Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host.Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host  transmission can reduce the number of susceptible host cells and cause pathogen extinction, while vertical (lysogenic) transmission impairs pathogen abundance. Scrutinizing transmission of temperate phage P22 throughout a bacterial population at single cell resolution now revealed that this phage is able to disseminate immunity factors that allow the emergence of transiently resistant subpopulations of host cells. The continued fostering and consumption of such subpopulations points to an entirely new strategy by which viruses could manage to sustain an active infection with their host. Viruses that infect microorganisms are ubiquitous in nature and often outnumber their hosts by an order of magnitude . Their pThe biology and life cycle of bacterial viruses (termed bacteriophages or phages) has been extensively studied and so far has revealed a plethora of phage–host interactions along the lines of two distinct reproductive strategies. In fact, it has long been established that upon infection the incoming phage chromosome can commit to replication and the production of new phage particles that are typically released by lysing the host and that enable further horizontal transmission . During Despite these current paradigms of phage biology, however, the function and necessity of most phage encoded proteins still remains obscure while often the ecological complexity of phage-host associations remains unresolved ,15,16. IOur current lack of understanding might stem from the fact that most of the insights into phage biology are primarily derived from bulk level approaches that tend to overlook more subtle but nevertheless deterministic phage-host interactions that are either transient or operative in only a fraction of the infected population. The improvement of single-cell analysis approaches, however, is bringing such elusive insights within reach, and has started to shed light on the dynamic spatiotemporal regulation and orchestration of phage replication inside the cell –23.Salmonella Typhimurium host at single-cell resolution. As such, we found evidence for a mechanism that imposes the dissemination of phage-borne immunity factors to transiently protect emerging subpopulations of host cells from phage infection.In order to expand this view towards phage infected populations, we adopted a live cell biology approach to scrutinize the dynamic transmission of the P22 temperate phage throughout a population of its S. Typhimurium LT2 host with time-lapse fluorescence microscopy, we (i) recombineered a parS sequence into the P22 chromosome , and  mutant  mutant . While t) mutant , indicatparS for four hours before the ongoing infection was monitored with time-lapse fluorescence microscopy  and P22-free (i.e. displaying a diffuse cytoplasmic GFP-ParB fluorescence) cells. In fact, closer examination of such emerging lineages revealed that often the first sister cell(s) of the phage carrier cell became P22-free, after which typically the phage carrier cell itself became lysogenized by an integration event and further gave rise to P22-lysogenic siblings , all phage carrier sister cells became P22-free and immune, indicating that the actual integration event was not essential to yield this immunity , as after a number of generations the lineage eventually became infected by P22 appeared . More spee cells . Furtherint parS phage carrier cell in general also seemed predisposed to be the first cells of the emerging lineage to lose their immunity and become infected, suggesting that an immunity determinant is originating from the phage carrier cell and cytoplasmically passed down and diluted to subsequent generations of P22-free siblings up to the point where it becomes insufficient to prevent infection.Interested in the molecular mechanism underlying this transient immunity of P22-free siblings, we noticed that the first sister cells spawning off a P22 ΔS. Typhimurium LT2 was screened for clones resistant to P22 infection upon cross-streaking on LB agar plates. As such, the gtrABC  attachment of the P22 particle to the host LPS by modifying the O-antigen (via gtrABC), (ii) entry of the P22 chromosome through a currently unknown mechanism (via sieA) and (iii) its lytic proliferation (via c2) [In order to identify P22-borne determinants that could potentially participate in conferring immunity against P22 infection, a shotgun plasmid library of the P22 genome in ication; ,33), sie factor; ) and c2 c cycle; ) loci we(via c2) ,32,34.gtrABC and sieA was made fluorescently tractable , however, it could be observed that the phage carrier cell expressed these factors to a much higher extent than established lysogens did  gave rise to P22-free siblings that could readily be infected with P22 parS, indicating that GtrABC and SieA factors both independently confer the observed immunity by preventing entry of the P22 chromosome in P22-free cells (gtrABC expression is invariably activated during the phage carrier state irrespective of the phase variability of the gtrABC promoter.To further confirm whether the cytoplasmic inheritance of these two factors indeed plays a role in the (transient) immunity of the P22-free cells, the phage carrier state was established with P22 er state  only phaee cells . NotablyΔint ΔsieA ΔgtrABC infected population the phage carrier cell and its sister cells were deprived of their transient immunity and readily got infected by P22 parS, we observed this incoming P22 chromosome to remain idle in the cytoplasm. The fact that the infecting P22 could not commit to lytic or lysogenic proliferation likely indicated its premature repression or silencing by C2 repressor proteins that are most probably cytoplasmically inherited from the phage carrier cell as well. Moreover, the observation that this apparent silencing did not become alleviated after a number of generations  suggested that the incoming P22 chromosome  itself became genetically toggled to sustained C2 repressor production  of P22 Δint ΔgtrABC ΔsieA with LT2, cultures typically got overtaken by a phage population harboring c2 mutations. Such dynamics were not observed with P22 Δint, indicating that the SieA and GtrABC mediated delay of P22-free cell infection abolishes the competitive advantage and thus enrichment of such c2 mutants.Moreover, in line with this ability of P22 Δint parS infection as well, indicating that GtrABC and SieA mediated prevention of this phenomenon is not absolute.Finally, it should be noted that premature entry and subsequent silencing of P22 chromosomes was sporadically observed in (GtrABC and SieA proficient) P22-free immune cells generated during P22 S. Typhimurium) cells, we used live cell biology to track the different possible fates and whereabouts of individual host cells and phage chromosomes during an active infection. Aside host cells engaged in horizontal (lytic) and vertical (lysogenic) P22 transmission  throughout a population of host  imposed by cytoplasmic dilution of GtrABC and SieA activity in transiently immune P22-free siblings would at first liberate only a limited number of P22 attachment or entry sites on the cell surface, so that even at a high multiplicity of infection the majority of freshly susceptible cells would tend to undergo lytic infection (contributing to phage particle production) initiated by the entry of a single phage chromosome rather than lysogenic conversion enforced by the simultaneous entry of multiple phage chromosomes.Although future research should proceed to address the exact molecular details of phage carrier state establishment and its prevalence in other phage-host systems, the iterative fostering and subsequent consumption of transiently immune subpopulations as revealed in this study could allow for a phage to sustain an active infection, and the concomitant production of phage particles, without compromising the co-existence with its host. Since horizontal and vertical phage transmission in principle tend either to deplete the population of susceptible host cells or to enrich the population in immune lysogens carrying the dormant prophage, respectively, the carrier state mediated supply of new susceptible host cells might constitute a bet-hedging mechanism that could adjust these dynamics.in silico models for homogenously mixed infected populations have since long proposed transient forms of phenotypic phage resistance to greatly stabilize phage-host coexistence [in vivo observations of a phage carrier state mediating such transient phenotypic phage resistance, although it remains to be established whether virulent phages could indeed embark in such a carrier state association. For temperate phages on the other hand, the carrier state mediated supply of new susceptible host cells could counterbalance the inevitable enrichment of prophage carrying lysogens with the production of free phage particles, in turn allowing proper bet-hedging between both repositories of the phage chromosome.In fact, virulent (i.e. strictly lytic) phages face a serious threat of extinction in the absence of fresh susceptible hosts ,18, and xistence . Furtherxistence –40. Thespid gene to be particularly expressed in the carrier state [Our work also more generally draws attention to the presence and importance of more subtle or transient phage-host interactions that defy