Sterkiella, i.e., S. tricirrata, based on two populations isolated from soil samples collected from the Colfiorito Regional Park, Umbria Region, Italy and from the Silent Valley National Park, India. Both populations showed a highly similar morphology, however different ontogenetic pattern in between. The study confirms the validity of the species S. tricirrata which was considered to be a species within the Sterkiella histriomuscorum complex. The main ontogenetic difference between S. tricirrata and other species of the genus Sterkiella is the different mode of formation of anlagen V and VI of the proter in the former. In the phylogenetic analyses, Sterkiella tricirrata clusters with Sterkiella sinica within the stylonychine oxytrichids, in a clade away from the type species (Sterkiella cavicola) of the genus Sterkiella. The study highlights the importance of ontogenetic as well as molecular data in shedding light on the polyphyletic behavior of the genus Sterkiella. A detailed description of S. tricirrata based on morphology, ontogenesis and molecular phylogenetic methods is presented. Further, the improved diagnosis has been provided for the genus Sterkiella and the poorly known species S. tricirrata.Present study, investigates a poorly known species of the genus  Fragmospina Foissner, 2016 is a recent example where morphology of cyst has been incorporated as a generic character. Morphologically, Fragmospina depressa Foissner, 2016, is rather similar in the arrangement of ciliature with Sterkiella histriomuscorum  Foissner, Blatterer, Berger & Kohmann, 1991; however, the undulating membranes are arranged in Australocirrus pattern [Metasterkiella Kumar et al., 2017, for a species isolated from the petroleum contaminated soil, which was morphologically similar to species of the genus Sterkiella Foissner, Blatterer, Berger & Kohmann, 1991. However, this species possesses a variant character in ontogenetic pattern, i.e., it is the only known stylonychid ciliate, thus far, where cirrus V/3 was incorporated during the anlagen formation.Recent studies, among the hypotrich and spathidiid ciliates, have shown that detailed observations of characters often resolve the discrepancy between the morphological and molecular analyses –4. This  pattern , 10. SimSterkiella is a non-monophyletic assemblage [Sterkiella histriomuscorum, a sibling species complex according to Berger [The relatedness between genera within the subfamilies Oxytrichinae and Stylonychinae is rather difficult to understand, despite being strongly reflected as monophyletic groups in the phylogenetic analyses –15. A prsemblage , 16, 17.o Berger , includeSterkiella tricirrata  Berger, 1999, which is probably a synonym of one of the species within the Sterkiella histriomuscorum complex together with S. terricola, according to the remarks of Berger [S. tricirrata were studied and found to be highly similar in morphology. Ontogenetic stages of both populations showed difference in the anlagen formation during the early divisional stages with respect to that of Sterkiella species [Sterkiella.In this study, we describe a poorly known species f Berger . The Ind species , 18, 19,Sterkiella tricirrata was established for both the populations as described in Kumar et al. [Chlorogonium elongatum as food source. Observations on the live specimens were made using a microscope with bright-field and differential interference contrast illuminations at a magnification of 100–1000×. The protargol staining method described by Kamra and Sapra [Soil samples were collected from the core zone of the Silent Valley National Park, India  in January, 2008 and from the plains of the Colfiorito and Plestini uplands, Umbria region, Central Italy  in July, 2009. Vegetative cells were excysted from resting cysts from two-weeks-dried soil samples (approximately 200 g) by employing the non-flooded Petri dish method [nd Sapra  was usednd Sapra .FW 5’-AACCTGGTTGATCCTGCCAGT-3’) and Euk B (RV 5’-TGATCCTTCTGCAGGTTCACCTAC- 30) [FW 5’-TAG AGG GAC TTT GTG TGC AAC C-3’) and Eup 18S (RV 5’-ATC TCC CTG AAA CAC ACG TTG G-3’) were used in combination with the universal primers for amplification and sequencing. The PCR program for 18S rDNA amplification included an initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, 55°C for 45 s and 72°C for 80 s, with a final extension step at 72°C for 10 min. After confirmation of the appropriate size, the PCR products were purified using the Nucleospin gel extraction kit (Qiagen) and were then directly sequenced on both strands at StarSEQ GMBH, Germany.Unfortunately, we could not perform the DNA extraction for the Indian population. Thus the methods described here, including the phylogenetic analyses of the SSU rRNA gene, exclusively refer to the Italian population. Five cells were collected from a clonal culture with the help of glass micropipettes and washed three times with autoclaved distilled water (same culture was used for live observation and protargol staining to study morphology and ontogenesis). Genomic DNA was extracted using the Norgen DNA Kit , following the manufacturer’s instruction . ExtractTAC- 30) . AdditioSterkiella tricirrata was aligned with 55 SSU rRNA gene sequences of hypotrich ciliates from GenBank using the MAFFT software v. 7.047 (choosing the iterative refinement methods Q-INS-I that considers the secondary structure of the SSU rRNA molecules) [For phylogenetic analyses, the SSU rRNA gene sequence of lecules) .Akaike Information Criterion corrected (AICc). Markov chain Monte Carlo (MCMC) simulations were run, with two sets of four chains using the default settings, for 1,000,000 generations with trees sampled every 100 generations and discarding the first 25% of the sampled trees as burn-in. The remaining trees were used to generate a consensus tree and to calculate the posterior probabilities (PP) of all branches using the majority-rule consensus approach. The previous alignment was also used to perform a Maximum Likelihood (ML) tree by means of the Molecular Evolutionary Genetic Analysis (MEGA) software, v.5.2.2 [http://tree.bio.ed.ac.uk/software/figtree/.Ambiguously aligned regions were identified and excluded from the phylogenetic analyses with GBlocks v.0.91b  using pa v.5.2.2  using thSterkiella tricirrata is available from the GenBank/EMBL databases (accession number: MG805314). Two neotype slides of the Italian population containing the protargol stained neotype specimen and relevant morphostatic specimens have been deposited at the Natural History Museum, London, UK, with registration numbers NHMUK 2014.3.20.1 and NHMUK 2014.3.20.2. Further, two slides of the Indian population is deposited at the Natural History Museum, London, UK, with registration numbers NHMUK 2011.7.4.2 and NHMUK 2011.7.4.3 and one at the type collection of the Zoological Survey of India, Kolkata, India, with registration number Pt 3067. The SSU rRNA gene sequence is deposited in GenBank (accession number: MG805314).The newly obtained SSU rRNA gene sequence of http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:DB29FEE1-22B6-48CC-9E8D-661AD15BBB06. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “Sterkiella tricirrata highly overlap  measurements and morphometric data in Cirral pattern and number of cirri rather constant. Invariably, 16 fronto-ventral-transverse cirri Figs . Three fInvariably six dorsal kineties with bristles about 2–3 μm long in protargol preparations. Kinety 1 and 4 shortened anteriorly, kineties 2–3 bipolar, kineties 5 and 6 distinctly shortened posteriorly Figs . Three cAdoral zone extends about 35% of body length, on average composed of 23 membranelles with about 15 μm long cilia in vivo, bases of largest membranelles about 5 μm wide in protargol preparations Figs . UndulatResting cysts (two-week-old) about 35 μm across in vivo; cyst surface with hyaline ridges, about 3.0 μm high . Cyst waSterkiella [The ontogenetic stages of Italian and Indian population show a common origin of anlagen II, III, V, and VI for the proter and the opisthe Figs . Differeerkiella , 19  Figs  and 9K. Nuclear division proceeds in the usual manner, i.e., in mid-dividers the macronuclear nodules fuse to form a single mass which divides twice to produce the typical four nodules in late dividers Figs . The micSterkiella tricirrata Italian population is 1,628 bp in length and has a GC content of 45.15%. It has been deposited in the NCBI database under the accession number MG805314. Phylogenetic trees inferred from the SSU rRNA gene sequences using ML and BI present similar topologies; thus, only the BI tree is shown here (Sterkiella tricirrata clusters with Sterkiella sinica (1.00 BI and 99% ML) within the stylonychine oxytrichids group.The SSU rRNA gene sequence of own here . SterkieSterkiella tricirrata can be compared with species of the genus Sterkiella having two macronuclear nodules, i.e., Sterkiella histriomuscorum  Foissner, Blatterer, Berger & Kohmann, 1991; S. nova Foissner & Berger, 1999; S. subtropica Chen et al., 2015; S. sinica Chen et al., 2016, and S. ecuadoriana Foissner & Heber in Foissner, 2016. Sterkiella tricirrata mainly differs from all the above mentioned species in having invariably three (vs. four or five) transverse cirri. Apart from transverse cirri, the Italian and Indian populations of S. tricirrata possess lower number of adoral membranelles (20–26 and 21–26 vs. 26–44 average from populations described) in comparison with S. histriomuscorum [Sterkiella histriomuscorum with four transverse cirri [Sterkiella tricirrata. This, supports the validity of Sterkiella tricirrata at species rank and out of the S. histriomuscorum complex; this separation is in agreement with Kumar et al. [Sterkiella species with four macronuclear nodules mainly on the basis of four transverse cirri. Sterkiella histriomuscorum and S. nova are indistinguishable based on the morphology and gene sequence data are required for their identification [Sterkiella subtropica can be separated from S. tricirrata Italian and Indian populations by the marine vs. terrestrial habitat, large body size in vivo 100–200 × 35–70 μm (vs. 70–90 × 30–50 μm), slightly higher number of adoral membranelles 25–39 (vs. 20–26 and 21–26), and number of right 19–27 (vs. 18–21 and 14–18) and left 18–26 (vs. 14–18 and 13–16) marginal cirri [Sterkiella tricirrata can be separated from S. ecuadoriana by the slightly larger body length (63–86 μm and 61–74 vs. 91–150 μm), adoral membranelles (20–26 and 21–26 vs. 32–49) and bristles in DK1 (13–18 and 17–22 vs. 27–45) [Sterkiella sinica can be distinguished from Italian and Indian populations of S. tricirrata in having an extra cirrus between the second and third frontal cirri (vs. no such cirrus), distance between cirri V/2 and V/3 24.5 μm (vs. 19.3 and 10.6 μm) and cirri V/3 and V/4 4.7% (vs. 9.4% and 16.1%) of the body length, number of cirri in left 19–23 (vs. 14–18 and 