The decreased activity has been attributed to obesity. In mice, we tested the hypothesis that leptin increases total locomotor activity but inhibits food anticipatory activity. We also sought to determine if leptin induced increases in total locomotor activity are independent of changes in body weight and obesity. We studied obob mice and also created a novel transgenic mouse where leptin is over-expressed in a tetracycline-off system and can be abruptly and non-invasively suppressed by doxycycline within few hours. The studies were performed using two independent behavioral assays: home cage activity (HCA) and running wheel activity (RWA). Systemic administration of leptin (150 ng/hr) to obob mice produced a 122%±30% (mean ± SEM) increase (p≤0.01) in locomotor activity within 2 days In addition, cerebroventricular administration of leptin (5 ng/hr) also produced an early and progressive increase in total locomotor activity beginning on the 1st day  and increasing to +69±23% on day 3 without a decrease in body weight during this time. The increase in activity was restricted to the dark phase. Conversely, in a tet-off transgenic obob mouse line, acute leptin suppression reduced spontaneous locomotor activity. To further define activities that are leptin regulated, we assayed food anticipatory activity (FAA) and found that it was markedly augmented in obob mice compared to wild type mice  and abolished by leptin. Although melanocortin-3 receptors (MC3R) reportedly mediate FAA, we found augmented FAA and preserved inhibitory effects of leptin on FAA in MC3R−/−obob mice. In summary, this study demonstrates that total activity and FAA are regulated independently by leptin. Leptin, acting in the central nervous system and at physiologic levels, produces early increases in locomotor activity before substantial weight loss. In contrast, leptin suppresses augmented food anticipatory activity in obob mice.Obese, leptin deficient  Humans with complete leptin deficiency display aggressive food seeking behavior While total activity in There are different types of locomotor activity that can be assayed in mice including home cage activity (HCA), running wheel activity (RWA), and food anticipatory activity (FAA), each of which has distinct behavioral significance.obob mice. We analyzed effects of changing leptin on both total locomotor activity and on food anticipatory activity (FAA). Leptin was administered systemically and into a lateral cerebral ventricle. The time course of changes in activity was compared with changes in body weight to determine if leptin altered activity independent of changes in body weight. Finally, we evaluated the role of melanocortin-3 receptors in leptin-induced influences on FAA in obob mice In mice, we tested the hypothesis that leptin increases total locomotor activity but inhibits food anticipatory activity. Using two independent assays of locomotor activity, we studied both a novel Tet-off transgenic mouse model in which circulating leptin can be acutely and non-invasively suppressed and leptin deficient obob mice showed profoundly decreased locomotor activity compared to wild type controls using both HCA and RWA assays  produced marked early increases in RWA and HCA  in adult wild type mice obob or wild type mice  beginning on the 1st day  and increasing to +69±23% on day 3  at 5 ng/h. ICV infusion of leptin in on day 3 A–B, wheon day 3 B. Thereon day 3 B. ICV ibob mice A.These findings suggest that the effects of leptin on spontaneous activity are mediated by a central neural action.A). LAP-tTa/TRE-hleptin (Tg) mice showed a “skinny” phenotype Generation of transgenic (Tg) mice chronically over-expressing leptin has been previously reported obob background to generate LAP-tTa/Tre-hleptin/obob (Tg obob) mice, a strain in which the absence of a functional endogenous leptin gene allows the study of the acute effect of leptin deprivation in mice that are not perturbed or manipulated other than for the administration of Dox  it increased total home cage and running wheel activity. This inhibitory effect of leptin on FAA in obob mice and its contrasting effect to increase total activity are also shown in the actograms of obob mice, physiologic doses of leptin abolished FAA, but increased total locomotor activity.We next performed a set of behavioral experiments to assess FAA. In these experiments, availability of food was restricted progressively to the first 8, 6, and 4 hrs of the onset of the dark phase and then shifted to 4 hrs in the middle of the light phase . With thbob mice A comparbob mice B. It isbob mice A,, but bob mice B. In obobob mice that displayed augmented FAA. Importantly, leptin suppressed FAA in obob mice . Furthermore, similar