacuolar (H+)-ATPases — nature's

most versatile proton pumps

http://www.cancertreatmentreviews.com/article/S0305-7372(03)00106-3/abstract Cellular pH

regulators: potentially promising molecular targets for cancer chemotherapy.

From Differential Expression of the “B” Subunit of the Vacuolar H+-ATPase in Bovine Tissues* “The

presence of a nucleotide binding site on the B subunit of the vacuolar ATPases, as well as the high degree

of amino acid conservation of the B subunits from different species (7- 12) underscore the importance of

this polypeptide. In addition, there is evidence that multiple forms of the B subunit may be present in

eukaryotic tissues . . . The observation that multiple forms of the B subunit are expressed in all non-brain

bovine tissues examined could indicate that different forms of the B subunit are expressed in specialized

cells within these tissues; however, the fact that multiple forms are expressed in uniform cell lines implies

that there may be differences in the B subunit expressed within the vacuolar ATPases of a single cell.

http://flybase.org/reports/FBgg0000111.html V-ATPases (vacuolar (H+)-ATPases) are ATP-dependent

proton pumps. Principally, they pump protons from the cytoplasm to the lumen of organelles or across

epithelial apical membranes to drive secondary active transport. (Adapted from PMID:20450191). The

VACUOLAR ATPASE SUBUNITS gene group represents individual V-ATPase subunit genes. The subunits

exist in multiple isoforms/genes which are often expressed in a tissue specific manner and determine the

localization and role of the particular V-ATPase complex.

The apical membrane of a polarized cell is the surface of the plasma membrane that faces inward to the

lumen. This is particularly evident in epithelial and endothelial cells, but also describes other polarized

cells, such as neurons

VATPases: 23 genes in the family. From Wiki: Vacuolar-type H+ -ATPase (V-ATPase) is a highly

conserved evolutionarily ancient enzyme with remarkably diverse functions in eukaryotic organisms.[1]

V-ATPases acidify a wide array of intracellular organelles and pump protons across the plasma

membranes of numerous cell types. V-ATPases couple the energy of ATP hydrolysis to proton transport

across intracellular and plasma membranes of eukaryotic cells. It is generally seen as the polar opposite

of ATP Synthase because ATP Synthase is a proton channel that uses the energy from a proton gradient

to produce ATP. V-ATPase however, is a proton pump that uses the energy from ATP hydrolysis to

produce a proton gradient.

References 14 website: JMR, http://fluoroquinolonethyroid.com

Proton gradients in particular are important in many different types of cells as a form of energy storage.

The gradient is usually used to drive ATP synthase, flagellar rotation, or transport of metabolites

From Marchansky paper: “V-ATPase shares similarities with ATP-synthase (FATPase) in subunit

structure and rotational catalysis, as schematically shown in Figure 1b. Homologous subunits, such as

the A-subunit of V-ATPase and the b-subunit of F-ATPase are shown in the same color.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409362/ Rational Identification of Enoxacin as a

Novel V-ATPase-Directed Osteoclast Inhibitor. “Based on the accumulated data, Brown and colleagues

proposed that, in addition to their enzymatic role as proton pumps, V-ATPases may also be able to coat

vesicles and direct the trafficking of the vesicles in the same manner as clathrin, caveolins and coatamer

protein complexes [25]. In this scenario, the various interactions with cytoskeletal proteins and

cytoskeletal regulators might be required to manage the trafficking of V-ATPase-containing vesicles to

their ultimate destinations in cells. Although this hypothesis requires further study, evidence now points

to interactions between V-ATPases and cytoskeletal elements as novel targets for drug design. Disruption

of key protein-protein interactions might yield unique and cell selective modulators of V-ATPase-

dependent functions including bone resorption [26], tissue invasion by cancer cells [27], multidrug

resistance [28] and acid-base homeostasis [29]. Unlike traditional inhibitors of enzymatic activity, such

inhibitors would function by preventing subsets of V-ATPases from reaching the cellular destinations

where they perform cell type specific functions. Here, we will focus on the direct interaction between V-

ATPases and microfilaments that is mediated by the B2-subunit. We will review efforts to understand the

function of the microfilament binding site in the B2-subunit, and to develop small molecule inhibitors of

the interaction as potential therapeutic agents using a knowledge-based approach. A product of these

studies was the identification of enoxacin, a novel inhibitor of osteoclast bone resorption [30]. Efforts are

now underway to test the potential of enoxacin and other inhibitors of the B2-microfilament binding

interaction for the treatment of bone disease in animal models. Recently, it was reported that enoxacin is

also a selective inhibitor of the virulence of Candida albicans [31], and of cancer growth and metastasis

[32]. The possible use of enoxacin and related molecules as anti-cancer chemotherapeutic agents

emphasizes the need to fully understand the detailed mechanisms by which enoxacin affects cells.

https://en.wikipedia.org/wiki/Microfilament Microfilaments, also called actin filaments, are

filamentous structures in the cytoplasm of eukaryotic cells and form part of the cytoskeleton. They are

primarily composed of polymers of actin, but in cells are modified by and interact with numerous other

proteins. Microfilaments are usually about 7 nm in diameter and composed of two strands of actin.

Microfilament functions include cytokinesis, amoeboid movement and cell motility in general, changes in

cell shape, endocytosis and exocytosis, cell contractility and mechanical stability. Microfilaments are

flexible and relatively strong, resisting buckling by multi-piconewton compressive forces and filament

fracture by nanonewton tensile forces. In inducing cell motility, one end of the actin filament elongates

while the other end contracts, presumably by myosin II molecular motors.[1] Additionally, they function

as part of actomyosin-driven contractile molecular motors, wherein the thin filaments serve as tensile

platforms for myosin's ATP-dependent pulling action in muscle contraction and pseudopod

advancement. Microfilaments have a tough, flexible framework which helps the cell in movement

References 14 website: JMR, http://fluoroquinolonethyroid.com

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889180/ Identification of Enoxacin as an Inhibitor

of Osteoclast Formation and Bone Resorption by Structure-Based Virtual Screening. “An interaction

between the B2 subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments is required for osteoclast

bone resorption. An atomic homology model of the actin binding site on B2 was generated and

molecular docking simulations were performed. Enoxacin, a fluoroquinolone antibiotic, was identified

and in vitro testing demonstrated that enoxacin blocked binding between purified B2 and

microfilaments. Enoxacin dose dependently reduced the number of osteoclasts differentiating in mouse

marrow cultures stimulated with 1,25-dihydroxyvitamin D3, as well as markers of osteoclast activity, and

the number of resorption lacunae formed on bone slices. Enoxacin inhibited osteoclast formation at

concentrations where osteoblast formation was not altered. In summary, enoxacin is a novel small

molecule inhibitor of osteoclast bone resorption that acts by an unique mechanism and is therefore an

attractive lead molecule for the development of a new class of antiosteoclastic agents . . . Bone quality is

maintained by a balance between bone resorption by osteoclasts and bone formation by osteoblasts.1

Excess bone resorption can occur systemically, where it leads to osteoporosis,2 or locally, where it is

associated with bone tumors,3,4 infections,5–7 and inflammatory responses.2,5,7 Although various

agents are available for the treatment of excess osteoclast activity, none are ideal for the treatment of

the associated pathologies. Osteoclasts are specialized cells of the hematopoietic lineage.8 Unusual

features of osteoclasts include overexpression of vacuolar H+-ATPases (V-ATPasesa) and the transport of

V-ATPases to the plasma membrane.9,10 While V-ATPases are expressed in all nucleated eukaryotic

cells, where they are responsible for acidification of compartments of the endocytic pathway, V-ATPases

are normally present at low levels and are forbidden to entry into the plasma membrane. The

mechanisms underlying the overexpression of V-ATPases and their transport to the plasma membrane

represent potential targets for therapeutic intervention that might be selective for osteoclast bone

resorption . . . The fact that inhibition of the interaction between the B subunit and F-actin in biochemical

assays and inhibition of osteoclast formation and bone resorption occurred in a similar concentration

range is consistent with the results emanating from the same activity. However, during the course of the

study, it was reported that enoxacin and some related fluoroquinolones stimulate RNA interference and

enhance microRNA activity, also in a similar dose range.19 Micro-RNAs are a recently identified class of

endogenously produced small RNAs that are thought to “fine tune” gene expression by binding to mRNAs

and preventing their translation by sequestering them or triggering their degradation.20 Evidence was

provided suggesting that enoxacin might interact with human immunodeficiency virus-1 transactivating

response RNA-binding protein, a protein involved in loading the RNA-induced silencing complex. It seems

unlikely (but not impossible) that stimulation of micro RNA activity could be the result of disruption of the

interaction between V-ATPase and F-actin. Stimulation of micro RNA activity, regardless of the

mechanism, could affect osteoclast differentiation . . . To explore this, we assayed several

fluoroquinolones for their ability to affect the interaction between V-ATPase and F-actin in vitro and to

inhibit osteoclast formation. These included norfloxacin, which was reported to stimulate microRNA

activity and others (pefloxacin and levofloxacin) that did not.19,21 We found that only pefloxacin had a

detectable effect on the interaction between B-subunit and F-actin. . . . Pefloxacin inhibited osteoclast

formation, with an IC50 of about 50 μM compared with 10 μM for enoxacin (Figure 7B). The fact that

norfloxacin, which also stimulates microRNA activity to similar levels as enoxacin,19,21 but does not

block V-ATPase–F-actin interactions, had no effect on osteoclast differentiation suggests that the

References 14 website: JMR, http://fluoroquinolonethyroid.com

microRNA stimulation activity is not important in inhibiting osteoclast differentiation at the

concentrations tested. This interpretation was further supported by the ability of pefloxacin, which does

not stimulate microRNA activity,21 to block the V-ATPase–F-actin interactions and inhibit osteoclast

formation. Taken together, these data are consistent with direct interference with the interaction

between subunit B2 of V-ATPase and microfilaments being responsible for the inhibition in osteoclast

formation. . . . The use of systemic enoxacin as an antibiotic has been linked to a number of adverse side

effects. 22–24 These include phototoxicity, neurological problems, severe tendinitis, adverse immune

activity, and renal failure due to distal renal tubular sensitivity. The capacity of enoxacin to block

interactions between V-ATPase and microfilaments could represent an underlying mechanism for these

adverse consequences. V-ATPases are key housekeeping enzymes, yet in certain specialized cell types,

subpopulations of V-ATPases are vital for cell type specific functions. For example, in both osteoclasts

and epithelial cells of the renal distal tubule, V-ATPases are expressed at high levels and inserted into the

plasma membrane in order to pump protons from the cytosol to the extracellular environment.25 In

neurons, V-ATPases are associated with loading neurotransmitters into vesicles and with the fusion of

those vesicles with the presynaptic plasma membrane.26 Further studies will be required to examine the

potential utility, as well as adverse effects, of enoxacin and related molecules. In summary, we report

the identification of enoxacin as an inhibitor of osteoclast formation and function by making use of a

rational structure-based approach, utilizing molecular docking of a large chemical library in combination

with biochemical and tissue culture assays. Our data suggest that enoxacin inhibits osteoclasts by a

novel mechanism, blocking a binding interaction between the V-ATPase and microfilaments. V-ATPase

activity is vital to osteoclast function, but because V-ATPase is expressed at low levels by all eukaryotic

cells and performs housekeeping functions, efforts to use inhibitors of the proton pumping activity of V-

ATPase to block bone resorption have not yet been successful. However, the interaction between V-

ATPase and microfilaments has not been observed in most cell types studies but appears crucial for

osteoclast function. We therefore reasoned that blocking the interaction might selectively inhibit

osteoclastic bone resorption. Our data to date support this concept and encourage us to advance

enoxacin as a lead molecule in the development of more potent and specific inhibitors of the interaction

between V-ATPase B2-subunit and microfilaments as a new class of antiresorptive agents. We aim to

identify safe and effective small molecules that represent a new class of antiosteoclastic agents that

block bone resorption by two distinct strategies. First, we will analyze a series of 20 structural variants of

enoxacin in structure–activity relationship studies. The most active compounds in this series will be

elaborated (derivatized) and compared with the activity of parent compounds in blocking the B-subunit–

F-actin binding interaction and in blocking osteoclast formation in vitro. A separate strategy involves

docking all FDA approved small molecules in the V-ATPase structural pocket comprised of Tyr68, Val89,

Thr268, Ile269, Glu308, and Arg314. Additional FDA approved drugs (approved for other purposes) are

expected to be identified. Because these compounds are approved for use, this strategy may rapidly

result in evaluation in clinical trials.”

https://www.ncbi.nlm.nih.gov/pubmed/22474295 Enoxacin directly inhibits osteoclastogenesis

without inducing apoptosis. “Enoxacin has been identified as a small molecule inhibitor of binding

between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone

resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly

References 14 website: JMR, http://fluoroquinolonethyroid.com

and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-

ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments . . . Our data

show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel

mechanism that involves changes in posttranslational processing and trafficking of several proteins with

known roles in osteoclast function. We propose that these effects are downstream to blocking the

binding interaction between a3-containing V-ATPases and microfilaments . . . Enoxacin is a

fluoroquinolone antibiotic that has been used extensively in humans for the treatment of urinary tract

infections and gonorrhea with minimal side effects (2). Recently, unexpected properties of enoxacin have

come to light. Our group, making use of a rational reverse chemical genetic strategy, identified enoxacin

as an inhibitor of vacuolar H+-ATPase (V-ATPase)3-microfilament binding and of osteoclast formation

and bone resorption in cell culture (1). Concurrently, others have identified enoxacin in screens for small

molecule stimulators of RNA interference and microRNA activity (3, 4). Very recently enoxacin was

reported to be a “cancer-specific” inhibitor that blocks the growth and metastases of human colorectal

cancers in a mouse model (5). Because of the therapeutic potential of enoxacin, it is vital to understand

the mechanisms by which it selectively affects osteoclasts. V-ATPases are essential “housekeeping”

enzymes in all eukaryotic cells that are necessary for the acidification of compartments of the endocytic

and phagocytic pathways (6, 7). Most cell types express only the low levels of V-ATPase required to carry

out housekeeping functions, but some cell types also contain an additional subset of V-ATPases that

plays a role in the unique functions of the cell. V-ATPases are composed of more than 10 subunits, and a

number of these subunits have multiple isoforms. Housekeeping V-ATPases are composed of a specific

subset of subunit isoforms, whereas non-housekeeping V-ATPases are marked by the inclusion of cell

type-restricted isoforms of one or more of the subunits. These subpopulations are targeted and utilized

differently than the housekeeping enzymes. Although it is well documented that particular isoforms of

certain V-ATPase subunits are found in V-ATPases that are targeted to atypical cellular locations, the

underlying mechanisms by which isoforms contribute to differential targeting and use of V-ATPases are

not understood ”

https://www.ncbi.nlm.nih.gov/pubmed/23958763 Bis-enoxacin inhibits bone resorption and

orthodontic tooth movement. “Enoxacin inhibits binding between the B-subunit of vacuolar H(+)-

ATPase (V-ATPase) and microfilaments, and also between osteoclast formation and bone resorption in

vitro. We hypothesized that a bisphosphonate derivative of enoxacin, bis-enoxacin (BE), which was

previously studied as a bone-directed antibiotic, might have similar activities. BE shared a number of

characteristics with enoxacin: It blocked binding between the recombinant B-subunit and microfilaments

and inhibited osteoclastogenesis in cell culture with IC50s of about 10 µM in each case. BE did not alter

the relative expression levels of various osteoclast-specific proteins. Even though tartrate-resistant acid

phosphatase 5b was expressed, proteolytic activation of the latent pro-enzyme was inhibited. However,

unlike enoxacin, BE stimulated caspase-3 activity. BE bound to bone slices and inhibited bone resorption

by osteoclasts on BE-coated bone slices in cell culture. BE reduced the amount of orthodontic tooth

movement achieved in rats after 28 days. Analysis of these data suggests that BE is a novel anti-

resorptive molecule that is active both in vitro and in vivo and may have clinical uses.”