13–16) marginal row, and by the lower number of bristles in DK1 (22–25 vs. 13–18 and 17–22), DK2 (20–24 vs. 14–21 and 14–20), DK3 (14–19 vs. 10–14 and 7–14), DK4 (13–19 vs. 8–13 and 11–15), DM1 (9–15 vs. 5–8 and 7–10), and DM2 (5–10 vs. 2–5 and 3–6) [Sterkiella sinica and Italian and Indian populations of S. tricirrata were observed in the body size 85–110 × 35–45 μm (vs. 70–90 × 30–50 μm) and number of cirri in right 18–22 (vs. 18–21 and 14–18) marginal row [muscorum . There ise cirri ; howeverfication . Sterkieand 3–6) . Minor dinal row .Sterkiella, Sterkiella tricirrata can be compared with Parasterkiella thompsoni  Küppers et al., 2011; Metasterkiella koreana Kumar et al., 2017; and Fragmospina depressa Foissner, 2016. Sterkiella tricirrata mainly differs from Parasterkiella thompsoni in having two (vs. three) macronuclear nodules, numbers of dorsal kineties 6 (vs. 5), and presence (vs. absence) of fragmentation of dorsal kinety 3 during dorsal morphogenesis [Metasterkiella koreana mainly in the number of transverse cirri (invariably 3 vs. 5) and in the ontogenesis, i.e., cirrus V/3 intact (vs. involved during anlagen formation) [Fragmospina depressa can be separated from S. tricirrata by having a paroral membrane close (vs. distant) to the adoral membranelles, number of transverse cirri 5 (vs. invariably 3) and the structure of resting cyst, i.e., spinous (vs. wrinkled) surface [Other than species of the genus Sterkiella tricirrata shows minor differences in size and ciliature with the Italian population as mentioned in the description section. The resting cyst of Indian population appears to be smooth ; however, additional data on the resting cyst of the Indian population is required to confirm this feature. The original population described by Buitkamp [S. tricirrata mentioned the presence of five (instead of six recorded in the present study) dorsal kineties. We agree with Berger [The Indian population of Buitkamp  could noBuitkamp  rely in h Berger  since thh Berger . A reinvSterkiella. Later, Foissner et al. [Sterkiella cavicola  Foissner, Blatterer, Berger & Kohmann, 1991, correcting the previous observations that the anlagen V and VI originate by disaggregation of the cirrus V/4. The same pattern is observed also for Sterkiella tricirrata where cirrus V/4 generates anlagen V and VI of the opisthe; however the anterior patches of both the anlagen move anteriorly and later form the proter anlagen V and VI  by the anlagen IV, V, and VI of the proter in late-early divider, indicates that the Indian population may represent a separate subspecies/species if the pattern mentioned is found to be stable in other populations with consensus of molecular data. As of now, we do not perform its separation from the Sterkiella histriomuscorum complex and wait for further data to resolve the phylogenetic status of the species within the complex. However, the different morphogenetic patterns, within the genus Sterkiella, as seen in the present study and Foissner et al. [Sterkiella. The Austrian population of Sterkiella histriomuscorum shows some similarity in anlagen formation with S. tricirrata [Berger and Foissner  reportedr et al.  providedV and VI . On the la Kahl, 35 Foissnla Kahl, 35 Foissnicirrata , 33; howSterkiella tricirrata clusters with S. sinica  of the genus Sterkiella; we assume that the molecular relatedness of S. tricirrata and S. sinica could be because of similarity in the formation of anlagen. However, a detailed investigation of the ontogenesis of S. sinica is needed to properly compare these genetically similar species. Our phylogenetic analyses also shows that S. histriomuscorum and S. cavicola behave as sisters of a larger clade containing, in addition to the aforementioned S. tricirrata + S. sinica sub-clade, a further high supported sub-clade including S. tetracirrata + Gastrostyla steinii . The other Sterkiella species are distributed across two more clades: i) S. nova with Tetmemena bifaria ; and ii) S. subtropica with Metasterkiella koreana . The monophyly of the genus Sterkiella is not supported in our phylogenetic analyses as also evident in other recent studies [S. histriomuscorum complex as well as from other Sterkiella species are required to obtain better resolution. Although in recent years, the situation has been slightly improved with the establishment of genera, namely, Parasterkiella Küppers et al., 2011 and Metasterkiella Kumar et al., 2017. Sterkiella subtropica and S. nova cluster away from the type species (S. cavicola); for the former species, Kumar et al. [Metasterkiella due to highly similar morphology and gene sequence; this interpretation is also supported by our phylogenetic analyses. The classification of Sterkiella nova has been widely debated among classical taxonomists and molecular biologists who established this species as model organism for analyzing various biological phenomena such as epigenetic inheritance, genome rearrangement, somatic differentiation and many others [S. histriomuscorum and S. nova in great detail from viable genetic systems (via frozen resting cysts) established by molecular biologists. They mentioned that both species are inseparable based on the morphological characters; though based on the differences in molecular sequences of actin I and DNA pol α genes, they proposed them as different species. Considering the complexity of identification it is unclear whether the gene sequences provided by Hewitt et al. [S. nova, is of same species described by Foissner and Berger [ studies , 15–17. y others . In thisy others  describet et al. , which id Berger .Sterkiella is the data on the ontogenetic pattern on the ventral and dorsal surface. In Sterkiella cavicola anlagen V and VI of the proter originate from cirrus IV/3 forming W-shaped anlagen [Sterkiella tricirrata and the genus Metasterkiella. In our phylogenetic tree, Metasterkiella forms a distant clade away from that of Sterkiella tricirrata the involvement of cirrus V/3 (vs. intact) during anlagen formation possibly justifies this distant relationships. Nonetheless, several examples exists like, Parasterkiella thompsoni, Fragmospina depressa, which would have been easily identified as Sterkiella species but separated based on detailed investigations on morphology and cyst structure. Parasterkiella thompsoni shows a different ontogenetic pattern on the dorsal surface and acquires a place distant from Sterkiella species [Fragmospina no gene sequence is available thus far. We believe that addition of related molecular sequences, e.g., Fragmospina, S. histriomuscorum populations, and gene sequences from other loci will further support the monophyly of the genus Sterkiella.As of now, only differences which seem most suitable to solve the polyphyletic behavior of the genus  anlagen , whereas species , for thePseudouroleptus plestiensis [Sterkiella tricirrata (present study).Ciliated protists are a highly diverse group of microbial eukaryotes that play a key role in soil microbial food webs by mediating the fluxes of nutrients and energy between different trophic levels . Neverthstiensis  and SterIn the end, our sampling effort has allowed us to contribute to strengthen the knowledge about soil ciliate diversity, providing hints about their biogeographic distributions and new distinguishing characters  among hypotrich ciliates that can be helpful in species identification within problematic (cryptic) species "complexes".Phylum Ciliophora Doflein, 1901Class Spirotrichea Bütschli, 1889Order Sporadotrichida Fauré-Fremiet, 1961Family Oxytrichidae Ehrenberg, 1838Oxytricha pattern. Opisthe's anlage II may contribute to proter's anlage II. Anlagen V and VI of the proter originate from cirrus IV/3 forming W-shaped anlagen or from anlagen V and VI of the opisthe.Body semi-rigid. Eighteen or less frontal-ventral-transverse cirri arranged in typical oxytrichid pattern. One right and one left row of marginal cirri. Six dorsal kineties including dorsomarginal rows, kinety 3 with simple fragmentation; caudal cirri present. Undulating membranes in Size about 80 × 40 μm in vivo; body elongate to broadly ellipsoidal. Nuclear apparatus composed of two macronuclear nodules and two micronuclei on average. Invariably, 16 frontal-ventral-transverse cirri, including three transverse cirri. Right and left marginal rows composed of an average of 15 and 14 cirri, respectively. Adoral zone 37% of body length and composed of an average of 23 membranelles. Three narrowly spaced, inconspicuous caudal cirri. Resting cyst with wrinkled surface. Soil habitat.Sterkiella tricirrata with sampling site of the Italian population being the type locality. Two neotype slides of Italian population containing the protargol stained neotype specimen  we propose neotypification of the men Figs  and 3A aSterkiella tricirrata from the soil collected from the burnt savannah in the Ivory Coast. The Italian population was identified from the ‘Molinaccio’ site during the summer (dry season), where it was moderately abundant in non-flooded Petri dish culture. For details on the soil physico-chemical parameters and other ciliate species identified in the same soil sample, refer to Bharti et al. [Buitkamp  isolatedi et al. . The Indi et al. . Feeds o


Polycystic ovary syndrome (PCOS) is a complex endocrine disorder and regarded as the leading cause of anovulatory infertility. PCOS is characterized by reproductive dysfunction and metabolic disorders. Baicalin (BAL) is one of the most potent bioactive flavonoids isolated from the radix of Scutellaria baicalensis. In the present study, we investigated the potential effects of BAL on PCOS in dehydroepiandrosterone-treated rats. We found that BAL notably reduced the serum levels of free testosterone, total testosterone, follicle-stimulating hormone, luteinizing hormone, progesterone, and estradiol in PCOS rats. The increase of serum insulin level and HOMA-IR was markedly inhibited by BAL. Moreover, BAL decreased body weights, increased the number of rats with the regular estrous cycle, and ameliorated ovarian histological changes and follicular development in the DHEA-treated PCOS rats. The increase of pro-inflammatory cytokines  and decrease of anti-inflammatory cytokine (IL-10) in PCOS rats were suppressed by BAL. BAL induced a significant decrease in the mRNA expression of steroidogenic enzymes, including 3β-HSD, CYP11A1, CYP19A1, StAR, in ovarian tissues in PCOS rats. Furthermore, BAL inhibited the decrease of AMPK protein level and phosphorylation, the decrease of Akt phosphorylation and the increase of 5α-reductase enzyme 1 expression in ovarian tissues in PCOS rats. The effects of BAL were inhibited by an inhibitor of AMPK, dorsomorphin. The upregulation of AMPK contributed to the beneficial effects of BAL. The results highlight the potential role of BAL for the intervention of PCOS. Polycystic ovary syndrome (PCOS) is a complex endocrine disorder