to its effects in obob mice, leptin inhibited FAA . These data indicate that MC3R do not mediate the influence of leptin on food anticipatory activity or total locomotor activity.We next tested whether the effect of leptin on FAA was abrogated in the absence of the melanocortin MC3 receptor. We were interested in testing the role of MC3R because of a previous report showing that melanocortin 3 receptor knockout mice (MC3R −/−) have decreased food anticipatory activity ted, FAA A while bob mice A. Furthited FAA B in MC3activity B. Theseobob mice with high doses of leptin for three weeks increased locomotor activity, this was observed at a time when body weight had decreased by approximately 40% making it difficult to determine if the increased activity reflected reversal of obesity or a direct effect of leptin. Our finding that both systemic and cerebroventricular administration of leptin produced early increases in activity in obob mice before substantial decreases in body weight provides evidence for a direct effect of leptin at least partially independent of reversal of obesity. The concept of a direct effect of leptin on activity independent of adiposity is supported by the decreases in activity with abrupt leptin suppression in hyperleptinemic, skinny, Tet-off transgenic obob mice. This result in the Tet-off transgenic mice, obtained non-invasively and without manipulation in food availability, expands on a previous study from our laboratory While Pelleymounter, et al obob mice with food restriction. This is consistent with previous reports that Zucker obese rats (affected by a loss of function mutation in the leptin receptor) have augmented food anticipatory or seeking activity compared to lean control rats obob mice have a loss of function mutation in the leptin gene. The nature of the relevant signal(s) for the potentiated FAA in the obob mice is unknown. Food intake is regulated by numerous short and long term hormonal and metabolic signals. The robust FAA in obob mice provides an assay for the identification of signals and neural pathways mediating this phenomenon independent of leptin. Based on previous observations that the absence of melanocortin-3 receptors (MC3R) abrogates FAA in MC3R deficient mice, we postulated that MC3R might mediate the augmented FAA in obob mice. To our surprise, in a new double mutant MC3R −/− obob mouse model, the augmented FAA was preserved and not attenuated by deletion of MC3R.A striking observation in this study was the robust increase in food anticipatory activity (FAA) in obob mice abolished FAA while increasing total locomotor activity. The contrast between the effects of leptin on FAA and total locomotor activity has implications for understanding the different components of locomotor activity by showing that they can be regulated independently by leptin. The leptin-induced suppression of FAA in obob mice is consistent with observations from humans by two groups of investigators Leptin replacement in obob mice fed ad lib. In contrast, leptin suppressed activity in fasted obob and wild type mice. Morton et al concluded that leptin is a physiological regulator of spontaneous physical activity, but that the nature of leptin's effect on activity is dependent on food availability obob mice, we demonstrated that acute non-invasive leptin suppression in hyperleptinemic, skinny, tet-off transgenic mice decreases locomotor activity. We also found that cerebroventricular administration of leptin promptly increased activity in obob mice supporting a central neural mechanism of leptin regulation of activity. Finally, we also performed experiments to evaluate food anticipatory activity (FAA) by reducing the time but not the amount of food availability. FAA was augmented in obob mice. In this protocol, leptin suppressed FAA while increasing total 24 hr locomotor activity. These observations indicate that leptin produces opposing effects on total locomotor activity and FAA within a 24 hr protocol.Morton et al dbdb mice with loss of leptin receptor function, restoration of LRb only in pro-opiomelanocortin (POMC) neurons virtually normalizes locomotor activity with only modest decreases in body weight The increase in total locomotor activity with cerebroventricular infusion of leptin in our study raises the question of the neural pathways and mediators involved in the locomotor actions of leptin. In leptin receptor (LRb) null mice, unilateral arcuate restoration of LRb substantially improves locomotor activity while only modestly decreasing body weight dbdb mice with complete loss of LRb signaling Mice with constituitive activation of signal transducer and activator of transcription 3 (STAT3) in agouti-peptide related (AgRP) neurons are lean and have increased locomotor activity While restoration of physiologic levels of leptin increased total locomotor activity, a higher supraphysiologic dose did not produce further increases. These findings are consistent with reports that locomotor activity does not increase when normoleptinemic, wild type control mice are given supplemental leptin Leptin is a highly pleiotropic hormone. Decreases in leptin, which are sensed as starvation, trigger a constellation of adaptive responses including increases in appetite, decreases in energy expenditure, infertility, reduced immune function, and development of a euthyroid sick state obob mouse model decreases activity. Second, leptin replacement in obob mice promptly increases activity before substantial decreases in body weight. Third, the leptin-induced increases in locomotor activity in obob mice occur with cerebroventricular in addition to systemic administration, implicating a central neural mechanism. Fourth, food anticipatory activity (FAA) is markedly augmented in leptin deficient mice and is abolished by leptin. Fifth, deletion of melanocortin-3 receptors in obob mice does not attenuate the augmented FAA produced by leptin deficiency and does not alter leptin-induced regulation of FAA or total locomotor activity. Sixth, leptin produces opposing effects on total locomotor activity and food anticipatory activity.In summary, these studies provide several insights into the behavioral actions of leptin. First, acutely and non-invasively suppressing leptin levels in a novel Tet-off hyperleptinemic transgenic obob mice 8 weeks old from Jackson Laboratories  were used. Mice were individually housed with food and water available ad libitum, and a 12∶12 h light dark cycle (lights off at 1900h). Mice were fed standard rat chow (Lab Diet 5053). All procedures were approved by The Rockefeller University Institutional Animal Care and Use Committee, and followed the Public Health Services Policy on Humane Care and Use of Laboratory Animals.Male C57/BL6 or For systemic administration, Alzet® osmotic pumps were filled with phosphate buffered saline (PBS) or murine leptin (Amylin) at 150 or 750 ng/h and were incubated in saline at 37°C overnight prior to implantation. Subcutaneous implantation of the pumps was performed under isofluorane anesthesia and at the end of the procedure the animals were returned to their home cages and behavior recorded. For cerebroventricular administration, a cannula was placed in a lateral cerebral ventricle under isofluorane anesthesia using a stereotaxic apparatus. Cannulae were implanted 0.75 mm lateral to the midline, 0.1 mm posterior to the bregma, and 3.5 mm ventral to the surface of the skull. The cannula was connected to an Alzet subcutaneous osmotic pump filled with PBS or murine leptin at 5 ng/h.ad libitum. RWA was recorded in 1 minute bins, and later summed into 3 h or 24 h totals.Animals were placed in individual running wheel cages [dimensions of cage: 32×17×14 cm and dimensions of wheel: 25 cm in diameter ] with food and water available Individually housed mice were placed in the arousal testing chamber. The chamber consists of a 3 dimensional infra-red photobeam system (AccuScan Instruments), where the beams are placed at 1 cm intervals. Voluntary motor activity is measured as general voluntary activity in numbers of beam breaks in the vertical axis [vertical activity (VACTV)] and horizontal axis [horizontal activity (HACTV)] and also as total distance traveled in cm . All these variables were collected in 1 min intervals using Versamax software (AccuScan Instruments), and were later summed into 3 h or 24 h totals.B). LAP-tTa/Tre-hleptin mice were then crossed in an obob background to generate LAP-tTa/Tre-hleptin/obob. Only male mice were studied.The generation of these mice is described in detail in the SI Appendix. In synthesis, LAP-tTA C57Bl6J mice were purchased from Jackson laboratories  and crossed with TRE-hleptin Tg mice generated in C57Bl6J background by pronuclear injection. The function of the Tre-hleptin construct was tested in vitro and in vivo B. LAP-tHleptin serum levels in Tg mice were measured by a specific human leptin Elisa (R & D systems). Blood sampling was performed by orbital bleeding under isofluorane anesthesia. Animals were maintained on autoclaved standard chow (Lab Diet 5053). Doxycycline was mixed to the standard chow at the concentration of 0.1, 0.5 and 2 g/kg (Bio-Serve).ad libitum. Animals were then implanted with osmotic pumps containing PBS or leptin (150 ng/h) and activity was recorded for 1 week. Following this period, food was removed at dark onset (1400h), and food availability was gradually decreased in the following