the traditional bifurcation of phage biology into lytic or lysogenic development, and that might draw on dedicated phage functions that account for part of the currently cryptic phage gene pool. As such, we could demonstrate that the phage carrier state expresses immunity factors previously thought to serve only superinfection exclusion of established lysogens. Moreover, we have previously found the cryptic P22-endoded er state . Furtherer state  with theS. Typhimurium host. The continued fostering and consumption of such subpopulations points to a new and population-level exploitation or farming strategy by which viruses could manage to sustain an active infection with their host.In summary, by scrutinizing viral transmission dynamics throughout a bacterial population at single cell resolution, this study revealed that P22 is able to disseminate immunity factors that allow the emergence of transiently resistant subpopulations of its 100), chloramphenicol , kanamycin  and tetracycline .Bacterial strains, phages and plasmids constructed and used throughout this study are listed in 4)2SO4, 6 g/l Na2HPO4, 3 g/l KH2PO4, 3 g/l NaCl, 0.1 mM CaCl2, 1.0 mM MgCl2, 0.003 mM FeCl3 [100 between 0.2 and 0.3 before commencing phage infection and microscopy analysis.Strains for microscopic analysis were typically grown overnight at 30°C in AB-glycerol medium (AB). Mininal AB medium contains 2 g/l (NHmM FeCl3  and was S. Typhimurium LT2 as plaques in LB soft-agar (0.7% agar) or as lysates in LB as described previously [g for 10 min and resuspended in 400 μl AB. Phage titers before and after PEG-precipitation were essentially similar.Phages were propagated on eviously . Phage seviously . BrieflyPhage infected liquid cultures were grown in a semi-continuous batch system to maintain exponential growth. To achieve this, bacterial cultures were diluted 1:2 every hour to keep cell counts constant (i.e. doubling time in AB is approximately 1 hour).frt sites are present in the same P22 chromosome, the high efficiency of pCP20 mediated flipping tends to recombine all present frt sites in a random order, leading to unviable P22 prophages. Therefore, in these occasions pCP20 was not used, but whenever possible we used the endogenous tendency of P22 to recombine two close frt sites in larger than wild type P22 chromosomes, which was recently described by our group [For more efficient recombination, strains with increased transformation efficiency TH6939 (containing pKD46) and TH8127 (containing pSIM5) were used when appropriate . Because of the temperate nature of P22, mutations were typically constructed in the P22 prophage in the appropriate recombineering strain, after which the P22 mutants were collected from the supernatants of the corresponding lysogen. In this context, it should be noted that when more than two ur group .S. Typhimurium LT2 strain lacking the pSLT plasmid was kindly provided by Josep Casadesús  and tra, Spain;  leading KD3-parS  with priineering , as descineering . Please c2 parS was constructed by oligo-mediated mutagenesis [parS lysogen, using olignucleotide Oligo_c2_Stop .Clear mutant P22 agenesis  of the P_c2_Stop . This olΔint parS was constructed by oligo-mediated mutagenesis [parS lysogen, using P22_Δint_Oligo .P22 agenesis  of the cnt_Oligo . All othineering  a PCR amineering . Howeverineering . After ig by P22 . The ressieA and gtrABC were constructed by recombineering [Superinfection exclusion mutants of P22 of ineering  using pKΔint ΔsieA ΔgtrABC, the efficiency of spontaneous removal of the antibiotic resistance cassette from gtrABC proved to be too low to detect [gtrABC operon in a P22 ΔsieA lysogen was first replaced by the tetA-sacB PCR amplified from the E. coli strain T-SACK  with prtrol_Rev  derived and sacB , leadingyfp fusions to sieA and gtrABC in P22 parS were constructed by recombineering [yfp-frt-cat-frt with respective primer pairs P22_sieA_yfp_Fw and P22_sieA_yfp_Rev, P22_gtrC_yfp_Fw and P22_gtrC_yfp_Rev . For the analysis of the GtrC-YFP and SieA-YFP expression levels, aliquots were taken from the appropriate cultures at indicated time points. YFP fluorescence intensities (arbitrary units) of single-cells were measured using the NIS-Elements (Nikon) software, corrected for background fluorescence and subsequently binned to create the fluorescence intensity distributions.Fluorescence microscopy and time-lapse fluorescence microscopy were performed with a temperature controlled  Ti-Eclipse inverted microscope  equipped with a TI-CT-E motorised condensor, a YFP filter , an mCherry filter , a GFP filter  and a CoolSnap HQ2 FireWire CCD-camera. For imaging, cells were placed between AB agar pads and a cover glass using Gene Frames (Life Technologies), essentially as described previously , and incubated at 30°C. Images were acquired using NIS-Elements (Nikon) and resulting pictures were further handled with open source software FIJI , the plasmids from resistant clones were extracted and again transformed to competent LT2 to confirm the resistance phenotype. Sequencings of relevant inserts were performed by Macrogen Europe .A P22 shotgun library created in plasmid pFPV-Promoter; ) was traS1 FigparS sequence. (B) Removal of pSLT from LT2 (resulting in LT2ΔpSLT/pALA2705) results in cells with a diffuse GFP-ParB distribution indicating the loss of aspecific binding sites for GFP-ParB. A 5 μm scale bar is shown at the bottom right of each panel.(A) LT2/pALA2705 showing discrete foci even in the absence of the phage P1 (TIF)Click here for additional data file.S1 MovieΔint parS (MOI = 0.1) and grown in a semi-continuous culture for 5 hours before plating on an agar pad seeded with additional P22 parS. Please note that phage entry can occur with either P22 Δint parS already present in the culture or the added P22 parS. Analysis of 33 similar lineages revealed that under these conditions transient resistance on average lasted for ca. 12 generations, based on a doubling time of ca. 55 min in AB-glycerol media. Phase contrast images (showing the cells) and GFP signal (reporting the P22 parS chromosomes) are merged. A 5 μm scale bar is shown in the bottom right and the timestamp indicates time after plating cells for microscopy analysis.An exponential phase population of LT2ΔpSLT/pALA2705 was infected with P22 (AVI)Click here for additional data file.


Ovarian hormones regulate GnRH and its receptor (GNRHR) through negative and positive feedback loops. In contrast, the role of ovarian hormones in regulating activin, activin receptors, and components of the activin signaling pathway, including SMAD4 and FOXL2, remains understudied. The widespread distribution of activin and many of its signaling intermediates complicates analysis of the effects of ovarian hormones on their synthesis in gonadotropes, one of five pituitary cell types. We circumvented this complication by using a transgenic model that allows isolation of polyribosomes selectively from gonadotropes of intact females and ovariectomized females treated with or without a GnRH antagonist. This paradigm allows assessment of ovarian hormonal feedback and distinguishes responses that are either independent or dependent on GnRH. Surprisingly, our results indicate that Foxl2 levels in gonadotropes decline significantly in the absence of ovarian input and independently of GnRH. Expression of the genes encoding other members of the activin signaling pathway are unaffected by loss of ovarian hormonal feedback, highlighting their selective effect on Foxl2. Expression of Gnrhr, a known target of FOXL2, also declines upon ovariectomy consistent with reduced expression of Foxl2 and loss of ovarian hormones. In contrast, Fshb mRNA increases dramatically post-ovariectomy due to increased compensatory input from GnRH. Together these data suggest that ovarian hormones regulate expression of Foxl2 thereby expanding the number of genes controlled by the hypothalamic-pituitary-gonadal axis that ultimately dictate reproductive fitness.Gonadotropin-releasing hormone (GnRH) and activin regulate synthesis of FSH and ultimately fertility. Recent  Reproductive fitness requires tight regulation of the genes encoding LH and FSH. A complex network of signals emanate from the hypothalamic-pituitary-gonadal axis defining a characteristic temporal pattern of synthesis and secretion of LH and FSH that occurs over the course of the estrous cycle in rodents –3.Peaks of LH and FSH that occur during late proestrus trigger ovulation . This coA second surge of FSH occurs independently of LH during estrus. This secondary surge of FSH is less dependent on pulses of GnRH, which are slower and lower in amplitude during estrus, and instead reflects autocrine/paracrine increases in activin from pituitary gonadotropes and folliculostellate cells accompanied by reciprocal paracrine and endocrine decreases in follistatin from gonadotropes and folliculostellate cells, and inhibin from ovarian granulosa cells , 10–12. Lhb and Fshb [Lhb mRNA [Fshb that underlies the secondary surge of FSH.Growing evidence indicates that the activin signaling pathway may be the primary trigger that drives the secondary surge of FSH , 10–12. Lhb mRNA . This reFshb promoter contains conserved and species-specific regulatory elements that bind complexes of SMADs and the forkhead transcription factor FOXL2 [in vitro render the Fshb promoter responsive to the members of the TGFβ superfamily [Foxl2 in