References 14 website: JMR, http://fluoroquinolonethyroid.com

http://www.jbc.org/content/279/9/7988.full.html Vacuolar H+-ATPase Binding to Microfilaments

REGULATION IN RESPONSE TO PHOSPHATIDYLINOSITOL 3-KINASE ACTIVITY AND DETAILED

CHARACTERIZATION OF THE ACTIN-BINDING SITE IN SUBUNIT B. “Vacuolar H+-ATPase (V-ATPase)

binds microfilaments, and that interaction may be mediated by an actin binding domain in subunit B of

the enzyme . . . We sought to precisely map the actin-binding sites in the mammalian B subunits and to

identify amino acid residues that are crucial for the interaction. We demonstrate that the binding sites

are in 44-amino acid sections of the B subunits and that a portion of the binding sites, which is similar in

sequence to the actin-binding site of mammalian profilin I, is required for the actin binding activities of

the B subunits.”

https://www.ncbi.nlm.nih.gov/pubmed/10506172 Interaction between vacuolar H(+)-ATPase and

microfilaments during osteoclast activation. “Vacuolar H(+)-ATPases (V-ATPases) are multisubunit

enzymes that acidify compartments of the vacuolar system of all eukaryotic cells. In osteoclasts, the cells

that degrade bone, V-ATPases, are recruited from intracellular membrane compartments to the ruffled

membrane, a specialized domain of the plasma membrane, where they are maintained at high densities,

serving to acidify the resorption bay at the osteoclast attachment site on bone (Blair, H. C., Teitelbaum,

S. L., Ghiselli, R., and Gluck, S. L. (1989) Science 249, 855-857). Here, we describe a new mechanism

involved in controlling the activity of the bone-resorptive cell. V-ATPase in osteoclasts cultured in vitro

was found to form a detergent-insoluble complex with actin and myosin II through direct binding of V-

ATPase to actin filaments. Plating bone marrow cells onto dentine slices, a physiologic stimulus that

activates osteoclast resorption, produced a profound change in the association of the V-ATPase with

actin, assayed by coimmunoprecipitation and immunocytochemical colocalization of actin filaments and

V-ATPase in osteoclasts. Mouse marrow and bovine kidney V-ATPase bound rabbit muscle F-actin

directly with a maximum stoichiometry of 1 mol of V-ATPase per 8 mol of F-actin and an apparent

affinity of 0.05 microM. Electron microscopy of negatively stained samples confirmed the binding

interaction. These findings link transport of V-ATPase to reorganization of the actin cytoskeleton during

osteoclast activation.”

https://www.ncbi.nlm.nih.gov/pubmed/27824360 Association of ATP6V1B2 rs1106634 with lifetime

risk of depression and hippocampal neurocognitive deficits: possible novel mechanisms in the

etiopathology of depression. “Current understanding and treatment of depression is limited to the

monoaminergic theory with little knowledge of the involvement of other cellular processes. Genome-

wide association studies, however, implicate several novel single-nucleotide polymorphisms with weak

but replicable effects and unclarified mechanisms. We investigated the effect of rs1106634 of the

ATPV1B2 gene encoding the vacuolar H+ATPase on lifetime and current depression and the possible

mediating role of neuroticism by logistic and linear regression in a white European general sample of

2226 subjects. Association of rs1106634 with performance on frontal (Stockings of Cambridge (SOC)) and

hippocampal-dependent (paired associates learning (PAL)) cognitive tasks was investigated in

multivariate general linear models in a smaller subsample. The ATP6V1B2 rs1106634 A allele had a

significant effect on lifetime but not on current depression. The effect of the A allele on lifetime

depression was not mediated by neuroticism. The A allele influenced performance on the PAL but not on

the SOC test. We conclude that the effects of variation in the vacuolar ATPase may point to a new

References 14 website: JMR, http://fluoroquinolonethyroid.com

molecular mechanism that influences the long-term development of depression. This mechanism may

involve dysfunction specifically in hippocampal circuitry and cognitive impairment that characterizes

recurrent and chronic depression.”

https://www.ncbi.nlm.nih.gov/pubmed/20038947 Novel loci for major depression identified by

genome-wide association study of Sequenced Treatment Alternatives to Relieve Depression and

meta-analysis of three studies. “We report a genome-wide association study (GWAS) of major

depressive disorder (MDD) in 1221 cases from the Sequenced Treatment Alternatives to Relieve

Depression (STAR*D) study and 1636 screened controls. No genome-wide evidence for association was

detected. We also carried out a meta-analysis of three European-ancestry MDD GWAS data sets:

STAR*D, Genetics of Recurrent Early-onset Depression and the publicly available Genetic Association

Information Network-MDD data set. These data sets, totaling 3957 cases and 3428 controls, were

genotyped using four different platforms (Affymetrix 6.0, 5.0 and 500 K, and Perlegen). For each of 2.4

million HapMap II single-nucleotide polymorphisms (SNPs), using genotyped data where available and

imputed data otherwise, single-SNP association tests were carried out in each sample with correction for

ancestry-informative principal components. The strongest evidence for association in the meta-analysis

was observed for intronic SNPs in ATP6V1B2 (P=6.78 x 10⁻⁷), SP4 (P=7.68 x 10⁻⁷) and GRM7 (P=1.11 x

10⁻⁶). Additional exploratory analyses were carried out for a narrower phenotype (recurrent MDD with

onset before age 31, N=2191 cases), and separately for males and females. Several of the best findings

were supported primarily by evidence from narrow cases or from either males or females. On the basis of

previous biological evidence, we consider GRM7 a strong MDD candidate gene. Larger samples will be

required to determine whether any common SNPs are significantly associated with MDD.”

https://www.ncbi.nlm.nih.gov/pubmed/27340853 Human ApoE ɛ2 Promotes Regulatory Mechanisms

of Bioenergetic and Synaptic Function in Female Brain: A Focus on V-type H+-ATPase. “Humans

possess three major isoforms of the apolipoprotein E (ApoE) gene encoded by three alleles: ApoE ɛ2

(ApoE2), ApoE ɛ3 (ApoE3), and ApoE ɛ4 (ApoE4). It is established that the three ApoE isoforms confer

differential susceptibility to Alzheimer's disease (AD); however, an in-depth molecular understanding of

the underlying mechanisms is currently unavailable. In this study, we examined the cortical proteome

differences among the three ApoE isoforms using 6-month-old female, human ApoE2, ApoE3, and ApoE4

gene-targeted replacement mice and two-dimensional proteomic analyses. The results reveal that the

three ApoE brains differ primarily in two areas: cellular bioenergetics and synaptic transmission. Of

particular significance, we show for the first time that the three ApoE brains differentially express a key

component of the catalytic domain of the V-type H+-ATPase (Atp6v), a proton pump that mediates the

concentration of neurotransmitters into synaptic vesicles and thus is crucial in synaptic transmission.

Specifically, our data demonstrate that ApoE2 brain exhibits significantly higher levels of the B subunit of

Atp6v (Atp6v1B2) when compared to both ApoE3 and ApoE4 brains, with ApoE4 brain exhibiting the

lowest expression. Our additional analyses show that Atp6v1B2 is significantly impacted by aging and AD

pathology and the data suggest that Atp6v1B2 deficiency could be involved in the progressive loss of

synaptic integrity during early development of AD. Collectively, our findings indicate that human ApoE

isoforms differentially modulate regulatory mechanisms of bioenergetic and synaptic function in female

brain. A more efficient and robust status in both areas-in which Atp6v may play a role-could serve as a

References 14 website: JMR, http://fluoroquinolonethyroid.com

potential mechanism contributing to the neuroprotective and cognition-favoring properties associated

with the ApoE2 genotype.”

https://www.ncbi.nlm.nih.gov/pubmed/2145275 An mRNA from human brain encodes an isoform of

the B subunit of the vacuolar H(+)-ATPase. “The B subunit (approximately 60 kDa) of the vacuolar

H(+)-ATPase is one of the two major subunits comprising the hydrophilic catalytic complex of the

enzyme. Using left and catalytic complex of the enzyme. Using left and right primers which bind two

highly conserved sequences of the B subunit, an 836-base pair fragment was amplified from human brain

cDNA by the polymerase chain reaction. The amplified fragment was used to probe a Northern blot and

to screen a brain cDNA library. A single RNA band, 3.2 kilobases (kb) in length, was detected on Northern

blots. A positive cDNA clone containing a 2.5-kb insert was isolated and sequenced. It included a long 3'-

untranslated region (greater than 1.2 kb) and was missing a minor portion of the 5'-end of the coding

region. The coding region of the brain cDNA sequence was 77% identical at the nucleotide level and 90%

identical at the amino acid level to the previously reported sequence for the B subunit of the vacuolar

H(+)-ATPase from human kidney (Sudhof, T. C., Fried, V. A., Stone, D. K., Johnston, P. A., and Xie, X.-S.

(1989) Proc. Natl. Acad. Sci, U. S. A. 86, 6067-6071). Within the coding region of the brain cDNA, which is

6 amino acid residues shorter at the 3'-end than the kidney sequence, an 11% difference in the GC

content was calculated. The 3'-noncoding sequence of the brain cDNA was completely unrelated to that

of kidney and was three times longer. We conclude that the B subunit cDNAs from human kidney and

brain represent different isoforms. This is the first demonstration of an isoform of a vacuolar H(+)-ATPase

subunit.”

https://www.clinicalkey.com/#!/content/journal/1-s2.0-S1568163716300800 Disorders of lysosomal

acidification—The emerging role of v-ATPase in aging and neurodegenerative disease. (2016). Good

paper, but could not download.

https://www.ncbi.nlm.nih.gov/pubmed/1371275 Differential expression of the "B" subunit of the

vacuolar H(+)-ATPase in bovine tissues.

https://www.ncbi.nlm.nih.gov/pubmed/17497178 Differential expression of a subunit isoforms of the

vacuolar-type proton pump ATPase in mouse endocrine tissues. “Vacuolar-type proton ATPase (V-

ATPase) is a multi-subunit enzyme that couples ATP hydrolysis to the translocation of protons across

membranes. Mammalian cells express four isoforms of the a subunit of V-ATPase. Previously, we have

shown that V-ATPase with the a3 isoform is highly expressed in pancreatic islets and is located in the

membranes of insulin-containing granules in the beta cells. The a3 isoform functions in the regulation of

hormone secretion. In this study, we have examined the distribution of a subunit isoforms in endocrine

tissues, including the adrenal, parathyroid, thyroid, and pituitary glands, with isoform-specific

antibodies. We have found that the a3 isoform is strongly expressed in all these endocrine tissues. Our

results suggest that functions of the a3 isoform are commonly involved in the process of exocytosis in

regulated secretion.”

http://www.jbc.org/content/266/7/4392.long ATP-dependent uptake of 5-hydroxytryptamine by

secretory granules isolated from thyroid parafollicular cells. “The current study was done to test the

References 14 website: JMR, http://fluoroquinolonethyroid.com

hypotheses that parafollicular granules contain a vacuolar ATPase (V-ATPase) similar to that found in

chromaffin granules, that the transport of H+ into granules mediated by this enzyme drives the granular

uptake of 5-hydroxytryptamine (5-HT, serotonin), and that secretagogues stimulate both the

acidification of parafollicular granules and their ability to take up 5-HT by opening an anion channel in

the granular membrane. Our studies indicate that parafollicular granules contain a V-ATPase that is

antigenically similar to that of the V-ATPase of adrenal chromaffin granules; however, the parafollicular

granular membrane differs from that of chromaffin granules in permeability to Cl- and K+. The

membranes of granules derived from resting parafollicular cells appear to be relatively impermeable to

Cl- but permeable to K+. Parafollicular granules (and ghosts derived from them) manifest ATP-dependent

transmembrane transport of 5-HT. This transport is more dependent on the pH difference (delta pH) than

on the membrane potential component of the proton electrochemical gradient across the granular

membrane. Transport of 5-HT is thus inhibited more by exposure of parafollicular granules to agents,

such as nigericin, that collapse delta pH than by those, such as valinomycin, that decrease

transmembrane difference in potential. ATP-dependent uptake of 5-HT by granules isolated from

secretagogue-stimulated parafollicular cells is greater than that into granules isolated from

unstimulated cells. Since secretagogues open a Cl- channel in parafollicular granule membranes, which

enhances acidification of the granules, the facilitation of 5-HT uptake by secretagogues is probably due

to an increase in delta pH.

https://www.ncbi.nlm.nih.gov/pubmed/2252332 Serotonin-storing secretory vesicles. “Advances

have been made in the characterization of 5-HT-storing organelles of neurectodermal cells. The

parafollicular cell of the thyroid has been used as a model. This cell stores 5-HT, shares many properties

with neurons, and can be induced to change its phenotype from endocrine to neuronal by exposure in

vitro to NGF. The membranes of isolated parafollicular 5-HT storage vesicles appear to contain a chloride

channel that is gated in response to stimulation of the cells by secretogogues. Opening of this channel

permits the interior of the vesicle to acidify in response to the action of a H+ ATPase in the vesicular

membrane. Development of a delta psi appears to limit acidification of the vesicular interior when the

chloride conductance is low. Transmembrane transport of 3H-5-HT into parafollicular vesicle is inhibited

by dissipating the delta pH across the granular membranes. The physiological significance of the ability

of parafollicular vesicles to modify the internal pH of their 5-HT-storing organelles remains to be

determined. Like the synaptic vesicles of central and peripheral serotonergic neurons parafollicular

vesicles contain a specific 5-HT binding protein, SBP. 5-HT storage organelles and SBP have been found in

medullary thyroid carcinoma (MTC) cells, a tumor line derived from parafollicular cells. The cell biology of

SBP is now under study utilizing the MTC cells.”

https://www.ncbi.nlm.nih.gov/pubmed/12740220 Thyroid hormone stimulates Na-K-ATPase activity

and its plasma membrane insertion in rat alveolar epithelial cells. “Na-K-ATPase protein is critical for

maintaining cellular ion gradients and volume and for transepithelial ion transport in kidney and lung.