and regarded as the leading cause of anovulatory infertility that affects 8–12% of reproductive-aged women . PCOS isScutellaria baicalensis Georgi (known as huang qin) is a traditional Chinese herb, that has been shown to exhibit antioxidant, anti-inflammatory, anti-bacterial and anti-viral, antidiabetic and anti-dyslipidemic activities , 10. BaiIn the current study, we aimed to evaluate the effects of BAL on the dysregulation of hormones, histological changes of the ovary, and metabolic disorder in PCOS rats. Previous literature and our preliminary studies have suggested that activation of AMP-activated protein kinase (AMPK) is associated with the beneficial effects of BAL . Thus, wBaicalin and dehydroepiandrosterone (DHEA) were purchased from Sigma-Aldrich, USA. Antibodies against AMPK, P-AMPK, Akt, and P-Akt were acquired from Cell Signaling Technology Inc., USA. Antibodies against 5α-R1 and β-actin from Santa Cruz Biotechnology, USA. RIPA lysis from Beotime Corporation, Beijing, China. BCA TM protein assay kit from Pierce Chemical Company, Rockford, USA.All animal experiments were approved by the Institutional Animal Care Committee of The Second Hospital of Hebei Medical University and in strict accordance with the Guidelines for the Care and Use of Laboratory Animals . Forty Wistar female rats  were purchased from the Animal Centre of Hebei Medical University. The animals were housed under a temperature-controlled condition (22 ± 2 °C), with a 12 h/12 h light/dark cycle and had free access to water.Rats were randomly divided into 4 groups (10 rats in each group): Control group, PCOS group, BAL group (50 mg/kg in PCOS rats), BAL + dorsomorphin group (50 mg/kg BAL+ 10 mg/kg dorsomorphin in PCOS rats). Rats in the PCOS, BAL, and BAL + dorsomorphin groups were subcutaneously injected with DHEA  for 20 consecutive days to induce PCOS model with excessive androgen as previously described , 17. AftAfter the treatment, rats were fasted overnight and blood was centrifuged at 3000 rpm for 10 min to collect serum. Serum levels of insulin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), progesterone (P) were detected using ELISA kits . Free testosterone and total testosterone levels were measured using ELISA kits . These assays were carried out according to the manufacturer’s instructions. The homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated according to the following formula: plasma glucose (mmol/l) x serum insulin (mIU/l)/22.5.After the sacrifice of the rats, ovarian tissues were collected followed by the removal of coagulation and appendage. 10% ovarian tissue homogenates in precooled saline were centrifuged at 3000 rpm for 10 min to collect the supernatant. Levels of TNFα, IL-1β, IL-10, and IL-18 in ovarian tissue homogenates were measured using ELISA kits .Ovarian tissue was immediately cut, fixed and embedded with paraffin. The embedded tissue was cut into 5-μm sections, followed by classical HE staining procedure. Pathological changes were observed under an optical microscope .The percentage of different follicles per ovary section was counted. Follicles were classified as cystic follicles, primary follicles, early antral follicles, and atretic follicles according to the previous study .Bodyweight was monitored daily throughout the experimental procedure. The estrous cycle was measured through the microscopic determination of cell types on vaginal smears.Fifty milligrams of ovarian tissues were homogenized in RIPA lysis buffer followed by centrifuging at 12000×g for 10 min at 4 °C and collection of supernatant. The protein concentration was determined by the BCA protein assay . An equal volume of supernatant was mixed with 2 × SDS buffer. Mixtures were separated by electrophoresis via 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein was transferred to a nitrocellulose (NC) membrane . Blocking of membranes was conducted using incubation with 5% fat-free milk at 37 °C for 1 h. Then the membranes were incubated with indicated antibodies at 4 °C overnight. After 4-time washing using TBST, membranes were incubated with goat anti-rabbit-HRP secondary antibody  at room temperature for 1 h. β-actin served as an internal control. The immunoreactive bands were visualized by chemiluminescence and quantified by densitometry using a Quantity One Analysis Software (Bio-Rad).One hundred milligrams of frozen ovarian tissues were homogenized using Trizol to extract total RNA. The RNA concentration was detected using a NanoDrop ND-2000 and RNA quantification was performed by spectrophotometric analysis of OD values at 260/280 nm. RNA was reversely transcribed into cDNA using RT reagent Kit with gDNA Eraser  according to the manufacturer’s protocols. Quantitative RT-PCR detection of target genes was conducted using SYBR Green Master Mix  on a Bio-Rad system. GAPDH was used as an internal reference. Amplification condition: an initial denaturation at 95 °C for 10 min; 95 °C for 15 s, 62 °C for 60 s, 40 cycles.2-tests  was used which was followed by the non-parametric Kruskal–Wallis test for comparison of two groups. P < 0.05 was considered as the limit for statistical significance.Data were expressed as means ± standard deviation (SD). GraphPad Prism software was used to perform statistical analysis. The power analysis of the study was performed before the experiment. Enough numbers of animals were included for all the determinations. Statistical significance was evaluated using one-way analysis of variance followed by Student-Newman-Keuls test. For statistical analysis of the estrous cycle, χThe disorder of hormone secretion was a feature of PCOS. In the current study, we showed that serum levels of free testosterone (B), total testosterone (C), LH (D), FSH (E), progesterone (F), and estradiol (G) were significantly increased in PCOS rats Fig. . As expeInsulin resistance is a feature of PCOS. We examined the effect of BAL on insulin resistance in PCOS rats. As shown in Fig. The bodyweights of rats in PCOS group were significantly higher than those in control group Fig. a. BAL deWe also detected the effect of BAL on histological changes and follicular development in ovarian tissues in PCOS rats. We observed corpus luteum and follicles in control rats and there were oocytes in nearly mature follicles and fewer follicular cell layers Fig. c. In DHEMoreover, we also evaluated the effect of BAL on the percentage of different follicles per ovary section. We showed that the percentages of cystic follicles and primary follicles were significantly increased in PCOS rats Fig. d and e. Inflammation is considered to be associated with the pathological outcomes of PCOS. We also evaluated the effect of BAL on the content of pro-inflammatory and anti-inflammatory cytokines. As shown in Fig. The effect of BAL on the mRNA expression of steroidogenic enzymes in ovarian tissues was also determined. In Fig. We next tried to explore the mechanism of BAL-exhibited effect on PCOS. The activation of AMPK by BAL was confirmed by protein expression of total and phosphorylation of AMPK Fig. a and b. PCOS is a complex syndrome that is associated with abnormal secretion, insulin resistance, and inflammation. Importantly, hyperinsulinemia and insulin resistance are critical hallmarks and contributors to the progression of metabolic and reproductive dysfunction in women with PCOS. Hyperinsulinemia and insulin resistance could promote mutually and result in hyperandrogenism, leading to abnormal reproductive function. The pituitary response to gonadotropin-releasing hormone (GnRH) is promoted by hyperinsulinemia, resulting in enhancement of luteinizing hormone and androgen secretion, which, in turn, affects the function of the hypothalamus-pituitary-ovarian gonadal axis (HPO axis) . HyperanIn the study, we evaluated the potential protective effects of BAL against PCOS. We revealed that BAL significantly inhibited the development of PCOS, as evidenced by a significant decrease of serum hormone levels, including free testosterone, total testosterone, LH, FSH, progesterone, and estradiol, an improvement of insulin resistance, and an attenuation of functional and pathohistological changes and follicular development, and a decrease of pro-inflammatory cytokines and an increase of anti-inflammatory cytokines in PCOS rats.It is well-known that AMPK functions as an “energy sensor” in a cell . The dysPI3K/Akt signaling is a major downstream of insulin signaling that mediates the effect of insulin on glucose and lipid metabolism –30. UndeIn addition to AMPK/PI3K/Akt signaling, sirtuin 1 (SIRT1) is possibly involved in the BAL-exhibited effects on PCOS. A natural polyphenolic compound, resveratrol, has been shown to promote the expression of SIRT1 in rat ovarian granulosa cells, which implicated the important role of SIRT1 in ovarian function . It has There are still several issues to be addressed. Considering that PCOS is a multi-system reproductive metabolic disorder, activation of AMPK signaling may not be the only mechanism responsible for the beneficial effects of BAL . We trieIn summary, we identified that BAL could attenuate the dysfunction of endocrine and reproduction in PCOS rats. BAL ameliorated the complications of PCOS, including abnormal secretion of hormones, insulin resistance, pathohistological changes, and inflammation. Among those mechanisms, the upregulation of AMPK contributed to the beneficial effect of BAL Fig. . The res