mice impairs fertility in both male and female mice due to FSH deficiency caused by diminished transcription of Fshb mRNA [Fshb mRNA [The proximal promoter-regulatory region of the or FOXL2 –22. Theserfamily –25. Condshb mRNA . The impshb mRNA .Foxl2 is restricted to developing eyelids, ovarian granulosa cells, and pituitary thyrotropes and gonadotropes [Foxl2 may be controlled by sex steroids. For example, estrogen up regulates expression of Foxl2 in Southern catfish [Gobiocyprus rarus [Foxl2 in gonadotropes may be subject to regulation by the hypothalamic-pituitary-gonadal axis (HPG).Expression of dotropes , 27. Eme catfish , rainbow catfish , and theus rarus . In bovius rarus . Togethein vivo is challenging given the comingled arrangement of at least five cell types in the pituitary. Gonadotropes make up 5–15% of the cells in the pituitary [LHB-cre) [Examining gonadotrope gene expression ituitary , 33. Rouituitary . Other sLHB-cre)  with RibLHB-cre)  thereby in vivo data suggest that ovarian hormones regulate expression of gonadotrope Foxl2 thereby expanding the number of genes controlled by the HPG axis that ultimately dictate reproductive fitness.Herein we report the use of GRT mice, in conjunction with an ovariectomy (OVX)/GnRH antagonist paradigm, to interrogate the impact of loss of ovarian function on a battery of genes that define the activin signaling pathway. Our 2.All animal procedures were approved and carried out with strict accordance to guidelines from the Institutional Animal Care and Use Committee  at Washington State University. Surgeries were performed under avertin anesthesia. Ketoprofen was used as the post-operative analgesic and all efforts were made to minimize animal suffering. Animals were euthanized using CORpl22HA/HA) mice were generously provided by Dr. Paul S. Amieux (University of Washington) and have been described previously [Rpl22 that encodes a component of the 60S ribosomal subunit. This allele was engineered to express RPL22 with a hemagglutinin (HA) tag upon CRE-mediated recombination. LhbCre (Tg(Lhb-cre)1Sac) mice were kindly provided by Dr. Sally Camper (University of Michigan) and have been previously described [Tg(Lhb-cre)1Sac mice express Cre recombinase in gonadotropes due to Lhb promoter driven expression of Cre recombinase. This was confirmed by crossing Tg(Lhb-cre)1Sac mice with either a RosaGFP cre-reporter line or the Rosa26LacZ cre-reporter line. Both reporter genes were expressed primarily in gonadotropes and minimally in the testes of male mice along with a few cells in the cortex of the brain and the kidney [Rpl22HA/HA mice were mated with Tg(Lhb-cre)1Sac mice to produce GRT mice.RiboTag mice . OfOvariectomies were performed on 8 to 18 week old mice. Fifteen-21 age matched GRT females were split into sham + vehicle, OVX + vehicle, or OVX + antide groups. Starting on the day of surgeries, female mice were injected with the vehicle propylene glycol or 60 μg Antide in propylene glycol every other day for 10 days at which point pituitaries were harvested and pooled from 5 to 7 animals based on treatment group with each group containing the same number of pituitaries within a given experiment. Pooled samples were divided into aliquots for analysis of total versus immunopreciptated RNA by real time PCR. Each point on the graph represents a pooled sample from a single experiment.et al. [Immunoprecipitations were performed as described in Sanz et al.  with minet al. , and cenRpl19 was used as the endogenous control and cDNA from LβT2 cells was used as the calibrator in the 2-ΔΔCt calculation [-ΔCt or 2-ΔΔCt values were graphed for experiments performed on different days with the median with interquartile range shown. Although infrequent, amplicon values with high Ct values that exceeded the detectable range as determined by standard curve analysis were excluded. Thus “n” values ranged from 3 to 5 for any given primer set.cDNA was synthesized using qScript cDNA SuperMix (Quanta Biosciences) following the manufacturer’s protocol using the input fraction  or the immunoprecipitated fraction (gonadotrope) RNA. The synthesized cDNA was diluted 1:15, and 5 μL was used in each subsequent real time reaction using Fast SYBR Green Master Mix (Applied Biosystems) in a 20 μL final volume reaction on a 7500 Fast real time machine (Applied Biosystems). Cycling conditions were incubation at 95°C for 20 sec followed by 40 cycles of 95°C for 3 sec then 60°C for 30 sec followed by melt curve analysis. Primers were optimized for primer concentration, and primer efficiency was determined. Only primer sets with primer efficiencies between 95% and 105% were used. Primer sequences and concentrations are listed in culation . qPCR foData from the separate experiments were combined and analyzed with the assumption that there was no significant interaction between the “experiment” and treatment group and no “effect of experiment.” While this approach introduces additional independent variables that can increase biological variability, it allows for a more conservative estimate of statistical significance.Statistical analysis was performed using one-way parametric ANOVA followed by Tukey’s multiple comparison test  with GraphPad software.Lhbcre transgene and serve as a control. Lysates were subject to immunoprecipitation with either HA or Myc antibody. Gonadotrope specific transcripts  were enriched  in HA pulldowns from GRT pituitary lysate (Gonadotrope specific RiboTag (GRT) mice express HA-tagged RPL22 protein exclusively in gonadotropes. To verify isolation of gonadotrope specific transcripts, pituitaries were harvested from GRT mice as well as RiboTag mice, which lack the y lysate . The enry lysate .Lhb, Fshb, and Cga mRNAs in murine pituitaries increase after castration [Gnrhr mRNA levels decrease [Lhb, Fshb, and Cga mRNA [Levels of stration  while Gndecrease , 40. TreCga mRNA , 42, proLhb, Fshb, and Cga) in total pituitary RNA mirrored changes of the three mRNAs in HA-tagged polyribosomal mRNA from gonadotropes  were 3.6- vs. 2.6-fold for Lhb, 9.4- vs. 8.0-fold for Fshb, and 2.6 vs. 2.0-fold for Cga. Similar mirrored changes in levels of Lhb, Fshb, Cga mRNAs from OVX + Antide treated mice were also observed when measured in either total RNA or in polyribosomes from gonadotropes. Indeed, Antide almost completely blocks the post-OVX rise in Lhb, Fshb, and Cga, which is expected if their increase is dependent primarily on GnRH.As expected, mRNAs for Lhb, Fshb, or Cga mRNA, Gnrhr mRNA exhibited a post-OVX decline that was similar when measured either in total pituitary RNA or in polyribosomes from gonadotropes . Treatment of OVX animals with Antide led to an even further significant decrease in Gnrhr mRNA (30%) compared to OVX alone in samples of total pituitary RNA.In contrast to Gh mRNA and the lactotrope specific Prl mRNA were also examined. Gh mRNA exhibits a post-OVX rise whereas Prl mRNA declines . Nevertheless, the putative enrichment of Inhbb mRNA is consistent with previous reports indicating that activin B is expressed at higher levels in gonadotropes compared to other cells of the pituitary [Results from the GRT mice indicate that OVX has a minimal impact on the levels of activin and inhibin subunit mRNAs in total pituitary extracts, with the exception of  OVX Fig  and 4C.  OVX Fig  and 4F. ituitary .Fshb expression [Together, our results suggest that even though activin and inhibin are important regulators of pression , 3, 12, Foxl2 and Smad3 [Attempts were made to measure the level of follistatin, another target of nd Smad3  and itsend Smad3 .Smad3-deficient male mice have lower levels of Lhb and Fshb [Fshb [Fshb expression. Like activin and inhibin, SMADs are widely expressed in cells and tissues, thus the GRT mice allow for assessment of effects of the ovarian hormones and GnRH on Smad mRNAs specifically in gonadotropes.Activin signals through SMAD proteins and and Fshb . AP1 andhb [Fshb , offerinSmad3, Smad4, or Smad7 when sampled from total pituitary RNA  further supports the notion that SMAD3 may have a greater role in gonadotropes than in other pituitary cell types.Removal of the ovary with or without Antide treatment has little impact on  RNA Fig  and 5D. h Antide , the chaFoxl2 in polyribosomes from gonadotropes.Both gonadotropes and thyrotropes synthesize FOXL2 as evidenced by its immunohistochemical co-localization with both LHβ and TSHβ , 52. HenFoxl2 mRNA levels were enriched approximately five-fold in polyribosomal mRNA samples prepared from gonadotropes versus total pituitary RNA . Ovariectomy reduced Foxl2 mRNA ~42% in total RNA samples and ~76% in polyribosomal RNA from gonadotropes. This differential suggests that the decrease in Foxl2 mRNA occurs to a greater degree in gonadotropes than in thyrotropes. Antide had no impact on Foxl2 levels in either total pituitary RNA or total gonadotrope polyribosomal RNA, suggesting that ovarian hormones play an important role in maintenance of Foxl2 mRNA in gonadotropes. Interestingly, this decrease in Foxl2 levels after OVX appears to be specific to females as Foxl2 mRNA levels in intact males are not significantly different from levels in castrated males treated with vehicle or Antide  as transcriptional targets of the HPG axis that play key roles in regulating gonadotrope gene expression.FOXL2, in partnership with SMAD4, has been characterized recently as an essential master regulator of fertility via