Thyroid hormone, 3,3',5-triiodo-l-thyronine (T3), given for 2 days to adult rats, increases alveolar fluid

resorption by 65%, but the mechanism is undefined. We tested the hypothesis that T3 stimulates Na-K-

ATPase in adult rat alveolar epithelial cells (AEC), including primary rat alveolar type II (ATII) cells, and

determined mechanisms of the T3 effect on the Na-KATPase enzyme using two adult rat AEC cell lines

References 14 website: JMR, http://fluoroquinolonethyroid.com

(MP48 and RLE-6TN). T3 at 10-8 and 10-5 M increased significantly hydrolytic activity of Na-K-ATPase in

primary ATII cells and both AEC cell lines. The increased activity was dose dependent in the cell lines (10-

9-10-4 M) and was detected within 30 min and peaked at 6 h. Maximal increases in Na-K-ATPase activity

were twofold in MP48 and RLE-6TN cells at pharmacological T3 of 10-5 and 10-4 M, respectively, but

increases were statistically significant at physiological T3 as low as 10-9 M. This effect was T3 specific,

because reverse T3 (3,3',5'-triiodo-l-thyronine) at 10-9-10-4 M had no effect. The T3-induced increase in

Na-K-ATPase hydrolytic activity was not blocked by actinomycin D. No significant change in mRNA and

total cell protein levels of Na-K-ATPase were detected with 10-9-10-5 M T3 at 6 h. However, T3 increased

cell surface expression of Na-K-ATPase alpha1- or beta1-subunit proteins by 1.7- and 2-fold, respectively,

and increases in Na-K-ATPase activity and cell surface expression were abolished by brefeldin A. These

data indicate that T3 specifically stimulates Na-K-ATPase activity in adult rat AEC. The upregulation

involves translocation of Na-K-ATPase to plasma membrane, not increased gene transcription. These

results suggest a novel nontranscriptional mechanism for regulation of Na-K-ATPase by thyroid

hormone.”

https://www.einstein.yu.edu/uploadedFiles/LABS/vlad-

verkhusha/Champa%20et%20al%20Oncotarget%202016.pdf Obatoclax kills anaplastic thyroid cancer

cells by inducing lysosome neutralization and necrosis. “We demonstrate that Obatoclax does not

induce apoptosis, but rather necrosis of thyroid cancer cells, and that non-transformed thyroid cells are

significantly less affected by this compound. Surprisingly, we show that Obatoclax rapidly localizes to the

lysosomes and induces loss of acidification, block of lysosomal fusion with autophagic vacuoles, and

subsequent lysosomal permeabilization. . . . Finally, we show that also other lysosome-targeting

compounds, Mefloquine and LLOMe, readily induce necrosis in thyroid cancer cells, and that Mefloquine

significantly impairs tumor growth in vivo, highlighting a clear vulnerability of these aggressive,

apoptosis-resistant tumors that can be therapeutically exploited . . . We have now characterized the

mechanism of action of Obatoclax in thyroid cancer cells and present data supporting the surprising

conclusion that its efficacy in our model systems is independent of BCL2 family member targeting.

Instead, Obatoclax appears to localize to and functionally disrupt lysosomes, thus uncovering a critical

vulnerability of thyroid cancer cells, which we show can be also targeted using additional, molecularly

distinct, lysosome-disrupting small molecules. . . . Given the acidic environment of lysosomes, we

wondered whether Obatoclax was only fluorescent at low pH conditions, and, as a consequence, whether

we might just be unable to detect its presence in other cellular compartments due to a loss of

fluorescence. Thus, we measured Obatoclax’ fluorescence emission spectrum at different pH values and

found that fluorescence of Obatoclax is indeed dependent on pH (Figure 3E). The fluorescence intensity

changed 2-fold with the pH changes in the range of 2–12 (Figure 3F). Highest fluorescence was observed

in acidic environment. However, while acidic conditions increased Obatoclax fluorescence emission, the

difference between fluorescence intensity at cytoplasmic and lysosomal pH values was less than 25%

(Figure 3F), suggesting that, in fact, Obatoclax was rapidly and exclusively trapped in lysosomes. . . .

These results strongly suggest that Obatoclax causes a late block in lysosomal function by rendering

lysosomes unable to fully digest their content. It is also tempting to hypothesize that the large vesicles

engulf damaged lysosomes in an attempt to isolate them from the intracellular environment. . . .

Lysosome membrane permeabilization (LMP) causes cell death through the acidification of the cytosol

References 14 website: JMR, http://fluoroquinolonethyroid.com

and/or the release of active cathepsins in the cytoplasm. Therefore, we sought to determine whether

lysosomal acidity is important for the cells’ necrotic response to Obatoclax. We first tested the ability of

Bafilomycin A1 (BafA1), a V-ATPase inhibitor, to prevent the acidification of lysosomes. D445 cells were

incubated with Lysotracker in the presence or absence of BafA1 and analyzed by microscopy. BafA1 was

indeed able to prevent the acidification of lysosomes, as determined by the loss of Lysotracker signal

(Figure 5A). Thus, we pre-treated D445 cells with BafA1 for 2 hours before exposing them to Obatoclax

and assessing their cell number after 16 hours. Lysosome neutralization significantly reduced cell

proliferation in cells not treated with Obatoclax, suggesting that full lysosomal function is required for

proper cell growth. Strikingly, Obatoclax ability to kill thyroid cancer cells was severely reduced in the

presence of BafA1 (Figure 5B). . . . Therefore, it appears that an intact lysosomal acidic environment is

critical for the induction of cell death by Obatoclax. . . . If Obatoclax induces thyroid cancer cells necrosis

through its ability to neutralize and permeabilize lysosomes, then other well-established lysosomotropic

agents should be able to produce similar effects . . . We first tested the ability of Mefloquine to mimic

Obatoclax activity. Mefloquine is an anti-malarial compound that has been shown to localize to and

disrupt lysosomes in acute myeloid leukemia cells [32].

http://link.springer.com/article/10.1007/BF02899539 Functional and molecular analysis of

mitochondria in thyroid oncocytoma. “We report a functional and molecular analysis of nine oncocytic

tumors of the human thyroid. In all the abundance of mitochondria observed ultrastructurally was

accompanied by an increase in enzymatic activities of respiratory complexes I (NADH dehydrogenase), II

(succinate dehydrogenase) IV (cytochrome c oxidase), and V (ATPase). Western blot analysis failed to

detect uncoupling protein in the tumors. The elevated respiratory enzyme activities were paralleled by an

increase in the mitochondrial DNA content. Restriction analysis of mitochondrial DNA gave no indication

of heteroplasmy or other gross alterations. We conclude that the mitochondrial proliferation in oncocytic

tumors is probably not the result of a compensatory mechanism for the deficiency in enzyme complexes

of the mitochondrial respiratory chain.”

https://www.researchgate.net/profile/Masamitsu_Futai/publication/5340168_The_V-type_H-

ATPase_in_vesicular_trafficking_targeting_regulation_and_function._Curr_Opin_Cell_Biol/links/0deec5

2e4dfa781b6d000000.pdf The V-type H+-ATPase in vesicular trafficking: targeting, regulation and

function. Overview

https://www.ncbi.nlm.nih.gov/pubmed/10221984 Vacuolar and plasma membrane proton-

adenosinetriphosphatases. “The vacuolar H+-ATPase (V-ATPase) is one of the most fundamental

enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles

and membranes. V-ATPases have similar structure and mechanism of action with F-ATPase and several

of their subunits evolved from common ancestors. In eukaryotic cells, F-ATPases are confined to the

semi-autonomous organelles, chloroplasts, and mitochondria, which contain their own genes that

encode some of the F-ATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic

cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as

ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of

eukaryotic cells is utilized as a driving force for numerous secondary transport processes. The

mechanistic and structural relations between the two enzymes prompted us to suggest similar functional

References 14 website: JMR, http://fluoroquinolonethyroid.com

units in V-ATPase as was proposed to F-ATPase and to assign some of the V-ATPase subunit to one of

four parts of a mechanochemical machine: a catalytic unit, a shaft, a hook, and a proton turbine. It was

the yeast genetics that allowed the identification of special properties of individual subunits and the

discovery of factors that are involved in the enzyme biogenesis and assembly. The V-ATPases play a

major role as energizers of animal plasma membranes, especially apical plasma membranes of epithelial

cells. This role was first recognized in plasma membranes of lepidopteran midgut and vertebrate kidney.

The list of animals with plasma membranes that are energized by V-ATPases now includes members of

most, if not all, animal phyla. This includes the classical Na+ absorption by frog skin, male fertility

through acidification of the sperm acrosome and the male reproductive tract, bone resorption by

mammalian osteoclasts, and regulation of eye pressure. V-ATPase may function in Na+ uptake by trout

gills and energizes water secretion by contractile vacuoles in Dictyostelium. V-ATPase was first detected

in organelles connected with the vacuolar system. It is the main if not the only primary energy source for

numerous transport systems in these organelles. The driving force for the accumulation of

neurotransmitters into synaptic vesicles is pmf generated by V-ATPase. The acidification of lysosomes,

which are required for the proper function of most of their enzymes, is provided by V-ATPase. The

enzyme is also vital for the proper function of endosomes and the Golgi apparatus. In contrast to yeast

vacuoles that maintain an internal pH of approximately 5.5, it is believed that the vacuoles of lemon fruit

may have a pH as low as 2. Similarly, some brown and red alga maintain internal pH as low as 0.1 in

their vacuoles. One of the outstanding questions in the field is how such a conserved enzyme as the V-

ATPase can fulfill such diverse functions.”

https://www.ncbi.nlm.nih.gov/pubmed/24508215?dopt=Abstract Eukaryotic V-ATPase: novel

structural findings and functional insights. “The eukaryotic V-type adenosine triphosphatase (V-

ATPase) is a multi-subunit membrane protein complex that is evolutionarily related to F-type adenosine

triphosphate (ATP) synthases and A-ATP synthases. These ATPases/ATP synthases are functionally

conserved and operate as rotary proton-pumping nano-motors, invented by Nature billions of years ago.

In the first part of this review we will focus on recent structural findings of eukaryotic V-ATPases and

discuss the role of different subunits in the function of the V-ATPase holocomplex. Despite structural and

functional similarities between rotary ATPases, the eukaryotic V-ATPases are the most complex enzymes

that have acquired some unconventional cellular functions during evolution. In particular, the novel roles

of V-ATPases in the regulation of cellular receptors and their trafficking via endocytotic and exocytotic

pathways were recently uncovered. In the second part of this review we will discuss these unique roles of

V-ATPases in modulation of function of cellular receptors, involved in the development and progression

of diseases such as cancer and diabetes as well as neurodegenerative and kidney disorders. Moreover, it

was recently revealed that the V-ATPase itself functions as an evolutionarily conserved pH sensor and

receptor for cytohesin-2/Arf-family GTP-binding proteins. Thus, in the third part of the review we will

evaluate the structural basis for and functional insights into this novel concept, followed by the analysis

of the potentially essential role of V-ATPase in the regulation of this signaling pathway in health and

disease. Finally, future prospects for structural and functional studies of the eukaryotic V-ATPase will be

discussed.”

References 14 website: JMR, http://fluoroquinolonethyroid.com

https://www.ncbi.nlm.nih.gov/pubmed/18667600 V-ATPase expression in the mouse olfactory

epithelium. “The vacuolar proton-pumping ATPase (V-ATPase) is responsible for the acidification of

intracellular organelles and for the pH regulation of extracellular compartments. Because of the

potential role of the latter process in olfaction, we examined the expression of V-ATPase in mouse

olfactory epithelial (OE) cells. We report that V-ATPase is present in this epithelium, where we detected

subunits ATP6V1A (the 70-kDa "A" subunit) and ATP6V1E1 (the ubiquitous 31-kDa "E" subunit isoform) in

epithelial cells, nerve fiber cells, and Bowman's glands by immunocytochemistry. We also located both

isoforms of the 56-kDa B subunit, ATP6V1B1 ("B1," typically expressed in epithelia specialized in

regulated transepithelial proton transport) and ATP6V1B2 ("B2") in the OE. B1 localizes to the microvilli

of the apical plasma membrane of sustentacular cells and to the lateral membrane in a subset of

olfactory sensory cells, which also express carbonic anhydrase type IV, whereas B2 expression is stronger

in the subapical domain of sustentacular cells. V-ATPase expression in mouse OE was further confirmed

by immunoblotting. These findings suggest that V-ATPase may be involved in proton secretion in the OE

and, as such, may be important for the pH homeostasis of the neuroepithelial mucous layer and/or for

signal transduction in CO(2) detection.”

https://www.ncbi.nlm.nih.gov/pubmed/23028982 Loss of the V-ATPase B1 subunit isoform expressed

in non-neuronal cells of the mouse olfactory epithelium impairs olfactory function. “The vacuolar

proton-pumping ATPase (V-ATPase) is the main mediator of intracellular organelle acidification and also

regulates transmembrane proton (H(+)) secretion, which is necessary for an array of physiological

functions fulfilled by organs such as the kidney, male reproductive tract, lung, bone, and ear. In this study

we characterize expression of the V-ATPase in the main olfactory epithelium of the mouse, as well as a

functional role for the V-ATPase in odor detection. We report that the V-ATPase localizes to the apical

membrane microvilli of olfactory sustentacular cells and to the basolateral membrane of microvillar cells.