Peripheral neuropathy (neuropathy) is a common complication of obesity and type 2 diabetes in children and adolescents. To model this complication in mice, 5-week-old male C57BL/6J mice were fed a high-fat diet to induce diet-induced obesity (DIO), a model of prediabetes, and a cohort of these animals was injected with low-dose streptozotocin (STZ) at 12 weeks of age to induce hyperglycemia and type 2 diabetes. Neuropathy assessments at 16, 24 and 36 weeks demonstrated that DIO and DIO-STZ mice displayed decreased motor and sensory nerve conduction velocities as early as 16 weeks, hypoalgesia by 24 weeks and cutaneous nerve fiber loss by 36 weeks, relative to control mice fed a standard diet. Interestingly, neuropathy severity was similar in DIO and DIO-STZ mice at all time points despite significantly higher fasting glucose levels in the DIO-STZ mice. These mouse models provide critical tools to better understand the underlying pathogenesis of prediabetic and diabetic neuropathy from youth to adulthood, and support the idea that hyperglycemia alone does not drive early neuropathy.This article has an associated First Person interview with the first author of the paper. Summary: The mouse models described in this paper provide critical tools to better understand the underlying pathogenesis of prediabetic and diabetic neuropathy from youth to adulthood. Among children and adolescents ranging in age from 5 to 18 years old, obesogenic behaviors  (According to a recent study released by the Centers for Disease Control and Prevention (CDC) as part of the Youth Risk Behavior Surveillance System, 15.6% of adolescents in the US are overweight and an additional 14.8% are obese (ctivity)  underliectivity) . Of the ctivity) .Neuropathy is defined as the loss of peripheral nerve function in the extremities in a distal to proximal pattern that is characterized predominantly by sensory symptoms. There is a high degree of morbidity associated with the condition, including numbness, pain, poor balance and co-ordination, frequent falls, and ulceration in the lower extremities over time that can lead to amputation . Despitewww.diacomp.org) in 2000 to develop novel animal models of diabetic complications in order to understand disease pathogenesis so that effective therapies can be developed and translated to patients  simulates obesity-driven prediabetes and neuropathy , mice fed a high-fat (HF) diet to induce DIO (DIO mice), and DIO mice injected with STZ (DIO-STZ mice) to model T2D A. Mice wDuring the course of the study, all groups displayed a steady increase in weight  were then performed to assess glucose disposal and insulin resistance, respectively, both of which are hallmarks of T2D . DIO-STZAs emerging data support dyslipidemia as a major contributor to neuropathy, plasma cholesterol profiling was next assessed. At 36 weeks, total cholesterol was higher in DIO (232.9±20.4 mg/dl) and DIO-STZ mice (199.4±13.0 mg/dl) compared to SD mice (139.6±10.5 mg/dl) A. DIO anDIO and DIO-STZ mice had motor and sensory nerve conduction velocity (NCV) deficits at 16, 24 and 36 weeks when compared to SD mice A,B. InteA murine model of neuropathy early in the lifespan of the mouse, equivalent to adolescence in humans, can provide a unique and needed resource for the study of diabetes and its complications. In the current study, we longitudinally phenotyped neuropathy in non-genetic murine models of prediabetes (DIO) and type 2 diabetes (DIO-STZ) over a period spanning adolescence to adulthood. Our data revealed an early onset and similar degree of neuropathy among the two disease cohorts of adolescent mice that persisted to adulthood with similar severity in both models, despite the robust hyperglycemia of mice with the type 2 diabetes phenotype. Thus, these juvenile DIO and DIO-STZ mice uniquely parallel the progression of the human condition and represent a valuable tool to study how prediabetes and type 2 diabetes contribute to the development of neuropathy in youth.Given that an animal model of disease should recapitulate the human disorder as closely as possible, the Neurodiab Study Group of the European Association for the Study of Diabetes determined that the diagnostic criteria required for a murine model of diabetic neuropathy should include assessments of behavior, motor and sensory NCVs, and nerve structure . NeuropaUsing these established criteria, we confirmed neuropathy across a spectrum of ages corresponding to the transition between adolescence and adulthood in non-genetic prediabetic and diabetic animals. Animals in our study began a HF diet at 5 weeks of age and, by 12 weeks of age, HF-diet-fed animals weighed significantly more than animals fed normal chow, i.e. these animals had DIO. At 12 weeks, a cohort of DIO mice received STZ. By 16 weeks of age, the murine equivalent of adolescence , all aniOur study is the first to confirm neuropathy in both non-genetic DIO and DIO-STZ adolescent mice. The increasing prevalence of obesity, prediabetes and type 2 diabetes in children and adolescents is an immediate and serious concern, and neuropathy is a common complication in this population , 2017. TFor both prediabetes and diabetes, the ADA recommends lifestyle interventions, with an emphasis on diet and exercise. These recommendations come in light of the fact that glucose control has a modest or no effect on preventing the progression of type 2 diabetic neuropathy in multiple large clinical trials in man . These hWe next assessed glucose intolerance in all animals as they matured into young (24 weeks) adults , and obsRecent data strongly suggest that dyslipidemia significantly contributes to neuropathy . This idThere is no ideal murine model for most human diseases. In the current study, instead of recapitulating the hyperinsulinemia typically seen in type 2 diabetes, DIO-STZ mice instead develop hypoinsulinemia, which is attributed to STZ-induced loss of insulin-producing β-cells. However, the pathogenesis of type 2 diabetes is different in youth versus adult patients, with younger patients tending to be insulin deficient rather than insulin resistant . This feWe also acknowledge that we did not include a SD-STZ control group to determine whether STZ directly damages tissues other than the pancreas, including peripheral nerve tissue. The large doses of STZ used to model type 1 diabetes are known to have a pronounced effect on mice, which exhibit significant weight loss due to muscle atrophy . As no sIn conclusion, we show that DIO and DIO-STZ mice develop metabolic changes and neuropathy in a manner that closely reflects disease progression in adolescents to adults with prediabetes and diabetes, respectively. While DIO-STZ mice display a similar degree of neuropathy as DIO mice, they develop hyperglycemia, making them more representative of frank diabetes. Despite having elevated glucose levels, however, DIO and DIO-STZ mice had nearly identical disease development, further suggesting that factors other than hyperglycemia may play a role in neuropathy progression. Hence, it is possible that dyslipidemia and/or altered insulin signaling resulting from obesity may contribute to nerve pathology. Finally, our study provides new juvenile murine models to assess the neurological complications that commonly afflict children and adolescents with obesity, prediabetes and diabetes. These preclinical disease models are needed to understand disease pathogenesis and develop mechanism-based therapies for neuropathy to allow early intervention in humans from youth to adulthood.ad libitum access to drinking water and food. After 1 week of acclimation, cages were randomly allocated and mice were provided with a SD or HF diet. The SD contains 10% energy from fat, 70% energy from carbohydrates and 20% energy from protein , while the HF diet contains 60% energy from fat, 20% energy from carbohydrates and 20% energy from protein . For all metabolic and neuropathy assessments, mice were transported into designated procedure rooms within the animal facility. All assessments were performed during the day between the hours of 9:00 a.m.-5:00 p.m. All procedures complied with Diabetic Complications Consortium (www.diacomp.org) protocols and were approved by the University of Michigan Institutional Animal Care and Use Committee (IACUC). At study conclusion, mice were euthanized with 150 mg/kg body weight of pentobarbital  delivered by intraperitoneal (IP) injection.Thirty male C57BL/6J mice  were purchased at 4 weeks of age and housed in a specific pathogen-free suite kept at a temperature of 20±2°C with a 12:12 h light:dark cycle. Mice were housed five per cage with fine corncob bedding, provided nestlet as enrichment, and kept with littermates to avoid fighting and aggressive behavior. The animals had 3C6H5O7 per 50 ml ddH2O, pH 4.5). Mice were administered with 75 mg/kg body weight STZ solution (IP injection) using 1 ml disposable insulin syringes  in a fume hood. Once administered, mice were transferred back to their cages containing feed and, 72 h later, mice were injected with a second dose of STZ (50 mg/kg body weight STZ). To aid recovery post-STZ injection, animals were provided with DietGel , while drinking water was supplemented with sucrose (10%) for 2 days after STZ injections to prevent hypoglycemic shock. No adverse events were observed in STZ-injected mice during the course of the study.STZ was administered following the protocol described by the Diabetic Complications Consortium using the dosages described by www.mmpc.org). OxLDL was measured via ELISA . All procedures complied with the Diabetic Complications Consortium (www.diacomp.org) protocols and were approved by the University of Michigan IACUC.Fasting blood glucose levels were measured every 4 weeks from 8 weeks of age to document the development and duration of hyperglycemia. Following a 4 h fasting period, one drop of tail blood was analyzed using a glucometer  with compatible glucose test strips . For GTT and ITT, 1 g/kg body weight glucose  or 0.075 U/kg body weight insulin , respectively, was injected IP and blood glucose levels were serially recorded at 15, 30, 60 and 120 s. At the end of the experimental period, mice were fasted, euthanized and plasma was isolated for further testing. Mice injected with insulin who showed signs of hypoglycemic shock were rescued with 20% glucose solution (IP injection). Plasma insulin levels were assessed at the Mouse Metabolic Phenotyping Center (MMPC) at Vanderbilt , while cholesterol and triglyceride lipoprotein profiles were assessed by FPLC analysis at the MMPC located in Cincinnati . Hindpaw withdrawal latency and sural sensory and sciatic motor NCVs were measured at 16, 24 and 36 weeks, while IENFD was determined at 36 weeks.Neuropathy phenotyping was performed as previously described  in accorThermal hindpaw withdrawal latency was measured using an analgesia meter . Mice were placed in acrylic compartments on a warm (32°C) glass plate and allowed to habituate for 45 min. The light source was maneuvered under the hindpaw and the time of activation of the beam to the time of paw withdrawal was recorded . The ligMice were anesthetized using isoflurane  with a dose of 4-5% for induction and 1-2% for maintenance . Onset oPrior to perfusion, foot pads were collected from the plantar surface of the hindpaw, immersed in Newcomer Zamboni's fixative  overnight at 4°C, rinsed in 5, 10 and 20% sucrose in 0.1 M sodium phosphate buffer, cryoembedded, sectioned (30 μm), and processed for pan-axonal marker, PGP9.5, immunofluorescence  . Three in=8/group is adequately powered to detect significant differences. Analyses were performed using GraphPad Prism 7 , according to F-tests. For normally distributed data, statistically significant differences (P<0.05) were determined using one-way ANOVA with Tukey's post-test for multiple comparisons. For non-normally distributed data, datasets were log2 transformed and the Brown–Forsythe F-test re-run. When log2 transformation normalized distribution, a one-way ANOVA with Tukey's post-test for multiple comparisons was run. When log2 transformation did not normalize distribution, the non-parametric Kruskal–Wallis test with Dunn's post-test for multiple comparisons was run on the original, non-transformed dataset. Data are presented as mean±standard error of the mean (s.e.m.) unless otherwise stated. The statistical methods for individual analyses and P-values for all dataset comparisons are provided in Tables S1-S15.Based on previously published studies , our rep


Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in chronic kidney disease (CKD) patients. QT interval prolongation is a congenital or acquired condition that is associated with an increased risk of torsade de pointes (TdP), sudden cardiac death (SCD), and all-cause mortality in the general population. The prevalence of acquired long QT syndrome (aLQTS) is high, and various acquired conditions contribute to the prolonged QT interval in patients with CKD. More notably, the prolonged QT interval in CKD is an independent risk factor for SCD and all-cause mortality. In this review, we focus on the epidemiological characteristics, risk factors, underlying mechanisms and treatments of aLQTS in CKD, promoting the management of aLQTS in CKD patients. The QT interval represents electrical depolarization and repolarization of the ventricles. QT interval prolongation is a congenital or acquired condition that is associated with an increased risk of TdP and sudden cardiac death (SCD) in the general population. The prevalence of acquired long QT syndrome (aLQTS) is high and increases with the decline in kidney function in CKD patients . The QTcThe QT interval normally varies with heart rate. In this regard, many attempts  have been made to ‘correct’ the QT interval to a value that might be expected if the heart rate is 60 beats per minute. The commonly used correction formulas are the Bazett, Fridericia, Framingham, Hodges, and Rautaharju formulas. The comparison of the superiority among different kinds of correction formulas showed that the Fridericia and Framingham formulas had the best correction, but the Bazett formula was the worst . How lonn = 1452) had CKD, and 14.5% (n = 956) had prolonged QTc. Prolonged QTc was present in 12.9% (n = 658) of subjects without CKD and in 20.5% (n = 298) of those with CKD (p < .0001) [Prolonged QTc is common in patients with CKD . In a st< .0001) . In addi< .0001) ,11,33,34p = .016) [p = .011) interval was an independent predictor of mortality in ESRD regardless of age, sex, diabetes, left ventricular hypertrophy (LVH), left ventricular ejection fraction or the presence of coronary artery disease on angiography [The prevalence of all-cause and cardiovascular mortality was significantly increased in CKD patients with aLQTS. Hag et al. observed 280 patients who had been referred for kidney transplant, and 47% died before kidney transplant during the 40-month follow-up period. Patients with a prolonged QTc (39%) had 1-, 3-, and 5-year death rates of 12%, 36%, and 47%, respectively, vs 8%, 24%, and 36% for those with normal QTc, respectively  . Gen et p = .01; normoalbuminuria vs macroalbuminuria 425 ± 19 ms vs 442 ± 24 ms, p = .0005) [+ dysfunction, and it can cause a prolonged diabetic QTc interval and action potential duration (APD) [CaL caused by the decreased protein expression of Cav1.2 and calmodulin [Ks currents and led to prolongation of the ventricular APD, which was reflected as QT prolongation on ECG [Diabetes accounts for 30–50% of all cases of CKD and affects 285 million (6.4%) adults worldwide . The aLQ= .0005) . In diab= .0005) . Abnormaon (APD) ,46. In tlmodulin . Acute an on ECG .p < .05). The definition of prolong QT interval is >460 ms in female and >450 ms in males. However, this study uniformly defined QT prolongation for men and women was 440 ms, which undoubtedly overestimated the number of patients with prolonged QT interval in hypertensive [There is a bidirectional relationship between hypertension and CKD. Hypertension can occur as the result of CKD, and it is also an important risk factor for CKD progression. Myocardial hypertrophy induced by hypertension can result in reduced repolarization reserve. The prevalence of myocardial hypertrophy is between 16% and 31% in CKD . Adeseyertensive . The defrtensive . The prortensive ,53. QT irtensive . The prortensive . The dowrtensive . The chartensive .Kr and IKs [Ca-L in HF prolong the plateau of APD. In addition, the Na+/Ca2+ exchanger (NCX) also plays a prominent role in modulating the APD in myocytes in failing hearts [Na is an integral part of the sodium current, which persists long after the fast-inactivating component and could contribute to the prolongation of the APD [Na is significantly increased in myocardial cells from failing human hearts and selected inhibition of late INa is effective in shortening the QT interval and reducing ventricular arrhythmogenic activity [The prevalence of LQTS in patients with heart failure is 60–70.2% ,28. Abno and IKs  and the g hearts . Late IN the APD . Late INactivity .p = .0061, α = .01) [p for trend <.001) in NHANES III study. In ARIC, the average differences in QT interval duration in adjusted models comparing the highest versus the lowest quartiles of total phosphorus were 2.3 ms  [I channel activity and increase the APD, further prolonging the QT interval [p = .031). However, this study showed that the prolonged QT was related to both high and low homocysteine levels [Uremic retention compounds with harmful biological or biochemical activity are defined as ‘uremic toxins’, which include urea, creatinine, phosphorus, parathyroid hormone (PTH), fibroblastic growth factor-23 (FGF23), indoxyl sulfate (IS) and homocysteine . Multiplα = .01) . High coα = .01) . Hyperphd <.001) . Hyperphd <.001) . Evidencd <.001) . Patch-cd <.001) . Low serd <.001) ,68,69. Iinterval ,71. The interval . Plasma e levels . Hyperhoe levels . The relHemodialysis is a therapeutic procedure that uses the extracorporeal circulation of blood to ameliorate the azotemia, fluid, electrolyte, and acid–base abnormalities that are characteristic of uremic syndrome. In dialysis patients, a number of small studies using ambulatory electrocardiography showed that the QT interval was increased in the hemodialysis population compared with that in healthy controls during dialysis ,5–7,11. + is removed mainly from the extracellular space and only slightly from the intracellular space; thus, the extracellular potassium level decreases too abruptly, and the intracellular/extracellular potassium ratio increases rapidly [Hypokalemia is a risk factor for a prolonged QT interval, and its prevalence in CKD is 12%–18% ,76. Low  rapidly .4 can prevents the recurrence of TdP without changing the QT interval. The mechanism of action of MgSO4 is not well understood.Hypomagnesaemia is common in CKD patients and is linked to the increased risk of development of coronary artery disease as well as the major cardiovascular risk factors such as dyslipidemia, endothelial dysfunction, metabolic syndrome, atherosclerosis, diabetes, hypertension and elevated fasting insulin levels, suggestive of insulin resistance . Magnesiin vivo and can cause a variety of changes in Ca2+ regulatory mechanisms. Prevalence of hypocalcemia in CKD patients is high and is strongly correlated with prolong QTc [Calcium is crucial for the entirety of the APD. The inward calcium current is a depolarizing current that prolongs the ventricular APD, and it can influence calcium channels and electrogenic exchangers in myocardial cells . CKD canlong QTc . Calciumlong QTc .R = 0.44, p = .035) [R = 0.376, p = .045) [Hypertension is highly prevalent and difficult to control in patients with long-term dialysis. Although 89% of these patients undergo treatment, only 38% have adequate control of their hypertension . The ult = .035) . Calcium = .035) . ROS has = .035) . Elaine  = .045) .Cardiovascular autonomic neuropathy (CAN) affects both the heart rate and ventricular repolarization. A decrease in heart rate variability and an increase in QTc are the initial signs of CAN. Cardiac sympathetic overdrive decreases vagal control in CKD. The association between CAN and GFR is curvilinear . AutonomCKD patients are often prescribed many medications with the potential of prolonging the QT interval . Drug phThe elimination of QT prolonging risk factors is the cornerstone of aLQTS management. However, the risk factors for QT prolongation are sometimes difficult to avoid, in which case regular ECG monitoring and necessary interventions are important, especially in patients who have had TdP. Interventions with aLQTS with TdP include maintenance of high normal serum potassium levels, intravenous magnesium, isoproterenol infusion, and temporary transvenous ventricular pacing. It is worth noting that different interventions are needed depending on the presence of TdP.Typically, the development of severe QT prolongation or TdP requires several precipitating factors to occur in combination to overcome a ‘repolarization reserve’ generated by the presence of multiple cardiac channels. Patients with QT prolongation in the absence of observed TdP episodes should undergo continuous ECG monitoring. The frequency of QT monitoring depends on the clinical situation and the extent of QT prolongation. The necessary interventions need to be performed immediately when accompanied by ECG signs of impending TdP, such as QTc prolongation >500 ms, QT-U prolongation and distortion after a pause, onset of ventricular ectopy and couplets, macroscopic T-wave alternans (TWA), or episodes of polymorphic ventricular tachycardia that are initiated with a short-long-short R-R cycle sequence . ApproprMost TdP can terminate spontaneously. However, for patients with TdP that does not terminate spontaneously or that degenerates into ventricular fibrillation, immediate direct-current cardioversion should be performed . The treNa-L) can potentially cause pro-arrhythmic phenotypes [Na-L can be observed in acquired conditions, such as hypertrophic cardiomyopathy, heart failure and drug-induced arrhythmias [Na-L without affecting QRS duration [Mexiletine has a significant therapeutic effect on refractory TdP and that it can shorten the QT interval of the basic ECG. The enhanced late sodium channel currents (Ienotypes . Enhancehythmias ,113. Mexduration . We onceduration .CKD is a growing public health problem. QT interval prolongation is responsible for CVD, especially SCD, which is a major cause of death in the CKD population. Numerous factors such as diabetes mellitus, hypertension, heart failure, electrolyte disorder, uremic toxins, hemodialysis and drugs can influence the repolarization of cardiac cells in CKD. However, there is still a lack of how to improve the dilemma of QT prolongation in patients with kidney disease. Especially like a wide variety of uremic toxins, their effects on myocardial depolarization and repolarization are not well understood. The traditional treatment of aLQTS patients with TdP includes removal of the offending causes, the maintenance of a high-normal serum potassium level and the use of drugs. The management of aLQTS in CKD patients still needs further exploration.