its positive effects on  in vivo . Expresses FOXL2 . In vitrogestins , 24, 53.Fshb in gonadotropes increases by approximately eight-fold after OVX  in samples of total pituitary RNA. This result suggests that levels of Gnrhr mRNA are dependent on gonadal steroids in intact animals and that elevated levels of GnRH that occur after OVX may provide partial compensation rather than down regulate the receptor encoding mRNA. Finally, treatment of OVX mice with estrogen and progesterone partially rescued GNRHR levels even in the presence of GnRH antiserum, a result which is consistent with our evidence that Gnrhr expression is dependent on ovarian steroids [FOXL2 also regulates expression of ell line . Thus, tsteroids .Fshb mediate responsiveness to GnRH and activin [Fshb [Several regulatory elements within the proximal promoter of  activin , 24. Thein [Fshb , 24.Fshb promoter [Fshb promoter, recent in vitro studies indicate that cJUN interacts with FOXL2, with both proteins required for GnRH-responsiveness [Fshb over the estrous cycle.At least two regulatory elements bind FOXL2 , 19, 50.siveness , 27. TogFoxl2 in gonadotropes requires ovarian hormones, we posit that the concentration of gonadotrope Foxl2 mRNA changes during the course of the rodent estrous cycle. While both estradiol and progesterone have been reported to regulate Foxl2 expression [Foxl2 expression in gonadotropes. Since the secondary surge of FSH is more dependent on activin than GnRH [Fshb would be maximal at the time activin signaling becomes the primary driver of the secondary surge of FSH. As progesterone falls during estrus, FOXL2 levels may decline as well. One of the hallmarks of a synergistic interaction is that a small change in concentration of one of the interacting components can lead to a large biological effect. Thus, even modest changes in levels of Foxl2 expression and its cognate protein would be expected to have an impact on expression of Fshb given the known synergism between FOXL2 and members of the SMAD family of DNA-binding proteins [Foxl2 and activity of the progesterone receptor, both of which set the transcriptional tone of Fshb and ultimately fertility in females. Clearly future studies are needed to address the validity of this proposed model. Regardless of whether the model is correct, our collective data indicate that the HPG axis controls expression of Foxl2 in pituitary gonadotropes primarily through positive feedback from ovarian hormones.Given that maximal expression of pression –31, we shan GnRH –12, we pproteins . ProgestS1 FigFoxl2 and Rpl19, used for normalization. 2-ΔΔCt values were calculated using total RNA from LβT2 cells as the calibrator. The median with interquartile range is shown for A.) total pituitary Foxl2 levels and B.) gonadotrope Foxl2 levels. Statistical analysis was performed with a one-way ANOVA followed by Tukey’s multiple comparison test (*<0.05). N = 5, where each “n” represents 5–7 pooled pituitaries per treatment group)Male mice were either sham operated or orchiectomized and then treated with vehicle or Antide every other day for 10 days. Pituitaries were harvested and gonadotrope specific transcripts isolated. Real time PCR was performed with primers specific to (TIF)Click here for additional data file.


Here we attempted to establish DNA methylation differences in these three prakriti phenotypes.DNA methylation and its perturbations are an established attribute to a wide spectrum of phenotypic variations and disease conditions. Indian traditional system practices personalized medicine through indigenous concept of distinctly descriptive physiological, psychological and anatomical features known as prakriti  between the age group of 20-30years were subjected to methylated DNA immunoprecipitation (MeDIP) and microarray analysis. After data analysis, prakriti specific signatures were validated through bisulfite DNA sequencing.Following structured and objective measurement of 3416 subjects, whole blood DNA of 147 healthy male individuals belonging to defined Pitta prakriti) in individuals showed distinct promoter (34) and gene body methylation (204), followed by Vata prakriti which correlates to motion showed DNA methylation in 52 promoters and 139 CpG islands and finally individuals with structural attributes (Kapha prakriti) with 23 and 19 promoters and CpG islands respectively. Bisulfite DNA sequencing of prakriti specific multiple CpG sites in promoters and 5′-UTR such as; LHX1 (Vata prakriti), SOX11 (Pitta prakriti) and CDH22 (Kapha prakriti) were validated. Kapha prakriti specific CDH22 5′-UTR CpG methylation was also found to be associated with higher body mass index (BMI).Differentially methylated regions in CpG islands and shores were significantly enriched in promoters/UTRs and gene body regions. Phenotypes characterized by higher metabolism  contains supplementary material, which is available to authorized users. DNA methylation at cytosine bases of CpG dinucleotide is an epigenetic phenomenon widely used to regulate gene expression in humans . CpG islprakriti which can be identified as Vata, Pitta and Kapha phenotypes with discrete characteristics  /100 (where C is Cytosine and 5-mC is 5-methyl Cytosine)].Reverse-phase high performance liquid chromatography (RP-HPLC) method was employed with minor changes for global methyl cytosine (mC) estimation as described previously ,25. In bGAPDH and AX1 primers  [Briefly, 10 μg of whole blood genomic DNA was sonicated by using a 2 mm probe at amplitude 40 for 30 cycles with 15 seconds on and off, to obtain fragment sizes of 100–800 base pairs. About 3 μg of sheared DNA (INPUT) was immunoprecipitated overnight with anti-5-mathyl cytosine antibody  along with positive and negative control DNA provided in the Diagenode kit. Enriched methylated fragments were purified by standard phenol-chloroform-ethanol precipitation . The quaBelgium) . INPUT aBelgium) ,29. Equaprakriti  were used for data analysis. Feature extraction software v10.1 was used to obtain background corrected, loess normalized, and logarithmic converted intensity values for green and red channels for each probe. Logarithmic difference of red to the green signal is the measure of methylation, generally represented as log ratio or (INPUT/IP) or “M-value” (methylation) for a given probe. Subsequent quality analysis was performed using R v2.15 with Bioconductor package limma. Quantile normalization was adopted for between array correction to remove batch effect and to equalize the overall signal intensities. Statistical significance within and between the groups was evaluated by Benjamini-Hochberg false discovery rate (FDR), as reported in the literature [prakriti specific differentially methylated regions. Quantile normalization and statistical test with FDR correction was performed using Gene Spring v12.6.Total of 147 randomly selected male volunteers of dominant terature -32 FDR cprakriti using single sample t-statistics with Benjamini and Hochberg FDR correction to filter, most significant probes (p ≤ 0.05) with fold change of ≥1.5. Inter prakriti analysis was performed to find out the differentially methylated probes using analysis of variance with Tukey-post hoc test. Subsequently, differential methylated probes were identified with a fold change difference of ≥1.2 and 20% Benjamini and Hochberg FDR correction. The cross comparison of the intra-prakriti with inter-prakriti analysis was performed to find the prakriti specific differentially variable probes (mPSR). The differential methylated significant probes from the comparative analysis were hierarchically clustered using Pearson centered, Ward’s distance matrix. The identified mPSRs were analyzed across prakriti using Manhattan linkage and Ward’s distance matric. Intra-prakriti analysis by t-statistics with FDR correction and fold change analysis helped to find potential and, consistent methylated probes, whereas the ANOVA analysis identified differentially methylated probes. The identified mPSRs were analyzed with reference to CpG Islands and we designated differentially methylated if a) it contained at least one significant probe which fulfilled two criteria of probe level analysis and b) it should contain two or more methylated probes with fold change of ≥1.5 and p ≤ 0.05 in one of the three prakriti groups. For validation purpose, we have considered such differentially methylated CpG islands.The differential methylated regions were identified at the probe level by both intra and inter-group analysis using relevant statistical measure. Intra-array probe level analysis was performed for the three prakriti specific methylated gene sets was constructed by Gene Ontology (GO) term, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis by DAVID [http://www.funrich.org/) tools with default parameters. The biologically enriched significant terms were deciphered by analyzing the prakriti specific methylated genes against the whole genome with hyper geometric test and p ≤ 0.05 without FDR correction was considered.The probe sequences were annotated using Agilent e-array technology file and Galaxy genome browser into different genomic regions based on the distance from TSS of the gene. The probes were mapped to 5′-UTR and 3′-UTR regions using RefSeq gene coordinates. The enrichment of mPSRs at chromosomes, CpG Island, CpG shores and different genomic position was statistically analyzed by Chi-square test (p < 