Plasma membrane V-ATPases containing the B1 subunit isoform are not detected in olfactory sensory

neurons or in the olfactory bulb. This precise localization of expression affords the opportunity to

ascertain the functional relevance of V-ATPase expression upon innate, odor-evoked behaviors in B1-

deficient mice. This animal model exhibits diminished innate avoidance behavior (revealed as a decrease

in freezing time and an increase in the number of sniffs in the presence of trimethyl-thiazoline) and

diminished innate appetitive behavior (a decrease in time spent investigating the urine of the opposite

sex). We conclude that V-ATPase-mediated H(+) secretion in the olfactory epithelium is required for

optimal olfactory function.”

https://www.ncbi.nlm.nih.gov/pubmed/17898041 Compensatory membrane expression of the V-

ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice. “Mice deficient

in the ATP6V1B1 ("B1") subunit of the vacuolar proton-pumping ATPase (V-ATPase) maintain body acid-

base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory

mechanisms involving the alternate ATP6V1B2 ("B2") isoform were examined to explain the persistence

of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical

B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B1-/- mice than in

B1+/+ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2

mRNA or protein expression was detected in B1-/- mice compared with wild-type controls. We conclude

References 14 website: JMR, http://fluoroquinolonethyroid.com

that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the

cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was

confirmed by the intracellular accumulation of B1-deficient V-ATPases in response to the microtubule-

disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of "rod-

shaped" intramembranous particles seen by freeze fracture microscopy in apical membranes of normal

and B1-deficient A-ICs. Intracellular pH recovery assays show that significant (28-40% of normal) V-

ATPase function is preserved in medullary ICs from B1-/- mice. We conclude that the activity of apical B2-

containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B1-

deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to

metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their

urine under these conditions.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973240/ BAX inhibitor-1-associated V-ATPase

glycosylation enhances collagen degradation in pulmonary fibrosis. “Endoplasmic reticulum (ER)

stress is considered one of the pathological mechanisms of idiopathic pulmonary fibrosis (IPF). Therefore,

we examined whether an ER stress regulator, Bax inhibitor-1 (BI-1), regulates collagen accumulation,

which is both a marker of fibrosis and a pathological mechanism of fibrosis. The presence of BI-1

inhibited the transforming growth factor-β1-induced epithelial–mesenchymal transition of epithelial

pulmonary cells and bleomycin-induced pulmonary fibrosis in a mouse model by enhancing collagen

degradation, most likely by enhanced activation of the lysosomal V-ATPase through glycosylation. We

also found a correlation between post-translational glycosylation of the V-ATPase and its associated

chaperone, calnexin, in BI-1-overexpressing cells. BI-1-induced degradation of collagen through

lysosomal V-ATPase glycosylation and the involvement of calnexin were confirmed in a bleomycin-

induced fibrosis mouse model. These results highlight the regulatory role of BI-1 in IPF and reveal for the

first time the role of lysosomal V-ATPase glycosylation in IPF.”

http://www.sciencedirect.com/science/article/pii/S0006291X98993573 Involvement of V-ATPases in

the Digestion of Soft Connective Tissue Collagen. “The contribution of vacuolar H+-ATPases (V-

ATPases) to collagen degradation was investigated in soft connective tissue explants (periosteum).

Immunolocalisation showed faint to intense staining of cells throughout the periosteum. The V-ATPase

inhibitors, bafilomycin A1and folimycin, decreased overall collagen degradation by 40 and 50% after 24

and 48 h, respectively. The participation of V-ATPases in intracellular degradation of collagen was

demonstrated by the decrease of the amount of phagocytosed collagen in fibroblasts upon inhibition of

pump activity. The inhibition of degradation was not due to a reduction in activity of gelatinase A, an

enzyme previously found to mediate collagen degradation, as assessed by zymographic analysis of tissue

and conditioned medium. Bafilomycin A1 even induced an increase of gelatinase A and B levels in both

fractions. In conclusion, acidification by V-ATPases may represent an important mechanism in

extracellular and intracellular collagen degradation in soft connective tissue.”

http://dmm.biologists.org/content/6/3/689 Elevated expression of the V-ATPase C subunit triggers

JNK-dependent cell invasion and overgrowth in a Drosophila epithelium.

References 14 website: JMR, http://fluoroquinolonethyroid.com

http://cancerres.aacrjournals.org/content/76/14_Supplement/1570 Abstract 1570: Defective collagen

production by mammary epithelia leads to increased breast cancer metastasis: a novel role of

vacuolar ATPase.

https://www.ncbi.nlm.nih.gov/pubmed/1312570 Characterization of the P-type and V-type ATPases

of cholinergic synaptic vesicles and coupling of nucleotide hydrolysis to acetylcholine transport.

“Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in

cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal

antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by

assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two

overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein

isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and

activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by

hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed

two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be

a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and

Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine

(ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km

for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at

concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-

type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The

V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of

vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps

are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the

synaptic vesicle preparation is demonstrated.”

https://www.ncbi.nlm.nih.gov/pubmed/26404656 Alteration of complex sphingolipid composition

and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase. “In the

yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group and five

types of ceramide; however, the physiological significance of the structural diversity is not fully

understood. Here, we report that deletion of vacuolar H+-ATPase (V-ATPase) in yeast causes dramatic

alteration of the complex sphingolipid composition, which includes decreases in hydroxylation at the C-4

position of long-chain bases and the C-2 position of fatty acids in the ceramide moiety, decreases in

inositol phosphorylceramide (IPC) levels, and increases in mannosylinositol phosphorylceramide (MIPC)

and mannosyldiinositol phosphorylceramide [M(IP)2C] levels. V-ATPase-deleted cells exhibited slow

growth at pH 7.2, whereas the increase in MIPC levels was significantly enhanced when V-ATPase-

deleted cells were incubated at pH 7.2. The protein expression levels of MIPC and M(IP)2C synthases

were significantly increased in V-ATPase-deleted cells incubated at pH 7.2. Loss of MIPC synthesis or an

increase in the hydroxylation level of the ceramide moiety of sphingolipids on overexpression of Scs7 and

Sur2 sphingolipid hydroxylases enhanced the growth defect of V-ATPase-deleted cells at pH 7.2. On the

contrary, the growth rate of V-ATPase-deleted cells was moderately increased on the deletion of SCS7

and SUR2. In addition, supersensitivities to Ca2+, Zn2+ and H2O2, which are typical phenotypes of V-

References 14 website: JMR, http://fluoroquinolonethyroid.com

ATPase-deleted cells, were enhanced by the loss of MIPC synthesis. These results indicate the possibility

that alteration of the complex sphingolipid composition is an adaptation mechanism for a defect of V-

ATPase.”

http://www.genetics.org/content/187/3/771 A Genome-Wide Enhancer Screen Implicates

Sphingolipid Composition in Vacuolar ATPase Function in Saccharomyces cerevisiae. “The function of

the vacuolar H1-ATPase (V-ATPase) enzyme complex is to acidify organelles; this process is critical for a

variety of cellular processes and has implications in human disease. There are five accessory proteins

that assist in assembly of the membrane portion of the complex, the V0 domain. To identify additional

elements that affect V-ATPase assembly, trafficking, or enzyme activity, we performed a genome-wide

enhancer screen in the budding yeast Saccharomyces cerevisiae with two mutant assembly factor alleles,

VMA21 with a dysfunctional ER retrieval motif (vma21QQ ) and vma21QQ in combination with voa1D, a

nonessential assembly factor. These alleles serve as sensitized genetic backgrounds that have reduced V-

ATPase enzyme activity. Genes were identified from a variety of cellular pathways including a large

number of trafficking-related components; we characterized two redundant gene pairs, HPH1/ HPH2 and

ORM1/ORM2. Both sets demonstrated synthetic growth defects in combination with the vma21QQ

allele. A loss of either the HPH or ORM gene pairs alone did not result in a decrease in vacuolar

acidification or defects in V-ATPase assembly. While the Hph proteins are not required for V-ATPase

function, Orm1p and Orm2p are required for full V-ATPase enzyme function. Consistent with the

documented role of the Orm proteins in sphingolipid regulation, we have found that inhibition of

sphingolipid synthesis alleviates Orm-related growth defects.”

http://www.ncf-net.org/forum/outsideBox-F06.htm “In his patent, Dr. Gow expressed his interest in

the ATPase system. According to his patent, "At the cellular level, fatigue has been linked with

alterations in the cell membrane ion-channel traffic and ATPase system. ATPases are also linked with

neurotransmitter release (e.g . dopamine) and cellular energy metabolism via creatine phosphatase.

Increased ATPase activity has been reported in muscle biopsies from patients with CFS. Previous work

has raised the possibility that patients with CFS may have an ion channel dysfunction. This dysfunction

might be induced by changes in the ion channel function, neurotransmitters involved in "gating" the

channel or by a shift in the balance of the cellular "energy charge", i.e - the ratio between ATP and ADP

that is normally a function of the ATPase activity." Dr. Gow continued, "A group at the University of

UIm, Germany, has recently suggested that a pentapeptide (QYNAD) with Na+ channel-blocking function

could be a biological marker of certain inflammatory and immunological disorders of the nervous

system. The inventors asked whether or not the pentapeptide identified by the German group might

play a role in CFS. Samples of serum were sent to the University of UIm for analysis. The 15 samples

included 5 normal controls, 5 patients with CFS and 5 disease controls including two patients with MS.

Samples were numbered 1-15 and the German group were not informed what the samples were, or

which samples were which, until the experiment was concluded. When the code was broken, the results

showed that the disease control group had levels of the pentapeptide which were 2.3X those of the

normal controls (similar to the published data) and the CFS samples had levels which were 3X higher

than the healthy controls....The German group was unable to identify an endogenous gene which

encodes the pentapeptide. The inventors carried out NCBI BLAST and EMBL-Heidelberg Bioccelerator

References 14 website: JMR, http://fluoroquinolonethyroid.com

amino acid alignments for the pentapeptide QYNAD....A number of cloned nucleotide sequences were

found and when these were run through the nucleotide databases, only one clone showed full-length

homology to any human gene. This gene was a human ion-channel gene - the vacuolar proton pump H+

ATPase (v-ATPase)." Dr. Gow then stated "The inventors next asked whether the v-ATPase represents a

candidate gene for a diagnostic test for CFS....the patient samples have a significantly higher level of v-

ATPase mRNA than the healthy controls. Thus the v-ATPase gene appears to represent a genuine

biomarker for CFS/ME. . . . Furthermore, Dr. Gow states in his patent that "The v-ATPase is known to be

involved in regulation of a number of metabolic functions which are deranged in CFS/ME. v-ATPase

upregulation could therefore provide an explanation for a number of the symptoms observed. For

example, increased v-ATPase activity could explain the intracellular acidosis in exercising muscles, chest

pain (syndrome X), altered neurotransmitter (dopamine) function and abnormal regulation of

hypothalamic hormones. In addition, it could explain the increased energy expenditure and fatigue

associated with the condition."

http://www.jbc.org/content/278/31/28872.abstract Sphingolipid Requirement for Generation of a

Functional V1 Component of the Vacuolar ATPase. “There has been no previous indication that

vacuolar ATPases (V-ATPases) require sphingolipids for function. Here we show, by using Saccharomyces

cerevisiae sur4Δ and fen1Δ cells, that sphingolipids with a C26 acyl group are required for generating V1

domains with ATPase activity. Sphingolipids in sur4Δ cells contain C22 and C24 acyl groups instead of

C26 acyl groups whereas about 30% of the sphingolipids in fen1Δ cells have C26 acyl groups and the rest

have C22 and C24 acyl groups. sur4Δ cells have several phenotypes (vacuolar membrane ATPase, Vma–)

that indicate a defect in the V-ATPase, and vacuoles purified from sur4Δ cells have little to no ATPase

activity. These phenotypes are less pronounced in fen1Δ cells, consistent with the idea that the C26 acyl

group in sphingolipids is necessary for V-ATPase activity. Other results show that the two V-ATPase

domains, V1 and V0, are assembled and delivered to the vacuolar membrane in sur4Δ cells similar to

wild-type cells. In vitro assembly studies show that V1 from sur4Δ cells associates with wild-type V0 but

the complex lacks V-ATPase activity, indicating that V1 is defective. Reciprocal experiments with V0 from

sur4Δ cells show that it is normal. We conclude that sphingolipids with a C26 acyl group are required for

generating fully functional V1 domains.”

http://mmbr.asm.org/content/70/1/177.full The Where, When, and How of Organelle Acidification by

the Yeast Vacuolar H+-ATPase. “All eukaryotic cells contain multiple acidic organelles, and V-ATPases

are central players in organelle acidification. Not only is the structure of V-ATPases highly conserved

among eukaryotes, but there are also many regulatory mechanisms that are similar between fungi and

higher eukaryotes. These mechanisms allow cells both to regulate the pHs of different compartments

and to respond to changing extracellular conditions. The Saccharomyces cerevisiae V-ATPase has

emerged as an important model for V-ATPase structure and function in all eukaryotic cells. This review

discusses current knowledge of the structure, function, and regulation of the V-ATPase in S. cerevisiae

and also examines the relationship between biosynthesis and transport of V-ATPase and compartment-

specific regulation of acidification.”

https://www.ncbi.nlm.nih.gov/pubmed/26177453 SIRT1 Interacts with and Deacetylates ATP6V1B2

in Mature Adipocytes. “SIRT1 plays a key role in maintaining metabolic homeostasis in mammals by

References 14 website: JMR, http://fluoroquinolonethyroid.com

directly modulating the activities of various transcription factors and metabolic enzymes through lysine

deacetylation. White adipose tissue plays a key role in lipid storage and metabolism. To identify novel

molecular targets of SIRT1 in fat cells, we used a non-biased proteomic approach. We identified a

number of proteins whose acetylation status was significantly affected by SIRT1 modulator treatment in

3T3-L1 adipocytes. Among them, ATP6V1B2, a subunit of the vacuolar (H+)-ATPase, was further shown

to be associated with SIRT1 by co-immunoprecipitation assay. Moreover, SIRT1 deacetylates ATP6V1B2

in vitro and in vivo. Taken together, our study demonstrates that ATP6V1B2 is a molecular target of

SIRT1 in fat cells and the role of SIRT1 and ATP6V1B2 acetylation in the vacuolar (H+)-ATPase function

warrants further investigation.”

https://www.ncbi.nlm.nih.gov/pubmed/25529766 Proteome alterations in cortex of mice exposed to

fluoride and lead. “Both fluoride and lead can cross the blood-brain barrier and produce toxic effects

on the central neural system, resulting in low learning and memory abilities, especially in children. In

order to identify the proteomic pattern in the cortex of young animals, from the beginning of fertilization

to the age of postnatal day 56, pregnant female mice and pups were administrated with 150 mg sodium

fluoride/L and/or 300 mg lead acetate/L in their drinking water. Two-dimensional electrophoresis (2-DE)

combined with mass spectrometry (MS) was applied to identify differently expressed protein spots.

Results showed that there were eight proteins in the cortex that significantly changed, whose biological

functions were involved in (1) energy metabolism (Ndufs1, Atp5h, Atp6v1b2), (2) cytoskeleton (Spna2,

Tuba1a, Tubb2a), (3) glycation repair (Hdhd2), and (4) cell stress response (Hspa8). Based on the

previous and current studies, ATPase, Spna2, and Hspa8 were shared by fluoride and lead both as

common target molecules.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4220163/#sup1 De novo mutation in ATP6V1B2

impairs lysosome acidification and causes dominant deafness-onychodystrophy syndrome. “The

identification of ATP6V1B2 c.1516 C>T mutation in three independently identified DDOD patients

provides evidence that defect in ATP6V1B2 is the genetic etiology for DDOD syndrome.”

https://www.ncbi.nlm.nih.gov/pubmed/24418614 Lysosomal alkalization and dysfunction in human

fibroblasts with the Alzheimer's disease-linked presenilin 1 A246E mutation can be reversed with

cAMP.

http://www.jbc.org/content/275/49/38245.full Cytosolic Ca2+ Homeostasis Is a Constitutive Function

of the V-ATPase in Saccharomyces cerevisiae. “In conclusion, this work demonstrates that cytosolic

Ca2+homeostasis is a constitutive function of the V-ATPase in S. cerevisiae. In higher eukaryotic cells,

cytosolic [Ca2+] fluctuates transiently to regulate such diverse processes as neurotransmitter release,

muscle contraction, and T-cell activation (39-41). Recently, compelling evidence for synergistic action of

V-ATPase and calcineurin in the modulation of macrophage effector function and in Ca2+ homeostasis of

fibroblast cell lines and primary astrocytes has been reported (42). The work presented here provides

interesting insights into the synergistic action of calcineurin and V-ATPase in intracellular

Ca2+homeostasis with implications for future studies in yeast and higher eukaryotes.”