A number of in vitro and clinical studies have suggested potential antineoplastic effects of metformin. The impact of this medication on outcomes in head and neck cancer is less clear. Our aim was to determine the effect of metformin on outcomes within our large cohort of head and neck cancer patients with Type II Diabetes (T2DM).Retrospective cohort study.Tertiary Cancer Centre.A retrospective review of individuals with head and neck squamous carcinoma (HNSCC) and T2DM between January 2005 and December 2011 at Princess Margaret Cancer Centre was conducted. Medication history was obtained from surveys at initial presentation and electronic medical record review. Using Cox regression analyses, the association between metformin use and local, regional and distant failures was explored. Subgroup analyses were conducted for oral cavity, oropharynx and larynx.A total of 329 HNSCC patients with T2DM were identified, including 195 metformin users and 134 non-metformin users. Patients were well-matched in terms of clinical, pathologic, and treatment factors. No difference in local, regional, or distant failure was observed between diabetic metformin users and diabetic non-metformin users for the entire cohort or within subgroup analysis for subsite. No difference between the two groups was observed for overall survival, recurrence-free survival, and disease-specific survival at 5 years.No association between metformin use and oncologic outcomes were observed in this large cohort of HNSCC patients. Multicenter, prospective studies may be needed to verify previous studies identifying a potential anti-neoplastic effect of this medication. Metformin, an oral hypoglycemic agent, has been recognized for its potential antineoplastic effects. This low-cost, widely available, biguanide medication is indicated most commonly for the treatment of Type II diabetes mellitus (T2DM) and has a favorable adverse effect profile –3. In viThere have been a few studies assessing the impact of metformin use on outcomes in patients with head and neck squamous cell carcinoma (HNSCC) with mixed results , 16. We Following approval by the institutional Research Ethics Board, a retrospective chart review was performed of patients with a newly diagnosed, previously untreated, squamous cell carcinoma of the oral cavity, oropharynx, larynx and hypopharynx managed in our institution between January 2005 and December 2011. Patients were eligible for inclusion if they were > 18 years of age, had a diagnosis of T2DM and received curative intent treatment. Patients were identified through the institutional Cancer Registry and from a radiation oncology prospective anthology of outcomes database (Anthology Database) of all radiation oncology patients in our institution ) between the two cohorts of OPC patients on UVA. Given the differences in biology of HPV-related and HPV-unrelated OPC, we performed an additional exploratory analysis of outcomes in metformin and non-metformin group based on p16 status, acknowledging that the results need to be interpreted with caution given the limited numbers. There were a total of 67 OPC patients with known HPV status, including 47 p16+ patients (70%) and 20 p16 negative patients (30%). Within the p16+ cohort of T2DM OPC patients, there were no differences between the metformin and non-metformin patients in terms of OS (p = 0.59), RFS (p = 0.71), DSS (p = 0.62), local control (p = 0.74), regional control (p = 0.43) and distant control (p = 0.76). For p16 negative OPC diabetic patients, similarly, there were no differences between metformin and non-metformin groups in terms of OS (p = 0.14), RFS (p = 0.56), DSS (p = 0.32), local control (p = 0.56), regional control (p = 0.5), and distant control (p = 0.29). Of note, there were very few local and regional failures in this population.There were 44 oropharynx cancer (OPC) patients on metformin and 36 diabetic OPC patients not taking metformin. Metformin use in T2DM OPC patients was not significantly associated with improved LC , regional control ) or distant control . There were no significant differences in OS , RFS , and DSS (p = 0.73 HR 0.83 [95% CI 0.29–2.38]) between the two cohorts of laryngeal cancer patients upon univariate analysis  [p = .04); of note, there was no difference in RFS between the two groups. Given the limited number of patients and outcomes (i.e. death) the multivariable analysis must be interpreted with caution. It could not be excluded that the deaths were actually due to non-cancer reasons (e.g. cardiovascular disease) that could be linked to metformin use . Patients seeking care for their diabetes and taking metformin may seek out other healthy behaviors to modify their risk, potentially resulting in skewed survival results [n = 195) and metformin negative (n = 290) cohorts who subsequently developed head and neck cancer 16. In the most recent study, Spratt et al. performed a retrospective review of 184 diabetic OPC patients treated with primary radiation, of which 102 were on metformin [Given that metformin is a low cost, well-tolerated medication that has minimal toxicity, it would be an ideal anti-cancer agent. While some clinical studies have demonstrated that the use of metformin results in potentially increased overall survival and decreased incidence of cancer in multiple sites and cell types, such as colon, breast and liver , 29–31,  = 0.04) . However = 0.04) . They in results . In addietformin . They reIn terms of comparing clinical results with preclinical studies, the latter utilized much higher doses of metformin than the conventional dose used in the treatment of diabetes , 37, 38.The impact of a new agent with a favorable side effect profile for effective adjuvant therapy in HNSCC would be enormous. However, the clinical data thus far has failed to definitively demonstrate a significant effect on outcomes in metformin users. This study reinforces prior work that suggest this drug may not be useful in HNSCC patients overall or by subsite.In our HNSCC cohort, the use of metformin was not associated with improved overall survival, disease-specific survival, local, regional, or distant control overall or within any subsite. Additional prospective multi-centre studies are needed to further elucidate the role of metformin in this disease.


TNFRSF10A-LOC389641 and near GATA5 . A T allele at TNFRSF10A-LOC389641 rs13278062, a risk allele for CSC, is known to be a risk allele for AMD. This study not only identified new susceptibility genes for CSC, but also improves the understanding of the pathogenesis of AMD.The recently emerged pachychoroid concept has changed the understanding of age-related macular degeneration (AMD), which is a major cause of blindness; recent studies attributed AMD in part to pachychoroid disease central serous chorioretinopathy (CSC), suggesting the importance of elucidating the CSC pathogenesis. Our large genome-wide association study followed by validation studies in three independent Japanese and European cohorts, consisting of 1546 CSC samples and 13,029 controls, identified two novel CSC susceptibility loci:  Yoshikatsu Hosoda et al. present a genome-wide association study in Japanese and European cohorts, identifying genes related to central serous chorioretinopathy pathogenesis. They report two novel susceptibility loci, one of which is a known risk allele for age-related macular degeneration. Although retinal detachments in CSC eyes typically resolve spontaneously, some cases become chronic resulting in permanent retinal tissue damage5. Additionally, recent studies have shown that the occurrence of choroidal neovascularization (CNV) is a common complication, leading to severe vision loss7. Based on these findings, CSC is currently recognized as an important sight-threatening disease that can lead to legal blindness. Although clinical studies have shown that dysfunction of the retinal pigment epithelium and hyperpermeability of the choroidal vessels are essential in the etiology of CSC8, the exact mechanisms underlying CSC pathology remain unknown. Previous studies reported that the risk factors of CSC include glucocorticoid use, increased adrenergic hormones, stress, male gender, Helicobacter pylori infection, and type A personality16.Central serous chorioretinopathy (CSC) is a common eye disease characterized by serous retinal detachment of the macular regions and retinal pigment epithelium (RPE)19) often masquerades as AMD20, recent studies highlighted the importance of differentiating AMD and CSC25. Interestingly, although AMD and CSC show similar clinical manifestations, genetic studies revealed that they have contrasting characteristics with respect to complement factor H (CFH), an established AMD susceptibility gene26; the risk alleles for AMD at CFH Y402H and CFH I62V confer a protective effect against CSC29. Taken together, investigating the genetic background of CSC is currently of great importance and can improve the understanding of the etiology of both CSC and AMD.Recently, the pathological overlap between age-related macular degeneration (AMD), which is a major cause of legal blindness in developed countries, and CSC has received increased attention as they share similar clinical characteristics, including serous retinal detachment, pigment epithelium detachment, and CNV occurrence. Particularly, because CNV secondary to CSC  for CSC have been reportedTNFRSF10A-LOC389641 and rs6061548 near GATA5, which were significantly associated with the disease on a genome-wide scale. As these single-nucleotide polymorphisms (SNPs) showed a robust and homogenous effect among the Japanese and European cohorts, they may be important targets for further molecular biological evaluation and the development of new treatments.In the present study, we conducted a large-scale GWAS for CSC followed by replication analyses using three independent Japanese and European cohorts. Our analysis using 1546 CSC cases and 13,029 controls identified two novel CSC susceptible loci, rs13278062 at λGC) was 1.157. The quantile–quantile plot is shown in Supplementary Fig. After stringent quality control, 2,893,743 SNPs were included in the first-stage of the GWAS in a Japanese case–control cohort consisting of 610 CSC patients and 2850 controls. We included three principal components as covariates to adjust for possible population stratification, which provided an acceptable control; the genomic inflation factor lambda  for the current study using publicly available multiethnic genotype data from 1000 Genome project   and rs6061548 near GATA5 . The Manhattan plot, regional plots and linkage disequilibrium plots are shown in Figs. CHMP7 region showed relatively low P-values.In the first-stage, we identified two loci showing a suggestive GATA5 showed low P-values; the most significant association was found at rs13044490 within GATA5  and rs6061548 near GATA5  showed significant associations with CSC . Rs13278062 showed a trend towards an association with the same direction of effect . A meta-analysis of the Japanese and European cohorts also revealed strong association between CSC and rs13278062 at TNFRSF10A-LOC389641 , as well as rs6061548 near GATA5 . As shown in Fig. We conducted trans-ethnic replication analyses to confirm the association. Association analysis using 521 cases and 3577 controls of European descent revealed that rs13278062 and rs6061548 were significantly associated with CSC in the European cohort . SLC7A5 rs11865049 showed the same direction of effect as previously reported but did not reach a significant value . These results are