0.05). To understand the association of histone modification and CpG methylation, we used EpiExplorer and the statistical significance was estimated by the Fischer Exact test with FDR correction . Functioby DAVID  and Funrprakriti was treated sodium bisulfite and bisulfite specific PCR was performed. The amplicons were purified and sequenced using BigDye terminator cycle sequencing kit  [prakriti specific differentially methylated region, 20 dominant prakriti samples were chosen for validation. The primer list along with chromosomal coordinates and PCR conditions are provided in the supplementary information  according to manufacturer’s protocol. In brief, about 1.5 μg of total blood genomic DNA for each dominant BI, USA) . The quaBI, USA) . Two wayprakriti), using reverse phase HPLC methodology. We observed a significantly higher level in global methyl cytosine content (p < 0.05) in Vata when compared with Pitta and Kapha prakriti  was performed for 36 samples randomly selected (n = 12 for each prakriti methylated probes were identified by providing a cutoff of fold change (FC) ≥1.5 with 5% FDR, based on the previous reports [prakriti groups and observed a total of 23,101, 23,276 and 21,014 significantly methylated probes in Kapha, Pitta and Vata prakriti respectively. Venn analysis showed a total of 2523, 3547 and 1535 methylated probes uniquely representing Kapha, Pitta and Vata prakriti respectively  as compared to other two prakritis. A significant decrease in promoter methylated CpG probes in all three prakriti  was also observed  Kapha specific and 305 (262 genes) Vata specific methylated CpG islands, represented by two or more methylated probes respectively. Evaluation of multiprobe methylated CpG Islands in the prakriti across the genome with respect to extended promoter (−10000 bp to 500 bp from TSS) and gene body (inside the gene coordinates) showed variations in Kapha and Pitta prakriti , was performed in all three prakriti samples (n = 20). Results showed an average methylation level of about 40-60% in individual CpG sites across all the samples from three different prakriti  39 genes http://biogps.org/downloads/) [It is reported that 70% of CpG content of whole genome is represented in CpG Islands and about 50% of CpG islands encompasses promoter regulatory element to facilitate the expression variations among the individuals . We shownloads/)  revealedprakriti were subjected to quantile normalization. Analysis of variance (ANOVA) with Tukey post-hoc test at Benjamini Hochberg FDR of 20% correction was performed to enumerate differential methylated probes among prakriti samples. A total of 5447 differentially methylated probes were identified with fold change difference of ≥1.2 and FDR correction at P-value 0.2. These intra-prakrti probes were compared against significantly methylated probes of individual prakriti analysis. In all the, Kapha versus Pitta, Pitta versus Vata and Vata versus Kapha analysis, we observed 665 (Kapha vs Pitta), 755 (Pitta vs Vata) and 339 (Vata vs Kapha) probes to be differentially methylated. The supervised cluster analysis (Pearson’s distance metric and Ward’s linkage) of these differentially methylated probes , represented by 303 probes overlapping with 269 CpG islands in Pitta prakriti, followed by Vata prakriti with 175 probes representing 139 CpG islands and finally Kapha prakriti with 23 probes and 19 CpG islands  selection of normal healthy individuals with > 60% of one prakriti and b) variation due to the influence of second dominant prakriti phenotype.All 147 arrays irrespective of Further the mPSRs were significantly (p ≤ 0.05) enriched in the different genomic regions such as promoter and gene body associated with both CpG islands and shores , indicating that the identified prakriti specific regions were distinct and were not influenced by variation in the blood cell types tested  gene was selected for validation, to test the association of BMI group and prakriti.In a large population based study, we have previously shown an association between BMI and prakriti . Hence, l Figure A and B. prakriti specific methylated genes was analyzed using DAVID [prakriti. Although promoter methylation is known to show inverse correlation of expression, recent reports have suggested the significance of the gene body methylation and its role in gene expression regulation [prakriti specific methylation variation in the whole blood DNA. Considering these, we chose all the mPSRs genes and attempted to associate to different attributes as energy of structure, metabolism and motion for Kapha, Pitta and Vata respectively [The functional relevance of the identified ng DAVID . The mPSgulation . The ratectively ,43,44.HOXB1, LMX1B, LHX1, LHX5, EN2; p ≤ 0.05). In addition, we observed association of NFIX gene with low BMI which is one of the characteristic features of Vata prakriti. Pitta prakriti showed enrichment of mPSRs for metabolism related Gene Ontology terms and pathways. The biological process such as regulation of hormone secretion , regulation of nucleic acid metabolism  and several others were significantly enriched. Apart from these, signal transduction pathways such as electron transport system, NOTCH, SIP, AKT, BCR signaling associated genes were also enriched in Pitta prakriti. Interestingly, we have observed regulation of OCA2, MC1R genes, which are associated with white to blonde or red hair colour in Pitta phenotype. Kapha mPSR associated genes were significantly enriched in cell growth/maintenance , cytoskeleton anchoring activity  and cellular adhesion  function.Several methylated sequences of Vata prakriti were found to be represented in diverse biological processes such as cell communication, transcription, various signal transduction pathways and embryo morphogenesis. The associated genes are involved especially in neuronal development  higher methylation at CpG site 47, 55 and 65 in Vata prakriti  family of transcription factors mainly involved in the regulation of embryonic development and in the determination of the cell fate, present in the chromosome 2p25.2. The protein encoded is a transcriptional regulator possessing diverse functions namely, visual [Pitta prakriti governs body metabolism, temperature regulation and development/proper function of eyes. Although, these provide only a causal relationship between the methylation and phenotype, a functional experimental study is essential. We observed that overall average methylation at all CpG sites was found to be higher in the Pitta prakriti when compared to other two  was validated. The gene is mainly involved in the morphogenesis and tissue formation in neural and non-neural developmental process. The recent reports suggested that several known SNPs in the CDH22 were associated with type II diabetes and higher BMI [CDH22 amplicon was 301 bp in length, present at the 5′UTR of the gene, encompassing a total of 40 CpG sites out of which 33 CpG sites were analyzed in all the three prakriti. The fold change difference between prakriti underlined a higher methylation level for Kapha prakriti which was in concordant with the direct BS  (p<0.05), there by regulate structure and integrity of the cells. Further the concordance of CDH22 methylation validation was evident to find the association of Prakriti and BMI. The mechanisms and causes which sustain these changes in relation to prakriti determination needs further investigation and also demands for the case–control or retrospective large scale epigenome wide studies based on prakriti. However, we could able to demonstrate presence of the methylation signatures correlating to prakriti and its distinct phenotype specifically the BMI as an example. Thus, our results provide evidence for the identified genes for the specific prakriti attribute directly or indirectly to bring about defined phenotypes and suggest the impact of DNA methylation on specific prakriti.Gene ontology and pathway analysis for the mPSRs suggests the enrichment of gene related to characteristics of specific Prakriti may govern functions at cellular, tissue and at system levels maintaining equilibrium determined by genes which in turn are regulated by epigenetic cross talk especially through DNA methylation. These may provide basis to explore whether the DNA methylation programmed at the time of birth of an individual that will mimic the prakriti constitutions. This is suggested on the basis of genetic, biochemical, hematological or anatomical features already identified for the specific prakriti. Therefore, our study on the epigenetic basis of prakriti is a step towards deciphering diagnostic and prognostic utilities of traditional medicine by identifying genome-wide DNA methylation variants. In summary, we have identified DNA methylation signatures that distinguish the three major prakriti, which are responsible for imparting variations in humans. Taken together, DNA methylation is probably coupled to chromatin regulation as a contributor to different prakriti phenotypes such as Vata, Pitta and Kapha. The differentially methylated regions identified in our study needs further investigation as they may provide insight into epigenotype–phenotype correlation and that underlining molecular mechanisms of prakriti manifestations.Therapy in Ayurveda is based on the premise of maintaining human body or any living cell as an open system, which can partake with the external environment.