manner: day 1 - food available from 1400h to 2200h, day 2 - food available from 1400h to 2000h and from day 3 to day 10 food was available from 1400h to 1800h. On day 11, food availability was moved to the middle of the light period (0600h to 1000h), and this feeding schedule was maintained for an additional 5 days. Food was then provided ad libitum for 48 h, followed by no food for 48 h, and finally ad libitum food conditions for 48 h. RWA or HCA was recorded daily and was summed into 24 h total activity amounts, as well as the ratio of the activity displayed in the 3 h period preceding food presentation to the 24 h total activity (both preceding the dark onset feeding time and the middle of the light feeding period). Using this food anticipatory activity (FAA) paradigm with 48 h and refeeding periods, we are able to demonstrate that the mechanism behind the FAA is a food entrainable oscillator with a periodicity of 24 h rather than a hunger-derived, non-temporally regulated arousal signal.Mice were housed in individual cages  under a 12∶12 h light∶dark cycle for 1 week, with food and water available obob and obob controls.MC3R−/− mice were purchased from Jackson Laboratory and crossed with ob/+ mice to obtain double heterozygote for the MC3R and ob gene mutation. Double heterozygote mice were then crossed to obtain MC3R−/− obob and obob littermate control mice were housed in individual running wheel cages under a 12∶12 h light∶dark cycle for 2 weeks, with food and water available ad libitum. To detect food anticipatory activity food was removed during the light phase (0600h), and food availability was gradually decreased in the following manner: day 1 - food available from 0600h to 1400h, day 2 - food available from 0600h to 1200h and from day 3 to day 14 food was available from 0600h to 1000h. On day 15 mice were left with food ad libitum for 4 weeks. Running wheel activity was recorded daily and was summed into 24 h total activity amounts, as well as the ratio of the activity displayed in the 3 h period preceding food presentation to the 24 h total activity. After the recovery period, mice were implanted, under general anesthesia, with subcutaneous (Alzet) pumps filled with leptin (150 ng/h) or PBS. Six days after surgery mice were presented again with a protocol to detect food anticipatory activity. Again food was removed during the light phase (0600h), and food availability was gradually decreased in the following manner: day 1 - food was available from 0600h to 1400h; day 2 - food was available from 0600h to 1200h; and from day 3 to day 14 food was available from 0600h to 1000h. On day 14 mice were left with food ad libitum.MC3R −/− t-test post-hoc and using linear regression analysis. Statistical analyses were conducted using SigmaStat Software version 3.1, Systat, Chicago, IL and GraphPad Prism 5.Comparisons between groups of animals were made using a one-way analysis of variance (ANOVA) followed by unpaired Student's Figure S1Effect of a supraphysiological dose of leptin on locomotor activity. 24 hour running wheel activity (RWA) in obob mice treated with leptin, 150 ng/h (n = 6) and 750 ng/h (n = 6). As shown previously in (TIF)Click here for additional data file.Figure S2Food intake during vehicle, leptin, and pair feeding. As expected, food intake did not decrease during vehicle. The decrease in food intake tended to be greater during leptin 750 ng/h than during leptin 150 ng/h. Food intake in the pair fed group was matched to food intake in the group treated with leptin 150 ng/h. n = 6 each group. ***p≤0.001. *p≤0.05.(TIF)Click here for additional data file.Figure S3Effect of leptin (low dose) on activity of obob mice. RWA during infusion of a very low dose of leptin  or vehicle (n = 5) in obob mice. The low dose of leptin produced a progressive increase in RWA over four weeks.(TIF)Click here for additional data file.Figure S4Development of a hyperleptinemic Tet-off transgenic mouse line. Schematic of the transgenes used for generating Tet-off Tg obob mice: one line that controls the tetracycline trans-activator expression in the liver indicated as LAP-tTa (left) and the other containing the tetracycline responsive element together with human leptin cDNA (right). (B) The function of the Tre-hleptin construct was tested in vitro after transient transfection of the corresponding plasmid in a pK-15 Tet-off cell line (left) or co-transfecting it with an rtTA plasmid in HEK293T (Tet-on) cells (right). Our results show that, in vitro, the production of human leptin from the construct used to generate Tg mice can be efficiently induced or repressed in Tet-on and Tet-off settings respectively. (C) The specificity of the expression of the tTa in the liver was confirmed crossing the mouse line with a reporter EGFP mouse line and measuring fluorescence in the tissue extracts . (D) Pronuclear injection of the Tre-hleptin construct in C57Bl6 background resulted in 10 founder lines. (E) Lap-tTa/Tre-hleptin  mice showed a “skinny” phenotype as indicated by the lower body weight curve compared to the littermates controls , while mice carrying the Tre-hleptin gene alone  did not show body weight significantly different from the wild type mice. (F) The pattern of expression of the human leptin transgene was tested, using real time PCR, in different tissues  in Tg mice, Tre-hleptin and controls.