References 14 website: JMR, http://fluoroquinolonethyroid.com

http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2011.07234.x/pdf A Role for V-ATPase

Subunits in Synaptic Vesicle Fusion?

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009228 Vacuolar ATPase Regulates

Surfactant Secretion in Rat Alveolar Type II Cells by Modulating Lamellar Body Calcium. “Lung

surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from

alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme

responsible for pumping H+ into lamellar bodies and is required for the processing of surfactant proteins

and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown.

Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft

proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts

and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1

(Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was

blocked by the intracellular Ca2+ chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor,

staurosporine, and the Ca2+/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced

Ca2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion,

indicating cross-talk between lamellar body and endoplasmic reticulum Ca2+ pools. Stimulation of type II

cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled

the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant

secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion,

indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates

surfactant secretion via an increased Ca2+ mobilization from lamellar bodies and endoplasmic reticulum,

and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in

surfactant secretion.”

TOPO2A/B ATPase activity; Topo 2B (Topos necessary for transcription (not just cell replication): “transcriptionally active” genes

for rapid production of proteins additional targets for FQs ADR’s)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC140051/ Characterisation of the DNA-dependent

ATPase activity of human DNA topoisomerase IIβ: mutation of Ser165 in the ATPase domain reduces

the ATPase activity and abolishes the in vivo complementation ability. “We report for the first time

an analysis of the ATPase activity of human DNA topoisomerase (topo) IIβ. We show that topo IIβ is a

DNA-dependent ATPase that appears to fit Michaelis–Menten kinetics. The ATPase activity is stimulated

44-fold by DNA . . . Type II topoisomerases are essential enzymes which are required for the segregation

of intertwined daughter chromosomes following replication (1–4). Their ability to control DNA topology

is also important during transcription, replication and recombination (reviewed in 4). A number of

References 14 website: JMR, http://fluoroquinolonethyroid.com

anticancer drugs and antibiotics target the cleaved DNA intermediate of the topoisomerase (topo) II

reaction cycle, leading to the formation of double-strand DNA breaks which can result in cell death

(reviewed in 5,6). The catalytic cycle of type II topoisomerases is ATP dependent. The ATPase domain is

located at the N-terminus of the protein. DNA stimulates ATP hydrolysis by 4- to 20-fold, depending on

the source of the enzyme and the reaction conditions (7–10). Type II topoisomerases alter the topology of

DNA by transiently cleaving one DNA helix, termed the gate helix, and transporting a second helix

through the enzyme-bridged break (4,11,12). Extensive studies using pre-steady-state kinetic techniques

on yeast DNA topo II have enabled Lindsley and co-workers to deduce how ATP binding and hydrolysis is

coordinated with DNA cleavage and transport during the reaction cycle of eukaryotic topo II (13–18). The

dimeric enzyme binds the gate segment and cleaves it, generating an intermediate where each monomer

of topo II is covalently linked to one DNA strand via the active site tyrosine residue. The transported helix

is thought to bind between the two N-terminal ATPase domains, and ATP binding causes these two

domains to dimerise. One of the two bound ATP molecules is subsequently hydrolysed and then the

transported segment is passed through the break in the gate segment. The ATPs are hydrolysed

sequentially with the products of the first ATP hydrolysis reaction released prior to hydrolysis of the

second ATP molecule. The transported helix is thought to exit via the C-terminal dimer interface,

although it is not clear how this event correlates with hydrolysis of the second ATP molecule. Human

cells contain two isoforms of DNA topo II, α and β (reviewed in 19). The ATPase activity of human topo

IIα has been reported (10). To date no data have been published on the ATPase activity of human DNA

topo IIβ. In this paper we report the characterisation of the ATPase activity of human DNA topo IIβ. We

also describe a mutated protein with reduced ATPase activity which results in loss of complementation

activity”.

http://www.jbc.org/content/272/51/32696.long The DNA Dependence of the ATPase Activity of

Human DNA Topoisomerase IIα. “DNA topoisomerases are enzymes that catalyze topological changes

in DNA (1, 2). These enzymes have been found in all cell types and are essential for cell viability. Their

roles include maintenance of the level of intracellular DNA supercoiling, removing supercoils, which build

up ahead of and behind transcription and replication complexes, and the decatenation of daughter

chromosomes following replication. The topoisomerase reaction involves the breakage of DNA in one or

both strands, the formation of protein-DNA covalent bonds, and the passage of another segment of DNA

through the enzyme-stabilized break. In the case of type II enzymes, this DNA strand passage reaction

generally requires the hydrolysis of ATP. . . . On the basis of the alignment of their amino acid sequences,

DNA topoisomerases can be grouped into three subfamilies: type IA, type IB, and type II (8). All type II

enzymes are evolutionarily and structurally related, each possessing two distinct catalytic centers: a DNA

cleavage and rejoining site, and a site for ATP hydrolysis (9-13). The enzymes differ in their molecular

masses and subunit composition,e.g. DNA gyrase from Escherichia coli consists of two subunits GyrA and

GyrB of 97 and 90 kDa, which associate as an A2B2 complex (14). GyrA contains the DNA cleavage

activity, while GyrB catalyzes ATP hydrolysis. Eukaryotic type II enzymes are homodimers where each

monomer can be regarded as a fusion of a GyrB and GyrA subunit. Homology between eukaryotic and

prokaryotic enzymes is closest in the N-terminal region and the region containing the active site for DNA

cleavage (corresponding to the ATPase domain of GyrB and the N-terminal domain of GyrA,

respectively), but the C termini tend to be divergent. The molecular sizes of the eukaryotic enzymes show

References 14 website: JMR, http://fluoroquinolonethyroid.com

some diversity; the enzyme from Saccharomyces cerevisiae (yeast topoisomerase II) has a monomer

molecular mass of 164 kDa (15), whereas the two isoforms of the human enzyme α and β are 170 and

180 kDa (16). The mechanism of eukaryotic topoisomerase II is now understood in some detail as a

consequence of a number of structural and mechanistic studies (17-19). The enzyme binds a segment of

DNA (∼25 bp),1 which becomes the gate segment (or “G-segment”). The G-segment is cleaved in both

strands with a 4-base stagger between the break sites. This leads to the formation of covalent bonds

between the 5′-phosphates at the break site and the active-site tyrosines. Another segment of DNA (the

“T-segment”) is captured by an ATP-operated clamp (comprising the N-terminal domains of the two

subunits), which presents the T-segment to the double-stranded break in the G-segment and facilitates

the strand passage reaction. Resealing of the break in the G-segment leads to a change in linking

number of the DNA by 2, in the case of intramolecular reactions (e.g. DNA relaxation), or catenation or

decatenation in the case of intermolecular strand passage. Although the mechanism of topoisomerase II

is now understood in some detail, the role of ATP hydrolysis remains to be clarified. ATP hydrolysis is

normally required to drive reactions that are energetically unfavorable. Indeed, in the case of the

prokaryotic type II topoisomerase, DNA gyrase, the requirement for ATP hydrolysis is clear. Gyrase can

introduce negative supercoils into DNA, an energetically unfavorable reaction, which is coupled to ATP

hydrolysis. In this case, there appears to be an approximate correspondence between the free energy

available from the hydrolysis of ATP and the energy required to introduce supercoils (20-23). In the

absence of ATP, gyrase can catalyze the relaxation of negative supercoils (an energetically favorable

reaction) albeit less efficiently than the introduction of supercoils (24, 25). Eukaryotic topoisomerase II

cannot introduce supercoils into DNA but relaxes DNA in an ATP-dependent reaction. Given that this is an

energetically favorable reaction, it is unclear why ATP is required. . . . To further our understanding of

ATP hydrolysis by DNA topoisomerase II, we have studied the ATPase reaction of the HeLa enzyme. Given

the known effects of post-translational modifications on the activity of topoisomerase II, we have elected

in the first instance to isolate the enzyme from a human cell line rather than a yeast clone to establish

the properties of the native enzyme. . . . We have isolated to high purity human topoisomerase IIα from

HeLa cells and investigated the ATP hydrolysis activity of this enzyme. We considered it important to

obtain data on the human enzyme from HeLa cells as a prelude to work on enzyme overexpressed in

yeast. Proteins overexpressed in such clones are ubiquitously known as human, but some carry a

modified N terminus (41, 42) and may therefore differ in their biochemical characteristics from the native

enzyme. This is particularly true for the ATP hydrolysis of the enzymes, as this is known to be carried out

by the N-terminal domain (43). In addition, enzymes derived from yeast will not necessarily have

undergone the same post-translational modifications as those from human cells. This is important with

respect to phosphorylation of the enzyme, as this may play a major role in determining the ATPase

activity (29, 30). The N-terminal region contains a phosphorylation site at serine 29 (44), and the

availability of this residue for phosphorylation in enzyme cloned in yeast may be affected, as the N-

terminal portion of this enzyme contains a yeast-derived peptide followed by the human enzyme from

serine 29 onward (42). . . . In summary, we have found that the ATPase reaction of HeLa topoisomerase

IIα is stimulated by DNA in a length-dependent fashion and that hyperstimulation occurs at certain

enzyme:DNA ratios. The physiological relevance of these observations is unclear, and we do not know the

effects of phosphorylation on the DNA dependence of the ATPase reaction. Such issues are currently

under investigation.”

References 14 website: JMR, http://fluoroquinolonethyroid.com

https://www.researchgate.net/figure/10638629_fig1_Fig-1-The-catalytic-cycle-of-DNA-topoisomerase-

II-The-ATPase-domains-of-topoisomerase Figure 1 The Catalytic cycle of DNA-topoisomerase II The

ATPase domains of topoisomerase. “Fig. 1. The catalytic cycle of DNA topoisomerase II. The ATPase

domains of topoisomerase II are shown in light blue, the core domain in dark blue, and the active site

tyrosine residue in red. The C-terminal domain of the enzyme is not included in the diagram since its

orientation, with respect to the rest of the molecule, is not known. The catalytic cycle is initiated by

enzyme binding to two double-stranded DNA segments called the G segment (in red) and the T segment

(in green) (Step 1). Next, two ATP molecules are bound, which is associated with dimerization of the

ATPase domains (Step 2). The G segment is cleaved (Step 3) and the T segment is transported through

the break in the G segment, which is accompanied by the hydrolysis of one ATP molecule (Step 4). The G

segment is then religated and the remaining ATP molecule is hydrolyzed (Step 5). Upon dissociation of

the two ADP molecules, the T segment is transported through the opening in the C-terminal part of the

enzyme (Step 6) followed by closing of this gate. Finally, the N-terminal ATPase domains reopen,

allowing the enzyme to dissociate from DNA (Step 7). Data from Berger et al. (1996), Baird et al. (1999),

Brino et al. (2000), and Hu et al. (2002).”

References 14 website: JMR, http://fluoroquinolonethyroid.com

https://www.ncbi.nlm.nih.gov/pubmed/11574892 DNA topoisomerase IIalpha is required for RNA

polymerase II transcription on chromatin templates. “In the nucleus of the cell, core RNA polymerase

II (pol II) is associated with a large complex called the pol II holoenzyme (holo-pol). Transcription by core

pol II in vitro on nucleosomal templates is repressed compared with that on templates of histone-free

naked DNA. We found that the transcriptional activity of holo-pol, in contrast to that of core pol II, is not

markedly repressed on chromatin templates. We refer to this property of holo-pol as chromatin-

dependent coactivation (CDC). Here we show that DNA topoisomerase II alpha is associated with the

holo-pol and is a required component of CDC. Etoposide and ICRF-193, specific inhibitors of

topoisomerase II, blocked transcription on chromatin templates, but did not affect transcription on naked

templates. Addition of purified topoisomerase IIalpha reconstituted CDC activity in reactions with core

pol II. These findings suggest that transcription on chromatin templates results in the accumulation of

superhelical tension, making the relaxation activity of topoisomerase II essential for productive RNA

synthesis on nucleosomal DNA.”

http://www.nature.com/nrg/journal/v14/n2/full/nrg3418.html Transcription: A role for DNA

topoisomerase in activation. (2013). “DNA topoisomerases are thought to facilitate transcription by

removing excess topological strain induced by the tracking of the polymerase. A study in

Saccharomyces cerevisiae deficient for topoisomerases I and II has now suggested that in vivo these

enzymes are also involved in gene activation.”

https://www.ncbi.nlm.nih.gov/pubmed/27649880 Roles of eukaryotic topoisomerases in

transcription, replication and genomic stability. “Topoisomerases introduce transient DNA breaks to

relax supercoiled DNA, remove catenanes and enable chromosome segregation. Human cells encode six

topoisomerases (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α and TOP3β), which act on a broad range of DNA

and RNA substrates at the nuclear and mitochondrial genomes. Their catalytic intermediates, the

topoisomerase cleavage complexes (TOPcc), are therapeutic targets of various anticancer drugs. TOPcc

can also form on damaged DNA during replication and transcription, and engage specific repair

pathways, such as those mediated by tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 and by

endonucleases (MRE11, XPF-ERCC1 and MUS81). Here, we review the roles of topoisomerases in

mediating chromatin dynamics, transcription, replication, DNA damage repair and genomic stability, and

discuss how deregulation of topoisomerases can cause neurodegenerative diseases, immune disorders

and cancer.”

https://www.ncbi.nlm.nih.gov/pubmed/27630045 Topoisomerases and the regulation of neural

function. “Topoisomerases are unique enzymes that regulate torsional stress in DNA to enable essential

References 14 website: JMR, http://fluoroquinolonethyroid.com

genome functions, including DNA replication and transcription. Although all cells in an organism require

topoisomerases to maintain normal function, the nervous system in particular shows a vital need for

these enzymes. Indeed, a range of inherited human neurologic syndromes, including neurodegeneration,

schizophrenia and intellectual impairment, are associated with aberrant topoisomerase function. Much

remains unknown regarding the tissue-specific function of neural topoisomerases or the connections

between these enzymes and disease aetiology. Precisely how topoisomerases regulate genome dynamics

within the nervous system is therefore a crucial research question.”

https://www.ncbi.nlm.nih.gov/pubmed/27511624 Ribonucleotides and Transcription-Associated

Mutagenesis in Yeast. “High levels of transcription stimulate mutation rates in microorganisms, and

this occurs primarily through an enhanced accumulation of DNA damage. The major source of

transcription-associated damage in yeast is Topoisomerase I (Top1), an enzyme that removes torsional

stress that accumulates when DNA strands are separated. Top1 relieves torsional stress by nicking and

resealing one DNA strand, and some Top1-dependent mutations are due to trapping and processing of

the covalent cleavage intermediate. Most, however, reflect enzyme incision at ribonucleotides, which are

the most abundant noncanonical component of DNA. In either case, Top1 generates a distinctive

mutation signature composed of short deletions in tandem repeats; in the specific case of ribonucleotide-

initiated events, mutations reflect sequential cleavage by the enzyme. Top1-dependent mutations do not

require highly activated transcription, but their levels are greatly increased by transcription, which

partially reflects an interaction of Top1 with RNA polymerase. Recent studies have demonstrated that

Top1-dependent mutations exhibit a strand bias, with the nature of the bias differing depending on the

transcriptional status of the underlying DNA. Under low-transcription conditions, most Top1-dependent

mutations arise in the context of replication and reflect incision at ribonucleotides incorporated during

leading-strand synthesis. Under high-transcription conditions, most Top1-dependent events arise when

the enzyme cleaves the non-transcribed strand of DNA. In addition to increasing genetic instability in

growing cells, Top1 activity in transcriptionally active regions may be a source of mutations in quiescent

cells.”

https://www.ncbi.nlm.nih.gov/pubmed/27376333 Collision of Trapped Topoisomerase 2 with

Transcription and Replication: Generation and Repair of DNA Double-Strand Breaks with 5' Adducts.