A shear stress modifier was used to produce both advanced and more stable plaques in the carotid artery. After 4 weeks of ADT, increased areas of necrosis was observed in stable plaques from leuprolide-treated mice , 0.2% (IQR 0–4.4) and 11.0% (IQR 1.0-19.8), respectively). There was also evidence of increased inflammation as assessed by macrophage immunohistochemistry in the plaques from leuprolide-treated mice, but we found no evidence of such changes in plaques from control mice or mice treated with degarelix. Necrosis destabilizes plaques and increases the risk for rupture and development of acute cardiovascular events. Destabilization of pre-existing atherosclerotic plaques could explain the increased cardiovascular risk in prostate cancer patients treated with GnRH-R agonists.Androgen-deprivation therapy (ADT) for prostate cancer has been associated with increased risk for development of cardiovascular events and recent pooled analyses of randomized intervention trials suggest that this primarily is the case for patients with pre-existing cardiovascular disease treated with gonadotropin-releasing hormone receptor (GnRH-R) agonists. In the present study we investigated the effects of the GnRH-R agonist leuprolide and the GnRH-R antagonist degarelix on established atherosclerotic plaques in ApoE We used a shear stress-modifying cast to generate both advanced and more stable plaque characteristics in the carotid artery. The results show that treatment with leuprolide, but not degarelix, cause development of necrotic cores in the more stable plaques. This could provide an explanation to the observed increased cardiovascular risk of GnRH receptor agonists.Androgen deprivation therapy (ADT) is a well-established treatment for advanced prostate cancer. Treatment with gonadotropin-releasing hormone receptor (GnRH receptor) agonists is currently the most common approach for ADT. GnRH receptor agonists, such as leuprolide and goserelin, produce a decline in testosterone after an initial testosterone surge in the first 1–3 weeks of therapy2491113−/− mice fed a high-fat diet. In this model, a cast placement around the artery results in the formation of advanced inflammatory plaques in the region proximal to the cast and more stable plaques distal to the cast  in control mice, 0.2% IQR 0–4.4) in degarelix-treated mice, but as high as 11.0% (IQR 1.0–19.8) in leuprolide-treated mice C and 4A..4 in degTo investigate the possibility that the increased presence of necrosis in distal plaques of leuprolide-treated mice was due to a direct effect of the drug on the viability of smooth muscle cells, we incubated cultured human coronary artery smooth muscle cells with leuprolide or degarelix for 24 hours. However, none of the treatments induced smooth muscle cell apoptosis as assessed by analyses of caspase 3 activity or mRNA expression  or cell et al.A recent pooled analysis of six phase 3 randomized trials involving 2328 men with advanced prostate cancer show differences in cardiovascular risk among men treated with GnRH receptor agonists versus those treated with GnRH receptor antagonists, and provide additional evidence that treatment with GnRH receptor agonists is associated with increased incidence of cardiovascular events in patients with pre-existing CVDet al. have reported evidence for a suppressive effect of the GnRH receptor on macrophage functionThe mechanism through which treatment with a GnRH receptor agonist such as leuprolide may increase the risk for necrosis in atherosclerotic plaques remains to be fully characterized. The observation that no such effect was observed in response to treatment with the GnRH receptor antagonist degarelix suggests that activation of the GnRH receptor is important. Treatment with GnRH receptor agonists is known to down-regulate the expression of the receptor in the pituitary gland leading to a suppression of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone levels. However, before this down-regulation occurs there is an initial increase in LH, FSH and testosterone levels due to an increased stimulation of the receptor. In contrast, GnRH receptor antagonists lead to a rapid inhibition of the receptor and down-regulation of LH, FSH and testosterone levels. The observation that treatment with leuprolide did not reduce testosterone levels in the present study suggests that GnRH receptors had not yet been down-regulated and remained responsive to stimulation with the agonist. We used an immunofluorescence staining to determine the presence of GnRH receptor expressing cells in atherosclerotic plaques and found that almost all cells expressing this receptor were T cells. Presence of GnRH receptors has previously been demonstrated on cultured T cells and shown to stimulate the activity of these cells18An interesting and unexpected finding of the present study was that increased plaque necrosis occurred only in the distal stable lesions. These lesions are generally more smooth muscle cell-rich than the more inflammatory proximal low shear stress plaques2426−/− mice also need to be further characterized as well as why only certain types of plaques are affected. The possible role of the decreased body weight in degarelix treated mice should be further investigated to determine if this is of importance for the different effect of leuprolide and degarelix on atherosclerotic plaque stability. Previous studies suggest that the decreased body weight of degarelix-treated mice is explained by hormonal effects leading to loss of adipose tissueThere are some limitations of the present study that should be considered. Most importantly results from animal models of atherosclerosis are not directly transferable to the clinical situation and the observation that treatment with GnRH receptor agonists may induce necrosis in atherosclerotic plaques requires confirmation in human studies. The mechanisms through which leuprolide-treatment caused plaque necrosis in ApoEIn conclusion, the present study demonstrates that treatment with the GnRH receptor agonist leuprolide is associated with induction of necrosis in certain types of atherosclerotic plaques, while no such effect could be observed in mice treated with the GnRH receptor antagonist degarelix. The biological mechanisms involved in the plaque destabilizing effect of the GnRH agonist remains to be fully characterized but are likely to involve T cells since they were the most common GnRH receptor expressing cells in the plaque. Plaque necrosis is considered to increase the risk for plaque rupture, the major cause for development of acute cardiovascular events, such as myocardial infarction and stroke. It is possible that this effect may explain the difference in cardiovascular risk between GnRH agonists and degarelix that has recently been observed in retrospective pooled analysis of randomized intervention trials−/− mice  were fed an atherogenic high cholesterol-diet  for the duration of 14 weeks starting at the age of 16-weeks. Two weeks post-onset of the diet, a perivascular shear stress-modifying cast was placed around the right common carotid artery to alter the pattern of hemodynamic flow, as described by Cheng et al.Male ApoEThe mice were perfused with Histochoice, the right carotid arteries were taken out to determine the atherosclerotic burden and the left carotid arteries were used as controls. The carotid arteries were fixed in Histochoice, embedded in paraffin and sectioned (5 μm).Carotid artery sections were stained with Accustain trichrome (Masson) (Sigma-Aldrich) according to the manufacturer’s instructions. Flat preparations of descending aortas were stained with 0.3% Oil Red O for 50 minutes and mounted with Mountquick . Carotid artery sections were immunohistochemically stained using antibodies against Mac-2 , F4/80 (AbCam 60343), MMP-9 (AbCam 38898), Anti-smooth muscle myosin heavy chain 11 (AbCam 125884), Anti-Actin, α-Smooth Muscle (Sigma-Aldrich A2547), CD3 (AbCam 11089), CD45R/B220 (AbCam 64100) and the GnRH receptor (AbCam 183079). Sections were deparaffinized and rehydrated in xylene and a graded series of alcohols before heat-induced antigen epitope retrieval was performed . The ImmPRESS HRP anti-rat (mouse absorbed) and anti-rabbit Ig (Peroxidase) polymer detection kits (Vector Laboratories) were used for the staining procedures according to the manufacturer’s instructions. For immunofluorescence the same procedure was followed using a Cy3-conjugated secondary goat anti-rabbit rat IgG (Jackson) or a goat anti-rat IgG  or a biotinylated anti-mouse IgG (Vector Laboratories) in combination with fluorescein-streptavidin (Amersham) for detection instead of the HRP-rat secondary antibody from the ImmPRESS kit.Lesion size is expressed as absolute size and necrotic areas are defined as areas not stained by Masson’s Trichrome and are calculated as a percentage of the plaque; presented as the mean value of four sections collected where each lesion was at its largest. Immunohistochemically stained sections  were scanned and digitalized using an Aperio ScanScope digital slide scanner  and images of immunofluorescent stains were