(TIF)Click here for additional data file.Figure S5Schematic of food anticipatory activity (FAA) protocol. Schematic of the protocol for study of FAA. Clear bars represent periods when food was withdrawn. Shaded bars represent periods when food was available.(TIF)Click here for additional data file.Figure S6Representative actograms of FAA activity in obob mice and the effect of leptin treatment. (A) Representative actograms of RWA in an obob mouse treated with vehicle or (B) leptin, 150 ng/h, before and during exposure to the FAA protocol described in obob mouse treated with vehicle expressed marked FAA activity (increase in RWA activity in the light phase before feeding time in the dark phase). During the same protocol, leptin treatment in an obob mouse completely suppressed FAA while increasing RWA during the dark phase.(TIF)Click here for additional data file.Figure S7Food intake of obob mice during FAA protocol. Food intake (plotted as mean every two days ± SEM) in obob mice treated with vehicle and compared to the leptin treated group. As expected, in the group of obob mice treated with vehicle, food intake decreased, during the initial phase of the FAA protocol.(TIF)Click here for additional data file.Figure S8Role of melanocortin-3 receptors (MC3R) in locomotor activity of obob mice. (A) Total RWA was not significantly attenuated in obob mice with deletion of MC3R  compared to obob (n = 5). (B) Leptin increased (p≤0.05) total RWA in the MC3R−/−obob mice (n = 7) compared to controls treated with vehicle (n = 6).(TIF)Click here for additional data file.Supporting Information S1(DOC)Click here for additional data file.


POMP) gene. The deletion causes a relative switch in transcription start sites for POMP, predicted to decrease levels of POMP protein in terminally differentiated keratinocytes. To investigate the pathophysiology behind KLICK we created an in vitro model of the disease using siRNA silencing of POMP in epidermal air-liquid cultures. Immunohistochemical analysis of the tissue constructs revealed aberrant staining of POMP, proteasome subunits and the skin differentiation marker filaggrin when compared to control tissue constructs. The staining patterns of POMP siRNA tissue constructs showed strong resemblance to those observed in skin biopsies from KLICK patients. Western blot analysis of lysates from the organotypic tissue constructs revealed an aberrant processing of profilaggrin to filaggrin in samples transfected with siRNA against POMP. Knock-down of POMP expression in regular cell cultures resulted in decreased amounts of proteasome subunits. Prolonged silencing of POMP in cultured cells induced C/EBP homologous protein (CHOP) expression consistent with an activation of the unfolded protein response and increased endoplasmic reticulum (ER) stress. The combined results indicate that KLICK is caused by reduced levels of POMP, leading to proteasome insufficiency in differentiating keratinocytes. Proteasome insufficiency disturbs terminal epidermal differentiation, presumably by increased ER stress, and leads to perturbed processing of profilaggrin. Our findings underline a critical role for the proteasome in human epidermal differentiation.Keratosis linearis with ichthyosis congenita and keratoderma (KLICK) is an autosomal recessive skin disorder associated with a single-nucleotide deletion in the 5′untranslated region of the proteasome maturation protein ( POMP gene was recently identified in a homozygous state in patients with KLICK POMP, resulting in a 29-fold increase in the proportion of transcripts with long 5′UTR's in differentiated keratinocytes. Immunohistochemical analysis of skin biopsies from KLICK patients display altered expression of POMP, proteasome subunits and the epidermal differentiation marker filaggrin. Furthermore, differentiated epidermal layers of patient skin biopsies show increased staining of the unfolded protein response (UPR) protein CHOP, suggesting that ER stress is a pathophysiological mechanism in KLICK POMP 5′UTR mutation have been hampered due to difficulties in obtaining