“Topoisomerase 2 (Top2) is an essential enzyme responsible for manipulating DNA topology during

replication, transcription, chromosome organization and chromosome segregation. It acts by nicking

both strands of DNA and then passes another DNA molecule through the break. The 5' end of each nick is

covalently linked to the tyrosine in the active center of each of the two subunits of Top2 (Top2cc). In this

configuration, the two sides of the nicked DNA are held together by the strong protein-protein

interactions between the two subunits of Top2, allowing the nicks to be faithfully resealed in situ.

Top2ccs are normally transient, but can be trapped by cancer drugs, such as etoposide, and subsequently

processed into DSBs in cells. If not properly repaired, these DSBs would lead to genome instability and

cell death. Here, I review the current understanding of the mechanisms by which DSBs are induced by

etoposide, the unique features of such DSBs and how they are repaired. Implications for the

improvement of cancer therapy will be discussed. . . . The double-helical structure of DNA dictates that

topological strains are generated by replication forks and transcription machineries as they plow

References 14 website: JMR, http://fluoroquinolonethyroid.com

between the two intertwined strands [1]. An essential protein for managing DNA topology is

topoisomerase 2 (Top2) [2,3,4]. Top2 is a homodimeric enzyme that changes topology by nicking the two

strands of DNA to create a double-strand break (DSB), through which another DNA molecule then passes.

In theory, physical breakage of DNA poses a serious danger to genome integrity and cell survival. Top2

solves this problem by forming a covalent complex between the 5′ end of the broken DNA and the

tyrosine in the catalytic center (Top2cc). The strong interaction between the two subunits ensures that

the 5′ ends are juxtaposed to the 3′ ends and can be quickly and faithfully resealed in situ. Top2ccs are

normally transient, but can be trapped by various factors, such as DNA lesions, natural products in the

diet, chemicals in the environment and, most importantly, many anticancer drugs [2]. Prolonged

trapping allows cellular processes to convert Top2ccs into true DSBs, which, if not repaired or improperly

repaired, would lead to genome instability or cell death [5]. Trapped Top2ccs are an intrinsic aspect of

genome maintenance, and their significance is further amplified by the central role they play in

mediating the cytotoxicity of some of the most widely-used cancer drugs. This review will focus on how

trapped Top2ccs are converted into DSBs, the unique features of the resulting DSBs and what pathways

are employed to repair them. Emphasis will be on higher eukaryotes, and implications for cancer therapy

will be explored. . . . In mammalian cells, the fate of etoposide-trapped Top2ccs is strongly affected by

the isoforms of Top2. While lower eukaryotes have only one Top2, mammalian cells have two Top2

isoforms, Top2α and Top2β, sharing ca. 70% sequence identity [13,14,15,16]. Top2α promotes

replication, transcription, chromosome structure and chromosome segregation [17]. It is essential for cell

proliferation and expressed mostly in dividing cells during the S and G2 phases [18,19,20]. In contrast,

Top2β participates mainly in transcription and is expressed in both dividing and non-dividing cells [21]. It

is dispensable for cell proliferation, but required for neural development in mice [22,23,24]. The two

isoforms use the same catalytic mechanism and are equally inhibited by etoposide [25,26]. However,

their distinct biological functions and expression profiles have a profound impact on the mechanisms by

which etoposide-trapped Top2α and Top2β are processed to DSBs in cells . . . The finding that

transcription and Top2β play key roles in DSB induction is not surprising, but fully consistent with the

known properties of Top2β and etoposide. What is surprising is the apparent irrelevance of Top2α and

replication. Many studies have shown that Top2α, the major isoform in proliferative cells, rather than

Top2β is the dominant isoform mediating cytotoxicity of etoposide [31,35,36]. Even in the study showing

that Top2β is the isoform mediating DSB induction, Top2α is still the isoform responsible for the

cytotoxicity of etoposide [32]. Replication, which in principle can also collide with trapped Top2ccs, has

also been long known to partially mediate the cytotoxicity of etoposide [37,38]. In one study, inhibiting

replication is significantly more effective than inhibiting transcription in protecting cells from etoposide .

. . The Top2 molecules trapped on DNA by the cancer drug etoposide are converted to DSBs by two

mechanisms in cells. One mechanism is dependent on the collision with replication complexes, which

causes replication run-off, generating DSBs with an intact Top2 linked to the 5′ end. The other

mechanism is dependent on the collision with transcription complexes, which stimulates the degradation

of Top2, generating DSBs with a small Top2 peptide linked to the 5′ end. The replication-dependent

mechanism is mediated by Top2α and can occur even if only one subunit is trapped. In contrast, the

transcription-dependent mechanism requires both subunits to be trapped and is mediated by both Top2α

and Top2β.”

References 14 website: JMR, http://fluoroquinolonethyroid.com

http://www.ncbi.nlm.nih.gov/pubmed/25217229/ DNA topoisomerase IIβ as a molecular switch in

neural differentiation of mesenchymal stem cells. “Two isoforms of DNA topoisomerase II (topo II)

have been identified in mammalian cells, named topo IIα and topo IIβ. Topo IIα plays an essential role in

segregation of daughter chromosomes and thus for cell proliferation in mammalian cells. Unlike its

isozyme topo IIα, topo IIβ is greatly expressed upon terminal differentiation of neuronal cells . . . and

apparently genes involved in regulation of several ion channels and transporters, vesicle function, and

cell calcium metabolism were particularly affected by topo IIβ silencing suggesting that topoIIβ silencing

can significantly alter the gene expression pattern of genes involved in variety of biological processes and

signal transduction pathways including transcription, translation, cell trafficking, vesicle function,

transport, cell morphology, neuron guidance, growth, polarity, and axonal growth. It appears that the

deregulation of these pathways may contribute to clarify the further role of topo IIβ in neural

differentiation.”

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340998/ Target genes of Topoisomerase IIβ regulate

neuronal survival and are defined by their chromatin state.

http://www.ncbi.nlm.nih.gov/pubmed/16923961 Role of topoisomerase IIbeta in the expression of

developmentally regulated genes

https://en.wikipedia.org/wiki/TOP2B Wiki

http://www.nature.com/nrm/journal/v17/n5/full/nrm.2016.55.html Pol II and topoisomerase 1 hand-

in-hand. “The RNA polymerase II (Pol II) machinery generates DNA torsional stress, which, if not relieved

by topoisomerase 1 (TOP1) through DNA swivelling, can impede transcription. Baranello et al. developed

TOP1-seq to map the position of catalytically active TOP1 and found that it is enriched at sites of Pol II

pausing,…”

https://www.ncbi.nlm.nih.gov/pubmed/27100743 A critical role for topoisomerase IIb and DNA

double strand breaks in transcription. “Recent studies have indicated a novel role for topoisomerase IIb

in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear

receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such

strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such

transcription was associated with initiation of the DNA damage response pathways, including the

activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly

(ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in

repair of DNA breaks generated by topoisomerase IIb.”

https://www.ncbi.nlm.nih.gov/pubmed/27058666 RNA Polymerase II Regulates Topoisomerase 1

Activity to Favor Efficient Transcription. “We report a mechanism through which the transcription

machinery directly controls topoisomerase 1 (TOP1) activity to adjust DNA topology throughout the

transcription cycle. By comparing TOP1 occupancy using chromatin immunoprecipitation sequencing

(ChIP-seq) versus TOP1 activity using topoisomerase 1 sequencing (TOP1-seq), a method reported here to

map catalytically engaged TOP1, TOP1 bound at promoters was discovered to become fully active only

after pause-release. This transition coupled the phosphorylation of the carboxyl-terminal-domain (CTD)

References 14 website: JMR, http://fluoroquinolonethyroid.com

of RNA polymerase II (RNAPII) with stimulation of TOP1 above its basal rate, enhancing its processivity.

TOP1 stimulation is strongly dependent on the kinase activity of BRD4, a protein that phosphorylates

Ser2-CTD and regulates RNAPII pause-release. Thus the coordinated action of BRD4 and TOP1 overcame

the torsional stress opposing transcription as RNAPII commenced elongation but preserved negative

supercoiling that assists promoter melting at start sites. This nexus between transcription and DNA

topology promises to elicit new strategies to intercept pathological gene expression.”

https://www.ncbi.nlm.nih.gov/pubmed/26695634 STAT-5 Regulates Transcription of the

Topoisomerase IIβ-Binding Protein 1 (TopBP1) Gene To Activate the ATR Pathway and Promote

Human Papillomavirus Replication.

https://www.ncbi.nlm.nih.gov/pubmed/26616758 The distinctive cellular responses to DNA strand

breaks caused by a DNA topoisomerase I poison in conjunction with DNA replication and RNA

transcription. “Camptothecin (CPT) inhibits DNA topoisomerase I (Top1) through a non-catalytic

mechanism that stabilizes the Top1-DNA cleavage complex (Top1cc) and blocks the DNA re-ligation step,

resulting in the accumulation in the genome of DNA single-strand breaks (SSBs), which are converted to

secondary strand breaks when they collide with the DNA replication and RNA transcription machinery.

DNA strand breaks mediated by replication, which have one DNA end, are distinct in repair from the DNA

double-strand breaks (DSBs) that have two ends and are caused by ionizing radiation and other agents.

In contrast to two-ended DSBs, such one-ended DSBs are preferentially repaired through the homologous

recombination pathway. Conversely, the repair of one-ended DSBs by the non-homologous end-joining

pathway is harmful for cells and leads to cell death. The choice of repair pathway has a crucial impact on

cell fate and influences the efficacy of anticancer drugs such as CPT derivatives. In addition to replication-

mediated one-ended DSBs, transcription also generates DNA strand breaks upon collision with the

Top1cc. Some reports suggest that transcription-mediated DNA strand breaks correlate with

neurodegenerative diseases. However, the details of the repair mechanisms of, and cellular responses to,

transcription-mediated DNA strand breaks still remain unclear. In this review, combining our recent

results and those of previous reports, we introduce and discuss the responses to CPT-induced DNA

damage mediated by DNA replication and RNA transcription. . . . The two major DSB repair pathways are

homologous recombination (HR) and non-homologous end joining (NHEJ) (O’Driscoll and Jeggo, 2006;

Shibata and Jeggo, 2014) (Fig. 1), which are active in different stages of the cell cycle. HR is an error-free

DSB repair mechanism that requires a sister chromatid and is therefore restricted to the S and G2 phases

of the cell cycle. NHEJ is a fundamental mechanism to rejoin two DSB ends, and can occur throughout the

cell cycle. Unlike HR, the NHEJ process is error-prone, often resulting in the introduction of mutations at

the joining site.

miRNA

References 14 website: JMR, http://fluoroquinolonethyroid.com

http://onlinelibrary.wiley.com/doi/10.1002/wrna.121/abstract?systemMessage=Due+to+essential+mai

ntenance+the+subscribe%2Frenew+pages+will+be+unavailable+on+Wednesday+26+October+between+

02%3A00+-+08%3A00+BST%2F+09%3A00+%E2%80%93+15%3A00++SGT%2F+21%3A00-

+03%3A00+EDT.+Apologies+for+the+inconvenience. Posttranscriptional Upregulation by MicroRNAs.

“MicroRNAs are small non-coding RNA guide molecules that regulate gene expression via association

with effector complexes and sequence-specific recognition of target sites on other RNAs; misregulated

microRNA expression and functions are linked to a variety of tumors, developmental disorders, and

immune disease. MicroRNAs have primarily been demonstrated to mediate posttranscriptional

downregulation of expression; translational repression, and deadenylation-dependent decay of

messages through partially complementary microRNA target sites in mRNA untranslated regions (UTRs).

However, an emerging assortment of studies, discussed in this review, reveal that microRNAs and their

associated protein complexes (microribonucleoproteins or microRNPs) can additionally function to

posttranscriptionally stimulate gene expression by direct and indirect mechanisms. These reports

indicate that microRNA-mediated effects can be selective, regulated by the RNA sequence context, and

associated with RNP factors and cellular conditions. Like repression, translation upregulation by

microRNAs has been observed to range from fine-tuning effects to significant alterations in expression.

These studies uncover remarkable, new abilities of microRNAs and associated microRNPs in gene

expression control and underscore the importance of regulation, in cis and trans, in directing appropriate

microRNP responses.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403296/pdf/fpsyt-06-00044.pdf A new tool for in

vivo manipulation of brain microRNA levels: the work of Smalheiser et al. (2014). “A commentary on

Enoxacin elevates microRNA levels in rat frontal cortex and prevents learned helplessness. Smalheiser

et al. have observed reduced microRNA expression levels in the prefrontal cortex of depressed suicide

subjects (2, 3), but learned helplessness induced in a rat model of MDD was accompanied by muted

responses in specific microRNAs compared to significantly reduced expression in rats that did not

develop learned helplessness (4). As potent down-regulators of messenger RNA abundance and

translation, microRNAs target a majority of genes in the human genome and thus represent a global,

and potentially druggable (5, 6), regulatory mechanism capable of affecting most molecular networks.

But do alterations in microRNA expression precede and potentially cause depression severe enough to

result in suicide, comprise part of the response to some other triggering pathology, or represent both

cause and effect depending on which microRNA is involved (7)?