taken using a Zeiss Axiophot 2 with a Hamamatsu C4742-95 camera, an X-Cite series 120Q lamp (Lumen Dynamics) and Openlab 5 software (Improvision). Image analysis was performed using BioPix iQ software .Blood was collected by cardiac puncture and plasma retrieved by centrifugation at 3000 rpm for 15 min at 4 °C. The colorimetric assay Infinity Total Cholesterol  was used to quantify total plasma cholesterol and the Bio-Plex Pro Mouse Cytokine Assay (BIO-RAD) was used to quantify plasma cytokine concentrations of with IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A and tumor necrosis factor-α (TNF-α). Both analyses were performed according to instructions from the manufacturer.T) was determined and results of gene expression were expressed as fold change of treated cells compared to untreated after normalization to housekeeping gene GAPDH. The cell supernatants were saved to investigate possible necrosis using the Vybrant cytotoxicity assay , which was performed according to manufacturer’s advice.Human coronary SMCs (hCASMC) were maintained in medium 231 with smooth muscle growth supplement . For Caspase 3 activity measurements, 25 000 cells per well in a 48 well plate were grown for 1–2 days in supplemented medium. Cells were then serum starved for 24 hours before they were stimulated with the indicated concentrations of Degarelix or Leuprolide for 24 hours. Caspase 3 activity was measured in the protein lysate from each well using a fluorogenic caspase 3 assay kit according to the manufacturer’s instructions . Protein concentration was measured using Pierce BCA protein assay kit (ThermoFisher Scientific). For analysis of caspase 3 mRNA expression HCASMC were treated as above, washed with 4 °C PBS  and homogenized in TRI-reagent  using an Omni tissue homogenizer. SYBR Green PCR kit  and commercially available primers for CASP3 and the housekeeping gene GAPDH (Qiagen) was used for mRNA gene expression quantification. The reaction was carried out in a Viia7 Real-time PCR system  after activation for 5 min in 95 °C followed by 40 cycles in the conditions; denaturation for 10 s in 95 °C, annealing and extension for 30 s in 60 °C. The cycle number when fluorescence signal reached threshold (Cwww.graphpad.com).Error bars represent median with interquartile range. Statistical analyses were performed using Kruskal-Wallis analysis of variance, followed by Dunn’s multiple comparison tests when appropriate. Statistics on necrotic measurements were performed with logged values. GraphPad Prism version 6.0 was used for the statistical tests .


MicroRNAs (miRNAs) are important small non-coding RNA molecules that regulate gene expression in cellular processes related to the pathogenesis of cancer. Genetic variation in miRNA genes could impact their synthesis and cellular effects and single nucleotide polymorphisms (SNPs) are one example of genetic variants studied in relation to breast cancer. Studies aimed at identifying miRNA SNPs (miR-SNPs) associated with breast malignancies could lead towards further understanding of the disease and to develop clinical applications for early diagnosis and treatment.X2) and odds ratio (OR) analysis.We genotyped a panel of 24 miR-SNPs using multiplex PCR and chip-based matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis in two Caucasian breast cancer case control populations (Primary population: 173 cases and 187 controls and secondary population: 679 cases and 301 controls). Association to breast cancer susceptibility was determined using chi-square (p = 0.041 and p = 0.023).Statistical analysis showed six miR-SNPs to be non-polymorphic and twelve of our selected miR-SNPs to have no association with breast cancer risk. However, we were able to show association between rs353291 (located in MIR145) and the risk of developing breast cancer in two independent case control cohorts (Our study is the first to report an association between a miR-SNP in MIR145 and breast cancer risk in individuals of Caucasian background. This finding requires further validation through genotyping of larger cohorts or in individuals of different ethnicities to determine the potential significance of this finding as well as studies aimed to determine functional significance. MicroRNAs (miRNAs) are small non-coding single stranded RNA molecules (21 – 25 nucleotides) involved in negative regulation of gene expression , where pmiRNAs are very likely to play an important role in cancer biology due to their role in the regulation of important cellular processes including growth, differentiation and cell survival –16. SeveBreast cancer is the second most commonly diagnosed cancer worldwide  and the most common type of cancer for women in developed countries  . HoweverIn this study, we have investigated a panel of miR-SNPs present in miRNA genes previously identified to be involved in the pathophysiological mechanisms of breast cancer. Our initial selection included 24 variants located in 10 miRNA genes or in close proximity which were genotyped using multiplex PCR and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Genotyping results of the 19 miR-SNPs successfully genotyped and included in this study identified six previously reported microRNA SNPs as non-polymorphic. From the remaining polymorphisms twelve miR-SNPs showed no significant differences between cases and controls and one variant in MIR145 was identified to be associated with breast cancer susceptibility in both of our Australian Caucasian breast cancer case-control populations.Two independent Australian Caucasian breast cancer case populations were available for our study: The Genomics Research Centre Breast Cancer (GRC-BC) population and part of the Griffith University-Cancer Council Queensland Breast Cancer Biobank (GU-CCQ BB). We conducted single nucleotide polymorphism genotyping in the GRC-BC population initially. This consisted of DNA samples from 173 breast cancer patients from South East Queensland and DNA samples from 187 healthy age and sex matched females with no personal and/or familial history of breast, ovarian or any other type of cancer collected at the Genomics Research Centre Clinic, Southport, with research approved by Griffith University’s Human Ethics Committee  and the Queensland University of Technology Human Research Ethics Committee . Breast cancer samples comprised prevalent breast cancer cases diagnosed previous to their inclusion in this study. All participants supplied informed written consent. Average age of test population was 57.52 years and 57 years for cases and controls respectively.Further validation of genotyping results was performed on a subset of the GU-CCQ BB population. 679 DNA samples from breast cancer patients residing in Queensland with a diagnosis of invasive breast cancer confirmed histologically were used to validate genotyping of miR-SNPs. Patient samples had been collected by the Genomics Research Centre in collaboration with the Cancer Council of Queensland as part of a 5-year population-based longitudinal study since January 2010. Patients included in this study were between 33 and 80 years of age, with an average age of 60.16 and they were screened for personal and/or familial history of breast, ovarian or any other type of cancer. Control population for the GU-CCQ BB was established from 2 sources: The control group for this cohort was comprised of genotyping result data taken from 201 healthy females belonging to the phase 1 European population from the 1000Genomes project. Efforts were made to select a subgroup of individuals that were comparable to the case group in terms of age, ethnicity and sex .Genomic DNA was extracted from whole blood samples using a modified salting out method described previously , 36. DNAFigure Using the MassARRAY® Assay Design Suite v1.0 software  we were able to create a single multiplex PCR genotyping assay containing 24 miR-SNPs from our preliminary selection . Primer extension reactions were performed according to the instructions for the SEQUENOM linear adjustment method included in the iPLEX™ GOLD genotyping protocol . All reactions were performed using Applied Biosystems® MicroAmp® EnduraPlate™ Optical 96-Well Clear Reaction Plates with Barcode  and an Applied Biosystems® Veriti® 96-Well Thermal Cycler .n = 240) were validated by Sanger Sequencing to ensure accuracy of genotyping results. In all cases, the Sanger Sequencing confirmed the genotyping obtained using MassARRAY.A total of 12-16 nl of each iPLEX® reaction product were transferred onto a SpectroCHIP® II G96  using SEQUENOM® MassARRAY® Nanodispenser . SpectroCHIP® analysis was carried out by SEQUENOM® MassArray® Analyzer 4 and the SpectroAcquire software Version 4.0 . Finally data analysis for genotype determination was done using the MassARRAY® Typer software version 4.0 . In order to confirm the genotypes obtained, randomly selected samples (5 each for case and control cohorts) from each genotype (http://pngu.mgh.harvard.edu/purcell/plink/) [Statistical analysis of genotypes and alleles was conducted using Plink software version 1.07 (/plink/) . The α f/plink/) , 46. We /plink/) . FinallyMicroRNAs