summarized in Table Previous GWAS of CSC reported two susceptibility loci, TNFRSF10A expression , but not with any other genes nor CHMP7. A multi-tissue eQTL plot revealed that the normalized effect size (NES) of rs13278062 on TNFRSF10A expression was strongest in the adrenal gland  database revealed that rs13278062 was significantly associated with https://eyeintegration.nei.nih.gov/, v1.01) and The Ocular Tissue Database (https://genome.uiowa.edu/otdb/), the only databases that includes gene expression data in human retina and choroid. The Eyeintegration database showed that the expression of both TNFRSF10A and GATA5 in the adult human RPE/choroid (n = 48) was stronger than that in the adult human retina (n = 52) as summarized in Fig. TNFRSF10A in the adult human RPE/choroid was stronger than that in the adult human retina  and that of GATA5 in the adult human RPE/choroid was also stronger than that in the adult human retina . The Eyeintegration database revealed that the expression of both TNFRSF10A and GATA5 was also observed in other human tissues . In total, 9723 pathways were evaluated. The top ten pathways are shown in Supplementary Table P = 2.60 × 10−5). However, no pathways reached the genome-wide, pathway-based significant P-value of <1.0 × 10−535.We performed pathway analysis using VEGAS2Pathway  in the present study was higher than that of traditional AMD occurrence in a previous study 39. It is also possible that TNFRSF10A has pleiotropic effects on both AMD and CSC. The effects of TNFRSF10A on AMD should be further evaluated while stratifying the data for the presence of a pachychoroid background.TNFRSF10A is more strongly expressed in the RPE/choroid than in the retina. However, the exact role of TNFRSF10A in CSC occurrence or choroidal structure is unclear. Considering that the adrenergic hormones are established risk factors for CSC42, and that the eQTL showed that the genotype of rs13278062 at TNFRSF10A was strongly associated with its expression in the adrenal gland, TNFRSF10A may affect the risk of CSC by modulating hormone secretion from the adrenal glands.CNV accompanied by the characteristics of CSC was recently termed as pachychoroid neovasculopathy, as the thickened choroid is the most characteristic clinical feature of the condition. Gene expression evaluation in ocular tissue supports the importance of the RPE/choroid regarding the etiology of pachychoroid neovasculopathy and CSC, as GATA5 was associated with CSC. GATA5 is known to play an important role in vascular system development45. A recent study showed that GATA5 is expressed in the microvascular endothelial cells and that its inactivation in mice results in vascular endothelial dysfunction46. Considering the stronger expression of GATA5 in the RPE/choroid than in the retina, we speculate that GATA5 may affects the susceptibility to CSC through vascular endothelial dysfunction in the choriocapillaris, which constitutes the inner vascular layers of the choroid, composed largely of fenestrated capillaries. This hypothesis is compatible with previous reports showing that eyes with CSC had choriocapillary hypoperfusion48, and primary choroidal ischemia48. GATA5 is also known to play an important role in stomach development and gastric diseases51. Interestingly, GATA5 is reported to be upregulated by H. pylori infection52, which is one of the risk factors of CSC14. As described above, GATA5 may also be an important target for the further understandings of CSC.We additionally identified that rs6061548 near λGC in our discovery GWAS, which may have led to false-positive associations. This inflation may be related to the presence of a mild population substructure, as inflation was still observed even after conducting the GWAS using samples genotyped with a single-DNA microarray platform, OmniExpress (λGC = 1.098). However, the positive associations of both TNFRSF10A-LOC389641 rs13278062 and near GATA5 rs6061548 were still significant even after genomic control correction , Kagawa University Hospital (N = 104), Yamanashi University Hospital (N = 80), and Fukushima Medical University Hospital (N = 37). Control allele frequency data were extracted from the genome-wide dataset of healthy Japanese subjects from the Tohoku Medical Megabank Organization (N = 3498)56, Kyoto University (N = 3074)58, and Yokohama City University dataset (N = 1048). Detailed information is shown in the 31, a brief summary is also provided in the In the first replication stage, 278 CSC patients were recruited from across Japan, particularly Kyoto University Hospital  examination, including enhanced depth imaging, fundus autofluorescence, fluorescein angiography, and indocyanine green angiography CSC was diagnosed based on medical history, serous retinal detachment, thickened choroid with dilated choroidal vessels seen on OCT, choroidal vascular hyperpermeability on ndocyanine green angiography, and/or leakages on fluorescein angiography at the level of the RPE. Based on these findings, two retina specialists (M.M. and Y.H.) diagnosed the patients independently, and discrepancies were adjusted in a face-to-face consensus session. Patients with other causes of fluorescein leakage or serous retinal detachment unrelated to CSC  were excluded from the study. In the second and third replication stages, patients with CSC were diagnosed based on previously described criteriaN = 250) and Asian Screening Array (N = 360), were used for genotyping the CSC samples, while Human610-Quad BeatChip (N = 1194) and OmniExpress (N = 1656) were used for genotyping the control samples.Genomic DNA was extracted from peripheral blood samples according to standard laboratory procedures. A series of BeadChip DNA arrays , namely OmniExpress (https://imputationserver.sph.umich.edu/index.html#!pages/home) with the 1000 Genome dataset (phase3 v5 release) of East Asians as a reference panel for each dataset. In each dataset, SNPs with a call rate <90% or a minor allele frequency <1% were excluded before genotype imputation. Imputed SNPs for which R2 was <0.9 were excluded from the subsequent association analysis. Next, SNPs with a call rate <90%, a minor allele frequency <1%, or significant deviation (P < 1.0 × 10−5) from Hardy–Weinberg equilibrium were excluded from further statistical analysis, and samples with a call rate <90% were also excluded. We checked the allelic discrimination of SNPs showing a suggestive association with CSC in the discovery GWAS (P < 1.0 × 10−5) for each platform. We excluded SNPs with an insufficient quality of allelic discrimination and their proxy SNPs (R2 > 0.8). Finally, 2,893,743 SNPs from 610 CSC samples and 2850 control samples were used for discovery stage analysis.Genotype imputation was performed using the Michigan imputation server (N = 278) were determined using a commercially available assay . We extracted the allele frequency from existing healthy Japanese genome-wide datasets, which included the Tohoku dataset56, Kyoto University58, and Yokohama City University datasets. The details of the genotyping methods are summarized in the https://ijgvd.megabank.tohoku.ac.jp/) provides genomic reference panels obtained from 3554 normal Japanese subjects recruited from the Tohoku Medical Megabank Organization, Iwate Medical Megabank Organization, Nagahama Prospective Cohort for Comprehensive Human Bioscience, and National Hospital Organization Nagasaki Medical Center. All DNA samples were whole-genome-sequenced using the Illumina HiSeq 2500. The Human Genetic Variation Database is a database of genomic reference panels released from Kyoto University (http://www.hgvd.genome.med.kyoto-u.ac.jp/index.html). This database is a web-accessible resource of genetic variations in the Japanese population. Whole-genome SNV genotyping was performed for 3712 individuals, who formed a subset of participants of The Nagahama Prospective Genome Cohort for the Comprehensive Human Bioscience (the Nagahama Study), with the Illumina HumanHap610 quad, Omni 2.5 M and Human exome Beadarrays (Illumina). The Yokohama City University dataset includes 1048 Japanese healthy controls recruited from the Yokohama City University, Okada Eye Clinic, and Aoto Eye Clinic in Yokohama, Kanagawa Prefecture, Japan. Genotypes of samples from Yokohama City University were determined using BeadChip DNA arrays, namely Human OmniExpress chip (Illumina), with the standard protocol recommended by each manufacturer.In the first replication stage, genotypes of CSC samples (31. Genomic imputation was performed for the dataset using the BEAGLE 4.1 and 1000 genomes dataset (phase3 v5 release) as the reference panels. Imputed SNPs for which R2 < 0.7 were excluded from the imputed dataset. The association of SNPs with CSC was tested by logistic regression analysis with no adjustment. The European CSC case–control dataset, which was used in the third replication stage, was genotyped using OmniExpress-12 or OmniExpress-24chip. The data were imputed with the Haplotype Reference Consortium release 1.1.2016, and were stringent quality controls were performed as described previously30.The Kobe CSC case–control dataset, which was used in the second replication stage, was genotyped using the Human OmniExpress BeadChips  as described elsewhereP = 5.0 × 10−8. We carried SNPs with P-value of <1.0 × 10−6 forward to the replication stage. In the first and second replication stages, logistic regression was performed for the SNPs that showed suggestive association in the first-stage. All meta-analyses were performed using the fixed-effect model. Thereafter, differences were considered significant at P < 0.05. Deviations in genotype distributions from Hardy–Weinberg equilibrium were assessed with chi-square tests. These statistical analyses were performed with R ver 3.5.2 , GCTA ver. 1.25.3 (http://cnsgenomics.com/software/gcta/index.html#Overview) and PLINK ver. 2.0 (https://www.cog-genomics.org/plink/2.0/).Genome-wide logistic regression analysis was conducted for the CSC, adjusting for three principal components. As on age and sex was not available for 1656 out of the 2850 control samples, adjustment for these factors was not performed in the discovery GWAS. Experiment-wide significance in the discovery stage was set to https://genome.sph.umich.edu/wiki/EPACTS; University of Michigan), correcting for sex and the first two components of ancestry analysis.In the third replication stage using the European dataset, association analysis was performed using the Firth bias-corrected likelihood ratio test, implemented in EPACTS . Briefly, VEGAS2 successively prunes the list of variants with R2 criteria of 0.99, 0.90, 0.70, and 0.50, if a gene contains >1000 SNPs. After each pruning interval, VEGAS2 checks the number of pruned SNPs. If the number of pruned SNPs is <1000, VEGAS2 uses the pruned SNPs from that interval to perform gene-based analysis; otherwise, it iteratively applies an increasingly stringent R2 criteria on all SNPs in the gene. After applying a pruning criterion of R2 = 0.50, it uses all pruned SNPs for analysis irrespective of the number. The gene list was obtained from the VEGAS2 official site (https://vegas2.qimrberghofer.edu.au/glist-hg19). Obtained gene-based result was used to perform calculate pathway-based tests and empirical P-values to obtain the significance of each pathway. Regions +/− 0 kb outside of genes were defined as gene regions, and all SNPs were used for analysis. For the SNPs that were carried forward for to the replication stage, P-values from meta-analysis were used rather than GWAS P-values. The Biosystems gene-pathway annotation file was obtained from the VEGAS2 official site (https://vegas2.qimrberghofer.edu.au/biosystems20160324.vegas2pathSYM). The significance threshold of the empirical P-value in the pathway analysis was set to 1.0 × 10−535.Using the GWAS summary statistics and ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/) were used for PCA.To determine the existence of a population substructure and its influence on the GWAS results, publicly available genotype data from five populations, including African, South Asian, European, East Asian, and Japanese  revealed an association between the SNP genotypes and gene expression in multiple human tissues. NES is defined as the slope of the linear regression and is computed as the effect of the alternative allele relative to the reference allele.Searching of the publicly available quantitative trait locus analysis (eQTL) database (Genotype-Tissue Expression (GTEx) Portal: https://eyeintegration.nei.nih.gov/, v1.01. accessed 10 April 2019). The database lists the expression levels of genes given in transcripts per million. Gene expression levels were compared by Wilcoxon test. Expression of genes in the human retina and choroid was explored in The OCULAR TISSUE DATABASE . This database provides quantitative expression level of genes in ocular tissues determined by the PLIER (Probe Logarithmic Intensity Error) algorithm.Expression of genes in the adult human retina and RPE was explored in the eyeintegration database (Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting summaryPeer review file