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417715/ miRNA-based therapies: Strategies and

delivery platforms for oligonucleotide and non-oligonucleotide agents. In 1993, Lee et al [18] found

two small transcripts in Caenorhabditis elegans which would not code for a protein because their sizes

were only 22 and 60 bp, respectively. In 2001, the term microRNA for these short non-coding sequences

was used for the first time when Science published three accompanying publications on this topic [19,

20]. miRNAs belong to the heterogenous family of small endogenous non coding RNAs that also include

small nucleolar RNA (snoRNA) and small interfering RNA (siRNA). The coding regions for miRNAs lie

usually in the regions historically called “junk DNA”. Continuous research with these seemingly useless

DNA sequences revealed that the transcribed RNA is not genetic waste at all. [21] The localisation of

References 14 website: JMR, http://fluoroquinolonethyroid.com

miRNA sequences can be separated into intergenic and protein-coding intronic regions in the genome . .

. A number of fluoroquinolone antibiotics were shown to enhance the effect of siRNAs [133] and

miRNAs [134] through interaction with the RNAi machinery (Figure 3). The most potent compound,

enoxacin, was further studied for the molecular mechanism of this effect. Enoxacin was found to

increase the binding affinity of TRPB, an integral component of the RISC, to miRNA precursors. As such,

there is of course no specificity for certain miRNA sequences. Because the global miRNA expression is

significantly lowered in tumours, a general increase of miRNA activity may nevertheless be an attractive

option in oncology. Indeed, enoxacin reduced cell viability in cancer cell lines [134].

http://www.nature.com/cr/journal/v18/n10/full/cr2008287a.html Enhancement of RNAi by a small

molecule antibiotic enoxacin. Our longstanding interest in drug-nucleic acids interaction 5 led us to

search for potential small molecular regulators of RNAi. We hypothesized that inhibitors of RNA helicases

may increase the stability of double-stranded siRNA, so as to enhance RNAi efficiency. Since a large

family of fluoroquinolone antibiotics target bacterial DNA gyrase complexed with the targeted DNA

possibly in A-form (similar to RNA) 6 and since they also exhibit antiviral activity through interference

with Tat-TAR interaction 7, we decided to screen a library of commercially available fluoroquinolone

antibiotics, with the hope that some of the analogs may cross-inhibit relevant human RNA helicases.

Herein, we report that enoxacin, one of the fluoroquinolone antibiotics known to inhibit bacterial gyrase

and topoisomerase IV with minimal effects on their mammalian counterparts, can increase RNAi

efficiency. We have found that enoxacin can reduce the concentrations of siRNA by 2~5-fold for the same

RNAi knockdown efficiency . . . In summary, we have demonstrated that certain fluoroquinolone

antibiotics such as enoxacin, in addition to their powerful clinic use for the treatment of infections in

humans and animals 10, can be used to increase RNAi efficiency. Enoxacin can significantly reduce the

amount of siRNA (by 2~5-fold) to achieve the same RNAi efficacy. The precise mechanism of this RNAi

enhancement remains unclear at present. While our manuscript was in preparation, a similar finding

with more detailed analysis was reported by Jin and colleagues 11, who proposed that enoxacin acts by

potentially increasing RISC loading efficiency through a mechanism depending on the protein factor

TRBP. Nevertheless, one cannot rule out the possibility that the effect of enoxacin on RNAi is due to the

cross-interaction with human RNA helicases and the stabilization of RNAi molecules, especially in view of

the finding that human RNA helicase A (RHA) is an active RISC component and functions in RISC as an

siRNA loading factor.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3060242/ Small molecule enoxacin is a cancer-

specific growth inhibitor that acts by enhancing TAR RNA-binding protein 2-mediated microRNA

processing. “MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression at the

posttranscriptional level and are critical for many cellular pathways. The disruption of miRNAs and their

processing machineries also contributes to the development of human tumors. A common scenario for

miRNA expression in carcinogenesis is emerging that shows that impaired miRNA production and/or

down-regulation of these transcripts occurs in many neoplasms. Several of these lost miRNAs have

tumor-suppressor features, so strategies to restore their expression globally in malignancies would be a

welcome addition to the current therapeutic arsenal against cancer. Herein, we show that the small

molecule enoxacin, a fluoroquinolone used as an antibacterial compound, enhances the production of

References 14 website: JMR, http://fluoroquinolonethyroid.com

miRNAs with tumor suppressor functions by binding to the miRNA biosynthesis protein TAR RNA-binding

protein 2 (TRBP). The use of enoxacin in human cell cultures and xenografted, orthotopic, and metastatic

mouse models reveals a TRBP-dependent and cancer-specific growth-inhibitory effect of the drug. These

results highlight the key role of disrupted miRNA expression patterns in tumorigenesis, and suggest a

unique strategy for restoring the distorted microRNAome of cancer cells to a more physiological setting.”

http://link.springer.com/article/10.1007/s00774-016-0757-8 MiR-142-5p promotes bone repair by

maintaining osteoblast activity. “MicroRNAs play important roles in regulating bone regeneration and

remodeling. However, the pathophysiological roles of microRNAs in bone repair remain unclear. Here we

identify a significant upregulation of miR-142-5p correlated with active osteoblastogenesis during the

bone healing process. In vitro, miR-142-5p promoted osteoblast activity and matrix mineralization by

targeting the gene encoding WW-domain-containing E3 ubiquitin protein ligase 1. We also found that

the expression of miR-142-5p in the callus of aged mice was lower than that in the callus of young mice

and directly correlated with the age-related delay in bone healing. Furthermore, treatment with agomir-

142-5p in the fracture areas stimulated osteoblast activity which repaired the bone fractures in aged

mice. Thus, our study revealed that miR-142-5p plays a crucial role in healing fractures by maintaining

osteoblast activity, and provided a new molecular target therapeutic strategy for bone healing.”

http://www.nature.com/articles/srep24496 Circulating micro RNA profiles of Ebola virus infection.

https://www.ncbi.nlm.nih.gov/pubmed/25266153 Identification of Ebola virus microRNAs and their

putative pathological function. “Ebola virus (EBOV), a member of the filovirus family, is an enveloped

negative-sense RNA virus that causes lethal infections in humans and primates. Recently, more than

1000 people have been killed by the Ebola virus disease in Africa, yet no specific treatment or diagnostic

tests for EBOV are available. In this study, we identified two putative viral microRNA precursors (pre-

miRNAs) and three putative mature microRNAs (miRNAs) derived from the EBOV genome. The

production of the EBOV miRNAs was further validated in HEK293T cells transfected with a pcDNA6.2-

GW/EmGFP-EBOV-pre-miRNA plasmid, indicating that EBOV miRNAs can be produced through the

cellular miRNA processing machinery. We also predicted the potential target genes of these EBOV

miRNAs and their possible biological functions. Overall, this study reports for the first time that EBOV

may produce miRNAs, which could serve as non-invasive biomarkers for the diagnosis and prognosis of

EBOV infection and as therapeutic targets for Ebola viral infection treatment.”

https://www.eurekalert.org/pub_releases/2016-03/nuso-aev030116.php An Ebola virus-encoded

microRNA-like fragment serves as a biomarker for early diagnosis. “In a new study, Chen-Yu Zhang's

group at Nanjing University collaborate with Ze-liang Chen's group at academy of Military Medical

Sciences report that an Ebola virus-encoded microRNA-like fragment serves as a biomarker for early

diagnosis. It is published in Cell Research. Ebola virus disease (EVD) is a severe infectious disease caused

by Ebola virus species. EBOV caused an epidemic in West Africa in 2013-2015, and have resulted in at

least 24,000 suspected cases and 10,000 confirmed deaths. Early diagnosis of EVD is not only essential

for implementation of effective interventions but also critical for prevention of the spread of infection.

However, it is particularly difficult to diagnose EVD at an early stage. Ebola causes symptoms seen in

many other infections, including malaria, typhoid, and influenza, and some patients even developed

References 14 website: JMR, http://fluoroquinolonethyroid.com

illness without specific signs and symptoms. Current methods to diagnose suspected Ebola virus infection

include reverse transcription polymerase chain reaction, antigen-capture enzyme-linked immunosorbent

assay, and immunoglobulin M and immunoglobulin G ELISA. In previous studies, the same group along

with others had demonstrated that microRNAs (miRNAs) produced by eukaryotic cells and viruses are

present in human blood in highly stable, cell-free forms and these so called circulating miRNAs can serve

as non-invasive biomarkers for the early diagnosis of various diseases, including viral diseases. Since

there are insufficient feasible methods established to diagnose EVD at early stage, Zhang's trying to

make breakthroughs by speculating EVDV-specific small non-coding RNAs that could be detected in

human blood. A gratifying finding was reported that the group extrapolated a putative sequence of

miRNA-like fragment encoded by EVDV using the principle of miRNA production in eukaryotes. In cellular

environment, the existing and maturation of the putative miRNA-like fragment in the presence of a

cloned pre-miR sequence were then verified. With the collaboration of Academy of Military Medical

Sciences, they further identified the Ebola virus-encoded miRNA-like fragment in serum of EVD patients

by qRT-PCR, Northern blotting and TA-cloning/sequencing. Strikingly, subsequent results showed that

this miRNA-like fragment existed in acute phase but disappeared in recovery phase of EVD survivors.

With great clinical significance, this miRNA-like fragment was detectable in EVD patients before

development of viremia with detectable Ebola genomic RNA, suggesting that it is an earlier biomarker

than genomic RNA and could advance diagnosable window for EVD.”

https://www.researchgate.net/publication/220028008_Computational_Evidence_for_a_Viral_Encoded_

miRNA_in_the_5'_UTR_of_the_Zaire_Ebolavirus_Nucleoprotein_Gene_May_Explain_Its_Differential_Vi

rulence_the_Pandora_Element Computational Evidence for a Viral Encoded miRNA in the 5' UTR of

the Zaire Ebolavirus Nucleoprotein Gene May Explain Its Differential Virulence: the Pandora Element.

“The Zaire ebolavirus is one of the most virulent of human viral pathogens with a reported CFR of 90%

resulting from complete disruption of the host immune response. Despite the relatively minor genetic

differences between the species of the genera Ebolavirus, the case fatality rate (CFR) is significantly

different. The Zaire Ebolavirus (EBOV) historically is the most lethal, with its sporadic outbreaks resulting

in a CFR ranging from 60% (Minkébé, Gabon 1994) to 90% (Kéllé, Congo 2003). This is in sharp contrast

to Reston ebolavirus (RESTV) which, despite its genetic similarity to EBOV and serologic evidence of

human transmission, has yet to result in even minor symptoms of the disease. The viral protein, VP35,

has been shown to be a multifunctional virulence factor by antagonizing anti-viral signalling pathways

via its interferon inhibitory domain (IID). However, it is unlikely the VP35 pathways could fully account for

the observed differential virulence between the species. Viral encoded miRNAs have already been

identified in many species and shown to modulate the expression and antiviral function of interferons

(IFNs). This paper summarizes the ab initio identification of a probable miRNA within the 5' UTR of the

genomic Zaire ebolavirus nucleoprotein gene, termed the Pandora Element, and a possible mechanism

by which it may associate with VP35 to achieve the observed disruption of the immune response. A

structural characteristic of the RESTV Pandora domain will also be introduced that may also account for

its lack of virulence. The associated hsa-miR-4999, hsa-mir-1302, and hsa-miR-2054 targets include XCL1

chemokine, ubiquitin specific peptidase 3, multiple signalling factors and interleukin.”

References 14 website: JMR, http://fluoroquinolonethyroid.com

https://taggs.hhs.gov/Detail/AwardDetail?arg_AwardNum=R21AI125775&arg_ProgOfficeCode=104

MicroRNAs induced in response to Borrelia burgdorferi.

http://medicalxpress.com/news/2015-04-scientific-breakthrough-potential-tendon-therapy.html

Scientific breakthrough unlocks potential novel tendon therapy. “Scientists are investigating a new

therapy for the treatment of tendon injuries such as tennis elbow and Achilles tendinitis after gaining

new insight into the condition. Tendon injuries (tendinopathies) are common, accounting for 30-50% of

all sporting injuries, and are usually caused by repetitive strain or major trauma. While many people

recover after a period of rest, a significant number of people do not because the structure of the tendon

itself has permanently weakened. Healthy tendons, connecting muscles to bones, are primarily

composed of type-1 collagen, a very strong material. When injured the body responds by producing the

inferior type-3 collagen to quickly repair the damage. This type of collagen is not as strong as type-1 and

is more prone to damage. Normally, over time, type 3 is replaced by the stronger type-1. However, in

some people, repetitive damage means the body never replaces the weaker type-3 collagen, leaving

them with inherently weaker tendons and long-term symptoms, such as pain and reduced mobility.

Scientists at the University of Glasgow are trialling a new therapy (TenoMiR) for treating tendinopathy

after being awarded a High Growth Spinout grant from Scottish Enterprise. The trial will use injections of

microRNA – small molecules that help regulate gene expression – into the tendon to 'dial-down' the

production of type 3 collagen and switch to type-1. The Glasgow team have already been successful in

making the switch in cultured cells in the lab and in mice. They will now work with international

collaborators to trial the treatment on horses, which also frequently suffer tendon injuries, particularly in

racing. Following this trial, the team intends to commercialise the treatments through a spin-out

company called Causeway Therapeutics focussing on bringing safe and effective medicines to human and

veterinary markets. Neal Millar, an academic consultant orthopaedic surgeon and clinical senior

research fellow at the University of Glasgow, said: "Tendinopathy is essentially the result of an imbalance

between collagen type-1 and type-3 and we have discovered the molecular cause. This breakthrough has

allowed us to find a way to alter the levels of collagen type-3 in tendons, with the ultimate aim to get

patients with tendon injuries better quicker." Co–investigator and senior molecular biologist Dr Derek

Gilchrist commented that: "Our studies have revealed the previously unrecognised ability of a single

microRNA to cross-regulate important functions in the early biological processes that lead to tissue

repair." Results of the previous studies by the team, which also includes Professor Iain McInnes, Director

of the Institute of Infection, Immunity and Inflammation within the University are published in Nature

Communications and reveal the role of the microRNA 29a in tendon tissue repair.”

http://www.nature.com/articles/ncomms7774 MicroRNA29a regulates IL-33-mediated tissue

remodelling in tendon disease

https://www.findaphd.com/search/projectdetails.aspx?PJID=74738 The role of microRNA:target

interactions in tendon function deterioration “MicroRNAs are gene expression regulators, which

control tissue homeostasis in response to environmental and intracellular changes (Brown & Goljanek-

Whysall 2015). Their expression is dysregulated during human and animal ageing in a number of

different systems/tissues (Soriano et al, 2016). Regulation of specific microRNAs in tendons subsequent

to loading has been characterised (Mendias et al., 2012) whilst miR-29 has been shown to mediate

References 14 website: JMR, http://fluoroquinolonethyroid.com

tendon remodelling (Millar et al., 2015). However, currently, there is little known about the role of

microRNAs in maintaining tendon homeostasis. This project will characterise distinct dysregulation of

microRNA:target interactions in endotenon/IFM leading to defective ECM maintenance and turnover,

hence one of the underlying mechanisms of tendon dysfunction. This project will be the first to test the

functional relevance of dysregulated microRNA:target interactions resulting in decline in tendon

function, providing a basis for potential targeted interventions aimed at ameliorating this condition.”