are some of the small non-coding RNA molecules responsible for gene regulation at the translational level. They require a very complex series of nuclear and cytoplasmic processes for their synthesis and to achieve their functional effects on genes involved in key cellular functions like replication and cell differentiation , 10, 13.p > 0.05). Ultimately, however, chi-square analysis of genotyping results of 12 SNPs located in relation to seven miRNAs  showed no significant differences for genotype and allele frequencies between cases and controls in our GRC-BC population  p > 0.0. Ultimatp = 0.041) although no significant difference in genotype frequencies (p = 0.09) (See Table p = 0.006) and also statistically significant differences at the genotype level (p = 0.02). Finally we calculated odds ratios for alleles in the GRC-BC and GU-CCQ BB populations to be 1.37 (CI 95%: 1.01–1.84) and 1.31 (CI 95%: 1.04–1.70) respectively, suggesting the presence of the C allele at this locus increases the risk of developing breast cancer. However it should be noted that if we consider multiple testing, our finding for the analysis of allele frequencies for both populations for this variant is not significant if we determine Bonferroni correction of the α for p-value to be 2.08 x 10−3. However these results are still potentially interesting particularly considering the similarity of allelic significance in both independent case-control cohorts and point to the need for further genotyping in extended populations.In contrast, we were able to determine significant differences at the allelic level for rs353291 after chi-square analysis in our GRC-BC cohort (miR-SNP rs353291 is located 450 bp upstream from the MIR145 gene, inside the miRNA 143 host gene transcript, in the long arm of chromosome 5 region 32 at position 148,810,746. To the best of our knowledge there are no previous association studies in relation to this miR-SNP and breast cancer risk in Caucasians or any populations of other ethnicities. However, MIR145 has been previously reported to play a role in cancer biogenesis and progression. Downregulation of MIR145 is a common finding previously reported in colorectal , bladderWe were able to determine that the presence of a polymorphism in miR-SNP rs353291 is associated with an increased risk of developing breast cancer based on our findings. To the best of our knowledge, this is the first report on breast cancer risk association for this variant in individuals of Caucasian background. This finding could potentially explain the previously described role that MIR145 plays in breast cancer documented in the literature, but it requires confirmation via functional studies using cell culture or animal models. It also requires further validation on larger Caucasian population as well as in cohorts of individuals with different ethnical backgrounds before it can be translated into clinical applications used in breast cancer diagnosis or treatment.


Schizophrenia is a complex mental disorder that displays behavioral deficits such as decreased sensory gating, reduced social interaction and working memory deficits. The neurodevelopmental model is one of the widely accepted hypotheses of the etiology of schizophrenia. Subtle developmental abnormalities of the brain which stated long before the onset of clinical symptoms are thought to lead to the emergence of illness. Schizophrenia has strong genetic components but its underlying molecular pathogenesis is still poorly understood. Genetic linkage and association studies have identified several genes involved in neuronal migrations as candidate susceptibility genes for schizophrenia, although their effect size is small. Recent progress in copy number variation studies also has identified much higher risk loci such as 22q11. Based on these genetic findings, we are now able to utilize genetically-defined animal models. Here we summarize the results of neurodevelopmental and behavioral analysis of genetically-defined animal models. Furthermore, animal model experiments have demonstrated that embryonic and perinatal neurodevelopmental insults in neurogenesis and neuronal migrations cause neuronal functional and behavioral deficits in affected adult animals, which are similar to those of schizophrenic patients. However, these findings do not establish causative relationship. Genetically-defined animal models are a critical approach to explore the relationship between neuronal migration abnormalities and behavioral abnormalities relevant to schizophrenia. Several genetic linkage studies have shown Neuregulin1 (NRG1) as a strong candidate gene for schizophrenia  gene was discovered at the breakpoint of inherited balanced chromosomal translocation in a Scottish family suffering from major depression, schizophrenia, and bipolar disorder , lissencephaly-1 (LIS1), phosphodiesterase 4B (PDE4B), glycogen synthase kinase 3β (GSK3β) and DIX domain-containing 1 (DIXDC1) . This C-terminal truncated DISC1 functions as a dominant negative protein, and impairs microtubule dynamics by blocking interaction between DISC1 and dynein complex  astonishingly showed almost normal cytoarchitectures of the cerebral cortex and the HP  mutagenesis was utilized to generate missense mutations of Disc1. L100P mutant (Disc1L100P/L100P) mice showed reduced brain volume, reduced number of cortical neurons, altered distribution of cortical neurons, interneuonal migration deficits, and schizophrenia-like behavioral abnormalities, although the behavioral phenotypes were not confirmed by another group due to the difference in the genetic background  exhibited enlarged ventricle and working memory deficits  is the most frequent known genetic cause of schizophrenia  with Drosha, which is essential for miRNA production. Overexpression of Dgcr8 rescued interneuronal migration deficits of 22q11DS model mice, and the migration of hippocampal DG and interneuronal progenitors were also affected in+/−Dgcr8 mice (Toritsuka et al., Dgcr8 in the pathogenesis of 22q11DS. miRNA–mediated regulation network fine tunes the balance of signaling and confers robustness to the system (Herranz and Cohen, Dgcr8 causes 20–70% reduction of a specific subsets of mature miRNAs both in Dgcr8 heterozygous and 22q11DS model mice (Stark et al., Dgcr8 heterozygousity might uncover the effects of 22q11 microdeletion through the disruption of miRNA-mediated buffering effects. In mice, heterozygous deletion of Dgcr8 alone showed working memory deficits, sensory information-processing deficits and some of neurodevelopmental abnormalities such as reduced cortical neuronal densities (Stark et al., +/−Dgcr8 mice are similar but some of them are milder than those of 22q11DS model mice,(Stark et al., Among of genes deleted in 22q11DS, Elucidating the relationship between neurodevelopmental abnormalities and the pathogenesis of schizophrenia would be exceptionally difficult. In order to dissect the complex causal relations, more sophisticated genetic manipulation would be required. Combination of various techniques such as conditional knockout, inducible transgene expression and virus-mediated gene delivery will enable cell type-specific and developmental stage-specific knockout or rescue experiments. In the future comprehensive profile of neurodevelopmental deficits-behavioral abnormalities will provide significant insights into mental disease pathogenesis of all these neurodevelopmental genes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Over expression of miR 29a/b could reduce virus levels by targeting the Nef gene. We hypothesized that differential expression of the microRNA maybe a protective factor in AIDS progression.The events during host virus interaction pave the way to differential outcomes in HIV-1 disease progression rates. We have earlier shown that HIV-In a cohort of 75 HIV-1 infected individuals, who show differential disease progression, classified as (1) Long Term Non Progressors (LTNPs), (2) Regular Progressors and (3) Rapid Progressors. We isolated RNA and DNA from PBMCs collected from these individuals and healthy controls. We quantified miR 29a expression through Real Time PCR and performed statistical correlation of the miRNA levels with disease progression rate. We further analyzed miR 29a promoter and coding region from selected patients.Patients, including various sub classes like LTNPs, Regular Progressors and Rapid progressors, had higher miR 29a levels than healthy controls. A small subset of patients had unusually high levels of miR 29a. Three of these individuals were LTNPs while others were classified as regular progressors.The absence of known genetic causes of non progression like the CCR5 delta mutation, in this patient cohort suggests that there are novel mutations that confer protection. The absence of healthy control individuals with high miR 29a expression suggests that the induction of miRNA maybe happen in response to infection. However, larger sample sizes are required to confirm this. We conclude that miR 29a expression maybe one of the factors that in certain backgrounds provides a better prognosis.