terminally differentiated keratinocytes in vitro.KLICK genodermatosis (MIM #601952) is an autosomal recessive skin disorder characterized by ichthyosis, hyperkeratotic plaques, palmoplantar hyperkeratosis, circular constrictions around fingers and numerous papules in a linear pattern in armfolds and on wrists The proteasome is a large protein complex, step-wise assembled by α and β subunits forming hemiproteasomes which dimerize to form a complete proteasome in vitro knock-down of POMP expression in organotypic epidermal tissue constructs mimics the immunohistochemical phenotype of KLICK epidermis in terms of aberrant POMP, filaggrin and proteasome subunit distribution. Silencing of POMP expression in cell culture results in decreased amounts of proteasome subunits and increased ER stress. These results underline the importance of proteasomes in epidermal differentiation and link ER stress to aberrant epidermal differentiation in KLICK genodermatosis.In this study we present additional evidence for an association between decreased amounts of POMP, increased ER stress and KLICK genodermatosis. We show that POMP wild type 5′ UTR reduces POMP-GFP fusion protein levels compared to short 5′ UTR POMP constructs POMP cDNA with different 5′ UTR's; one with an 81-nt-5′ UTR, wild type, and one with a 181-nt-5′ UTR including c.-95DelC. The constructs were fused with GFP and transiently expressed in HeLa and HaCaT cells. Western blot analysis revealed a 23% reduction in POMP-GFP expression from the mutant 181-nt-5′UTR construct compared to the wild type 81-nt-5′UTR construct in HeLa cells (One-Sample (OS) t-test p = 0.0801, Mann-Whitney (MW) p = 0.0636) and a 31% reduction in HaCaT cells . Sections of the tissue constructs were analyzed together with skin biopsies derived from healthy controls and KLICK patients POMP-silenced organotypic epidermis showed a weak staining of POMP and a weak and patchy staining of α7 proteasome subunits compared to non-transfected and mock-transfected tissue cultures  and 5.6% (HaCaT) of levels in mock transfected cells at 48 h after transfection and to 5.7% (HeLa) and 6.9% (HaCaT) at 72 h after transfection  but not in HeLa cells  in three out of three experiments, while there were no changes in BiP or ATF4 levels  and BiP mRNA was increased in HaCaT cells  with the KLICK mutation produce 23–31% less protein than shorter 5′ UTR (81 nt) constructs. These findings indicate that KLICK patients, predominantly expressing long 5′UTR POMP transcripts in differentiated keratinocytes, have a decreased POMP translation rate in differentiated epidermis.Transcripts with long 5′ UTR's are generally associated with reduced translation rate in vitro model of human interfollicular epidermis POMP. Interestingly, after 10–12 days we observed an aberrant staining of POMP and proteasome subunit α7 in siRNA transfected tissue constructs where the patchy staining of α7 resembles that of KLICK skin biopsies. This indicates that the deviations observed in KLICK skin are specific effects of POMP insufficiency and a disturbed proteasome function. Moreover, the incoherent staining of profilaggrin and filaggrin in KLICK skin was apparent also in POMP siRNA transfected tissue constructs. Although the expression of profilaggrin in tissue constructs seems to be reduced when compared to skin of KLICK patients, we observe a clear effect on profilaggrin processing associated with POMP deficiency. Western blot analysis of the N-terminus of profilaggrin revealed an aberrant pattern with absence of the N-terminal cleavage product of profilaggrin comprising the profilaggrin A and B domains POMP-silenced tissue constructs. In combination, these findings indicate that the proteasome is involved in profilaggrin processing and that KLICK is associated with a disturbed epidermal expression of filaggrin.We next wanted to ensure that reduced POMP protein levels cause the abnormal epidermal differentiation seen in KLICK skin POMP models KLICK syndrome in vitro. We then studied the effects of knock-down of POMP in regular cell cultures. It has previously been shown that silencing of POMP expression by siRNA results in abolished incorporation of β5 and β5i subunits into proteasomes and hence a decreased amount of mature proteasomes POMP knock-down on proteasome subunits explains the weaker expression of α7 and β5 in KLICK epidermis. Interestingly, there was no decrease in α7 and β5 subunits in cells treated with the proteasome inhibitor MG132, indicating that the subunits remain stable if proteasomes are inhibited at a mature stage.From our results we concluded that siRNA silencing of POMP in both HeLa and HaCaT cells. CHOP is generally not expressed under physiological conditions, but