Despite the strong association of smoking with cardiovascular disease (CVD) and cerebral stroke, the consequences of smoking have not been elucidated among Iranian populations. This study aimed to assess sex-specific incidence of CVDs among an urban Iranian population with different smoking habits.Participants were recruited from the Tehran Lipid and Glucose Study (TLGS). Data on socio-demographic features and smoking habits from a sample of 10,400 individuals (4378 men and 6022 women), aged ≥20 years without prior CVD history were analyzed. Participants were followed up for 12 years for incidence of CVD/CHD events. Men were categorized in six groups, including never-, passive, ex-, passive and ex-, occasional and daily smokers. Women were categorized in three groups, i.e. never smokers, passive smokers and ever smokers. Using cox regression model, adjusted hazard ratios (HRs) of incident CVD/CHD were calculated for each group, given never smokers as the reference.In men, HR of CVD was 1.13 (95%CI: 0.80–1.59) in passive smokers, 1.23 (95%CI: 0.91–1.66) in ex-smokers, 1.46 (95%CI: 0.90–2.36) in passive and ex-smokers, 2.33 (95%CI: 1.25–4.33) in occasional smokers and 2.05 (95%CI: 1.57–2.67) in daily smokers. In smokers of ≥21 cigarettes/day, HR of CVD was 3.79 (95%CI: 2.25–6.37), with less risk observed in those who smoked lesser numbers of cigarettes/day. Quitters of ≥15 years were almost risk free. In women, none of the HRs of CVD/CHD were significant.An increased risk of incidence of CVD/CHD was found in current male smokers. To confirm and further elaborate these findings, more data of sex-specific studies are required from culturally diverse urban and rural areas of Iran. Cardiovascular diseases (CVDs) are among the most prevalent non-communicable diseases (NCDs) responsible for 31% of all global mortality . AccordiSmoking is the second major modifiable risk factor of CVDs  which diDespite the relationship between smoking and CVDs being well documented , becauseThe TLGS is an ongoing community-based study initiated in 1999, and designed to be continued for at least 20 years. The main goal of study is to assess risk factors and different aspects of life style in relation with NCDs among a representative urban Tehranian population . DetailsThe current analysis has been conducted on all TLGS participants who participated in the study between 1999 and 2002; those aged under 20 years, those with positive CVD history, those with missing smoking data and those lost to follow up were excluded. A final sample of 10,400 adult individuals (4378 men and 6022 women) was followed for a median of 12 years. The sampling frame of the current analysis is shown in Fig. 2). After participants rested for 15 min, a physician measured blood pressure using a mercury sphygmomanometer with appropriate cuff size for each individual. Systolic and diastolic blood pressure were measured twice, at least 30-s apart and the mean of the two measurements was recorded as the participant’s blood pressure. After 12 to 14 h of overnight fasting, blood samples were collected between 7:00 and 9:00 AM to be sent to TLGS research laboratory and biochemically analyzed on the same day. Levels of fasting blood glucose (FBS), total cholesterol, high density lipoprotein (HDL-c) and triglycerides (TG) were enzymatically measured. Low density lipoprotein (LDL-c) was calculated using Friedewald formula [Data on socio-demographic information , smoking habits and exposure to ETS and any familial history of NCDs were collected by trained interviewers. For physical activity, data was collected using a validated, reliable questionnaire entitled the Modifiable Activity Questionnaire (MAQ) . Psychom formula . The det formula , 23.n = 146) or past smokers (n = 94) and consequently the limited number of CVD events that occurred in these groups (n = 15) through the study period.Male participants were categorized into the six following groups: 1) never smokers, 2) passive smokers (exposure to ETS), 3) ex-smokers (quitter since at least a month before interview), 4) passive and ex-smokers (quitters exposed to ETS), 5) active occasional smokers (non-routine smokers) and 6) active daily smokers. In another grouping pattern, based on number of cigarettes smoked per day, active male smokers  have been further subdivided in to three groups: ≤10, 11–20 and ≥ 21 cigarettes per day. Ex-smokers were also subcategorized into those who quit smoking in less than 15 years, and those that had quit 15 years ago or earlier, regardless of passive smoking. Female participants were categorized as never smokers, passive smokers  and ever smokers (active and ex-smokers pooled together). Different categorization in men and women in the current study was mainly due to the limited number of women who reported themselves as active  (diagnostic electrocardiogram (ECG) and biomarkers), probable MI , sudden cardiac death, unstable angina pectoris  and angiographically proved coronary artery disease . The defp-values less than 0.2 were selected for multivariate regression. Adjustment of potential confounding factors was conducted in three cumulative models; the first model was adjusted for age, the second for age and cardio-metabolic risk factors  and the third model was adjusted for all the above mentioned variables, plus socio-economic factors . Within each model, adjusted hazard ratios (HRs) of outcome events were estimated in men and women with 95% confidence intervals (95% CIs) for each smoking pattern, given never smokers as reference. Proportionality assumption of Cox models was assessed using the Schoenfeld residual test and confirmed. Statistical analyses were performed by STATA software version 12, and SPSS software version 15. P-values below 0.05 were considered statistically significant.All analyses and baseline characteristics are classified by sex. Continuous variables are expressed as mean ± (SD) for normally distributed and as median (Q1-Q3) for skewed ones. To compare continuous variables between different CVD groups, Student t-test and Mann-Whitney test were used for normally distributed and skewed variables, respectively. In different smoking groups, continuous variables were compared by ANOVA in normally distributed- and by Kruskal-Wallis test in skewed ones. Categorical variables are expressed as number (%) and have been compared with Chi-square test, both within different smoking groups and CVD groups. Cox proportional hazard model was used to study the association between different smoking patterns and cardiovascular outcomes. The follow-up duration was defined as the period starting from entrance to study and ending in occurrence of any predefined CVD/CHD event or any attributed mortality or censoring, whichever happened first. Censoring was defined as loss to follow-up or mortality, not attributable to CVD or CHD. Univariate regression model was first performed for all potential confounding factors and those with A total of 10,400 CVD free participants, aged ≥20 years with complete data on smoking status, were followed during 12 years for occurrence of CVD or CHD events. Mean ages of participants at baseline were 42.73 ± 15.30 and 40.33 ± 14.00 years in men and women, respectively. Over a median 12 year follow up, incidence rate of CVD was 10.6 (95% CI: 9.6–11.7) and 5.1 (95% CI: 4.5–5.8) per 1000 person years, in men and women respectively. Incidence rate of CHD was 9.0 (95% CI: 8.1–10.0) in men and 4.4 (95% CI: 3.9–5.0) in women, per 1000 person years.Table Hazard ratios of CVD/CHD incidence among different smoking groups of men, are shown in Fig. Figure Figure Our results showed that different smoking habits and higher rates of cigarette smoking increased the risk of CVD/CHD in men. In women, however, the relation between different smoking patterns and the risk of CVD/CHD was not established with these data. According to our findings, smoking rates were significantly higher among men than women, which is consistent with previous findings from Iran and other Middle Eastern countries. In this regard, a recent meta-analysis indicated that the prevalence of smoking in Iran ranged between 19.8 and 21.7% in men, while it was estimated to be 0.94 to 3.6% in women . AccordiOur findings regarding the prognostic value of current smoking both occasionally and daily in the prediction of CVD/CHD incidence are in agreement with the findings of Birgitte et al. who reported an age-adjusted relationship of active- and passive smoking with MI incidence in males and females . ShieldsBased on our findings, no association between various smoking habits and risk of CVD/CHD was found in females, findings in contrast with those of previous studies, reporting that smoking is the leading contributing factor in development and progression of CVDs in both men and women . FurtherRegarding the strengths of the current study to the best of our knowledge, this is the first longitudinal study to assess the association of smoking habits with the risk of cardiovascular diseases among Iranian populations among women and passive smokers. Among the limitations of the study was the possible information bias of data collection using a questionnaire-based method. In addition the lack of significant findings in women which can be explained in light of the potential under-reporting of smoking as well as the lack of statistical power due to low number of current smokers; limitations which need to be further investigated in future studies in Iran.In conclusion, although an increased risk of CVD/CHD incidence was found in current male smokers, this finding was not supported by our data in women. This result might be attributed to under-reporting of smoking in female participants due to the existing social stigma regarding smoking of women in Iran. Our findings indicated the importance of smoking cessation to prevent cardio-vascular outcomes in an Iranian urban population. To confirm and further elucidate these findings, more studies are required in both urban and rural areas of Iran.