http://jap.physiology.org/content/113/1/56 Mechanical loading and TGF-β change the expression of

multiple miRNAs in tendon fibroblasts. “Tendons link skeletal muscles to bones and are important

components of the musculoskeletal system. There has been much interest in the role of microRNA

(miRNA) in the regulation of musculoskeletal tissues to mechanical loading, inactivity, and disease, but it

was unknown whether miRNA is involved in the adaptation of tendons to mechanical loading. We

hypothesized that mechanical loading and transforming growth factor-β (TGF-β) treatment would

regulate the expression of several miRNA molecules with known roles in cell proliferation and

extracellular matrix synthesis. To test our hypothesis, we subjected untrained adult rats to a single

session of mechanical loading and measured the expression of several miRNA transcripts in Achilles

tendons. Additionally, as TGF-β is known to be an important regulator of tendon growth and adaptation,

we treated primary tendon fibroblasts with TGF-β and measured miRNA expression. Both mechanical

loading and TGF-β treatment modulated the expression of several miRNAs that regulate cell proliferation

and extracellular matrix synthesis. We also identified mechanosensitive miRNAs that may bind to the 3′-

untranslated region of the basic helix-loop-helix transcription factor scleraxis, which is a master regulator

of limb tendon development. The results from this study provide novel insight into the mechanobiology of

tendons and indicate that miRNA could play an important role in the adaptation of tendons to growth

stimuli.”

https://www.ncbi.nlm.nih.gov/pubmed/22572093 Cytotoxic lymphocyte microRNAs as prospective

biomarkers for Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372601/ Identification of a MicroRNA Signature for

the Diagnosis of Fibromyalgia. “Diagnosis of fibromyalgia (FM), a chronic musculoskeletal pain

syndrome characterized by generalized body pain, hyperalgesia and other functional and emotional

comorbidities, is a challenging process hindered by symptom heterogeneity and clinical overlap with

other disorders. No objective diagnostic method exists at present. The aim of this study was to identify

changes in miRNA expression profiles (miRNome) of these patients for the development of a quantitative

diagnostic method of FM. In addition, knowledge of FM patient miRNomes should lead to a deeper

understanding of the etiology and/or symptom severity of this complex disease. . . . Microarray analysis

of FM patient PBMCs evidenced a marked downregulation of hsa-miR223-3p, hsa-miR451a, hsa-miR338-

3p, hsa-miR143-3p, hsa-miR145-5p and hsa-miR-21-5p (4-fold or more). All but the mildest inhibited

miRNA, hsa-miR-21-5p, were validated by RT-qPCR. Globally, 20% of the miRNAs analyzed (233/1212)

showed downregulation of at least 2-fold in patients. This might indicate a general de-regulation of the

miRNA synthetic pathway in FM. No significant correlations between miRNA inhibition and FM cardinal

symptoms could be identified. However, the patient with the lowest score for mental fatigue coincided

with the mildest inhibition in four of the five miRNAs associated with the FM-group. We propose a

References 14 website: JMR, http://fluoroquinolonethyroid.com

signature of five strikingly downregulated miRNAs (hsa-miR223-3p, hsa-miR451a, hsa-miR338-3p, hsa-

miR143-3p and hsa-miR145-5p) to be used as biomarkers of FM. Validation in larger study groups is

required before the results can be transferred to the clinic.”

https://www.ncbi.nlm.nih.gov/pubmed/24554510 microRNA expressions in CD4+ and CD8+ T-cell

subsets in autoimmune thyroid diseases. “Graves' disease (GD) and Hashimoto's thyroiditis (HT) are

the most common autoimmune thyroid diseases (AITD). MicroRNAs (miRNAs) critically control gene-

expression and play an important role in regulating the immune response. The aim of this study was to

prove significant variations of key immunoregulatory miRNAs in peripheral blood mononuclear cells

(PBMCs) and in CD4+ and CD 8+ T-cells of AITD patients. Selected miRNAs were amplified by a

semiquantitative SYBR Green PCR from PBMCs and purified CD4+ and CD 8+ T-cells of 59 patients with

GD, HT, and healthy controls. Both GD and HT showed significantly decreased miRNA 200a_1 and miRNA

200a2* in CD4+-T-cells (mean relative expression 12,57 in HT vs. 19.40 in control group (CG), p=0.0002;

12,10 in GD vs. 19.40 in CG, p=0.0002) and in CD8+-T-cells (13.13 in HT vs. 18,12 in CG, p=0.02; 11.66 in

GD vs. 18.12 in CG, p=0.0002). GD and HT showed significantly decreased miRNA 155_2 and miRNA

155*_1 in HT in CD8+-T-cells (10.69 in HT vs. 11.30 in CG, p=0.01; 10.40 in GD vs. 11.30 in CG, p=0.005).

This study confirms significant variations of miRNA200a and miRNA155 in patients suffering from GD

and HT in vivo in CD4+ T-cells and CD8+ T-cells. These data may help to better understand the gene

regulations in the causative cells causing these autoimmune processes. They extend our very limited

knowledge concerning miRNAs in thyroid diseases.”

http://onlinelibrary.wiley.com/doi/10.1111/cen.12432/abstract Circulating microRNAs in

autoimmune thyroid diseases. “Autoimmune thyroid diseases (AITDs), including Graves’ disease (GD)

and Hashimoto's thyroiditis (HT), are the most common autoimmune diseases. MicroRNAs (miRNAs) are

small noncoding RNAs, which can play pivotal roles in immune functions and development of

autoimmunity. Recently, it has been recognized that identification of circulating miRNAs can provide

important and novel information regarding disease pathogenesis and clinical condition. However, the

role circulating miRNAs in AITD has not yet been described. The aim of this study was to characterize the

different circulating levels of miRNA in patients with AITD. Sixty-four participants who met the criteria

for HT or GD and healthy subjects were recruited. Microarrays were used to analyse the expression

patterns of miRNA in serum obtained from patients with HT and GD and healthy subjects. After analysing

the microarray data, four interesting miRNAs (miR-16, miR-22, miR-375 and miR-451) were selected and

validated by quantitative real-time PCR. Several miRNAs were observed to be differently expressed in

serum from patients with AITD compared with healthy subjects by microarray analysis. Further analysis

consistently showed that serum levels of miR-22, miR-375 and miR-451 were increased in patients with

HT. On the other hand, the serum levels of miR-16, miR-22, miR-375 and miR-451 were increased in

patients with GD compared with healthy subjects. We revealed that different levels of serum miRNAs

were associated with GD and HT, which may play a role in the pathogenesis of these diseases.”

https://www.ncbi.nlm.nih.gov/pubmed/24990808 Associations of single nucleotide polymorphisms in

precursor-microRNA (miR)-125a and the expression of mature miR-125a with the development and

prognosis of autoimmune thyroid diseases. “It is important to search the biomarker to predict the

development and prognosis of autoimmune thyroid diseases (AITDs) such as Hashimoto's disease (HD)

References 14 website: JMR, http://fluoroquinolonethyroid.com

and Graves' disease (GD). MicroRNA (miR) bind directly to the 3' untranslated region of specific target

mRNAs to suppress the expression of proteins, promote the degradation of target mRNAs and regulate

immune response. miR-125a is known to be a negative regulator of regulated upon activation normal T

cell expressed and secreted (RANTES), interleukin (IL)-6 and transforming growth factor (TGF)-β;

however, its association with AITDs remains unknown. To clarify the association between AITDs and miR-

125a, we genotyped the rs12976445 C/T, rs10404453 A/G and rs12975333 G/T polymorphisms in the

MIR125A gene, which encodes miR-125a, using direct sequencing and polymerase chain reaction-

restriction fragment length polymorphism (PCR-RFLP) methods in 155 patients with GD, 151 patients

with HD and 118 healthy volunteers. We also examined the expression of miR-125a in peripheral blood

mononuclear cells (PBMCs) from 55 patients with GD, 79 patients with HD and 38 healthy volunteers

using quantitative real-time PCR methods. We determined that the CC genotype and C allele of the

rs12976445 C/T polymorphism were significantly more frequent in patients with HD compared with

control subjects (P < 0·05) and in intractable GD compared with GD in remission (P < 0·05). The

expression of miR-125a was correlated negatively with age (P = 0·0010) and down-regulated in patients

with GD compared with control subjects (P = 0.0249). In conclusion, miR-125a expression in PBMCs and

the rs12976445 C/T polymorphism were associated with AITD development and prognosis. . . . The

MiR125A gene, which encodes miR-125a, is located on chromosome 19q13.41 in a gene cluster

containing MIR99B and MIR7E. miR-125a is known to be a negative regulator of Kruppel-like factor 13

(KLF13) and the tumour necrosis factor α-induced protein 3 (TNFAIP3), inhibits the production of

regulated on activation normal T cell expressed and secreted (RANTES) and promotes the nuclear factor

kappa B (NF-κB) pathway [12,15]. It has been reported that miR-125a is down-regulated in systemic

lupus erythematosus (SLE) [15,19], breast cancer [20], gastric cancer [21], ovarian cancer [22] and

verrucous carcinoma [14], although the roles of miR-125a in AITD still remain unclear. Therefore, we

performed a quantification of miR-125a expression in peripheral blood mononuclear cells (PBMCs). A G/T

single-nucleotide polymorphism, named rs12975333, has been identified in the MIR125A gene, and its T

allele blocks the pri- to pre-miR-125a processing step [17]. There are also two polymorphisms in this

gene, rs10404453 A/G and rs12976445 C/T, which may be correlated with the expression of mature miR-

125a in patients with AITDs. In this study, we genotyped these three MIR125A SNPs in patients with

AITD.”

https://www.ncbi.nlm.nih.gov/pubmed/25863684 Decreased expression of microRNA-125a-3p

upregulates interleukin-23 receptor in patients with Hashimoto's thyroiditis. “Interleukin IL-23

receptor (IL-23R) is increasingly recognized as a key checkpoint in autoimmune diseases, including

Hashimoto's thyroiditis (HT). However, the molecular mechanisms regulating IL-23R expression are still

unknown. MicroRNAs have emerged as key regulators of various biological events via suppressing target

mRNAs at the posttranscriptional level. In this study, we found that the IL-23R mRNA expression was

increased in peripheral blood mononuclear cells from HT patients, and there was a positive correlation

between the level of IL-23R mRNA and the serum level of anti-thyroglobulin antibody (TgAb). The miR-

125a-3p expression was decreased and inversely correlated with elevated level of IL-23R in patients with

HT. MiR-125a-3p inhibited IL-23R expression through directly targeting 3'untranslated region of IL-23R.

An inverse correlation was observed between the level of miR-125a-3p and serum level of TgAb.

Furthermore, we also found upregulated IL-23R expression and downregulated miR-125a-3p expression

References 14 website: JMR, http://fluoroquinolonethyroid.com

in thyroid tissues from HT patients. Taken together, our results indicate that decreased expression of

miR-125a-3p was involved in the pathogenesis of Hashimoto's thyroiditis.”

https://www.researchgate.net/publication/230834962_MicroRNAs_miR-146a1_miR-155_2_and_miR-

200a1_Are_Regulated_in_Autoimmune_Thyroid_Diseases MicroRNAs miR-146a1, miR-155_2, and

miR-200a1 Are Regulated in Autoimmune Thyroid Diseases. “Graves' disease (GD) and Hashimoto´s

thyroiditis (HT) are the most common autoimmune thyroid diseases (AITD). The exact etiology of the

immune response to the thyroid is still unknown. MicroRNAs (miRNAs) critically control gene-expression.

It has become evident that some miRNAs play an important role in regulating the immune response, as

well as immune cell development. However, data on the role of miRNAs in autoimmune thyroid diseases

are lacking. Objective: The aim of this study was to determine levels of key immunoregulatory miRNAs in

thyroid glands of AITD patients and healthy controls Design: Several miRNAs were amplified by a

semiquantitative TaqMan PCR from fine needle aspiration biopsies of thyroid tissue of 28 patients with

GD, HT, and healthy controls. Results: miRNA 146a1 is significantly decreased in the thyroid tissue of GD

(mean relative expression 5,17 in GD group vs. 8,37 in controls, p = 0.019) whereas miRNA 200a1 is

significantly decreased (mean 8,30 in HT group vs. 11,20 in controls, p = 0.001) and miRNA 155 2 is

significantly increased (mean 12,02 in HT group vs. 8,01 in controls, p = 0.016) in the thyroid tissue of HT

compared to controls. Conclusion: Although limited by small sample size and some other limitations (e.g.

missing matching for age and medication), our preliminary data open up a new field of research

concerning miRNAs in thyroid diseases. Further studies in this interesting field are clearly warranted.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3432448/ MIR141 Expression Differentiates

Hashimoto Thyroiditis from PTC and Benign Thyrocytes in Irish Archival Thyroid Tissues.

“MicroRNAs (miRNAs) are small non-coding RNAs approximately 22 nucleotides in length that function

as regulators of gene expression. Dysregulation of miRNAs has been associated with initiation and

progression of oncogenesis in humans. Our group has previously described a unique miRNA expression

signature, including the MIR200 family member MIR141, which can differentiate papillary thyroid cancer

(PTC) cell lines from a control thyroid cell line. An investigation into the expression of MIR141 in a series

of archival thyroid malignancies [n = 140; classic PTC (cPTC), follicular variant PTC, follicular thyroid

carcinoma, Hashimoto thyroiditis (HT), or control thyrocytes] was performed. Each cohort had a

minimum of 20 validated samples surgically excised within the period 1980–2009. A subset of the HT and

cPTC cohorts (n = 3) were also analyzed for expression of TGFβR1, a key member of the TGFβ pathway

and known target of MIR141. Laser capture microdissection was used to specifically dissect target cells

from formalin-fixed paraffin-embedded archival tissue. Thyrocyte- and lymphocyte-specific markers

(TSHR and LSP1, respectively), confirmed the integrity of cell populations in the HT cohort. RNA was

extracted and quantitative RT-PCR was performed using comparative CT (ΔΔCT) analysis. Statistically

significant (p < 0.05) differential expression profiles of MIR141 were found between tissue types. HT

samples displayed significant downregulation of MIR141 compared to both cPTC and control thyrocytes.

Furthermore, TGFβR1 expression was detected in cPTC samples but not in HT thyrocytes. It is postulated

that the downregulation of this miRNA is due, at least in part, to its involvement in regulating the TGFβ

pathway. This pathway is exquisitely involved in T-cell autoimmunity and has previously been linked with

References 14 website: JMR, http://fluoroquinolonethyroid.com

HT. In conclusion, HT epithelium can be distinguished from cPTC epithelium and control epithelium based

on the relative expression of MIR141.”