is activated at later stages of the UPR POMP knock-down caused a slight increase in the ER chaperone BiP in keratinocyte derived HaCaT cells, but not in HeLa cells, supporting tissue specific sensitivity to ER stress, as suggested previously Proteasome degradation and the UPR are tightly regulated and connected systems of critical importance for the cell. The systems relieve the ER from protein overload, e.g. related to protein misfolding, high protein production or defect protein degradation POMP 5′ UTR mutation associated with KLICK genodermatosis results in reduced POMP expression, which in turn causes reduced levels of proteasome subunits and filaggrin as well as increased ER stress. Our results are consistent with different possible mechanisms contributing to perturbed epidermal differentiation in KLICK genodermatosis: Firstly, the cleavage products of profilaggrin have been shown to be indispensable in the formation of the epidermal water barrier Taken together, we show that the Further studies are required to clarify the exact role of the proteasome in epidermal differentiation and the molecular effects of increased ER stress in epidermis. Increased understanding of ER stress involvement in KLICK genodermatosis and epidermal differentiation may open up for new therapeutic strategies of potential importance for a large group of skin disorders.This project was approved by the local Research Ethics Committee Uppsala. The use of human samples is conducted according to the principles expressed in the Declaration of Helsinki.2. All cells were cultured in 37 degrees Celsius with 5% CO2 in a humid environment.HeLa cells 5 cells/insert in 1.5 mM CaCl2 supplemented medium. The keratinocytes were cultured as described previously For air-liquid cultures we used second to third passage epidermal keratinocytes expanded in EpiLife™ medium (Invitrogen) that were seeded the day after siRNA transfection on Millicell-PCF inserts  at a density of 3×10POMP cDNA were cloned into a fluorescent reporter vector  to produce POMP-GFP fusion proteins, as described previously POMP transcripts with an 81-nucleotide 5′ UTR (81-wt-5′UTR) and one clone is associated with KLICK genodermatosis having a 181-nucleotide 5′ UTR with a cytosine deletion (c.-95DelC). All plasmids were verified by sequencing. HeLa and HaCaT cells were transfected with either of the POMP-GFP vectors together with empty vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Cells were harvested after 48 hours by trypsinization for protein preparation. Statistical calculations were performed with results from three experiment replicates using both One-sample t-test  and Mann-Whitney's test , using R software.cDNA clones corresponding to two variants of full length POMP expression Invitrogen Stealth Select RNAi™ siRNA  was used. Cultured keratinocytes, HeLa and HaCaT cells were transfected with POMP siRNA using Lipofectamine RNAiMax (Invitrogen) according to the protocol provided by the manufacturer. HeLa and HaCaT cells were harvested at 48 h and 72 h after transfection for RNA and protein preparation. The two siRNA oligos worked equally well with a 92.0–94.4% reduction of POMP expression in HeLa and HaCaT experiments. Cells transfected with negative control (mock) siRNA , non-transfected cells and non-transfected cells treated with 1 µM proteasome inhibitor MG132  for 20 h were used as negative and positive controls respectively. mRNA and protein analyses of POMP siRNA and mock siRNA transfected samples were performed with results from three separate experiments using Student's t-test and MW. Air-liquid cultures were set up in triplicates in three separate experiments and mock siRNA transfected and non-transfected cells were used as controls.For silencing of HeLa and HaCaT cells were harvested by trypsinization and were after centrifugation resuspended in RIPA buffer ) and kept at +4 degrees Celsius for 1 hour for protein preparation. The cell suspension was centrifuged for 10 min at +4 degrees Celsius at 13000 rpm to remove cell debris and the lysate was stored at −70 degrees Celsius until use Total RNA was extracted from cultured HeLa and HaCaT cells and epidermal tissue constructs using Trizol (Invitrogen). The RNA was treated with DNase I (Sigma Aldrich) and reverse transcribed with RevertAid™ H Minus First Strand cDNA Synthesis Kit . Quantitative real-time PCR (qPCR) was run using Platinum SYBR Green qPCR supermix-UGD kit (Invitrogen) as described previously Epidermal tissue contructs were cut into 6 µm sections for protein visualization using primary antibodies against POMP (Abcam), α7 proteasome subunit, β5 proteasome subunit (both BIOMOL),filaggrin  and profilaggrin . The same antibody was used for immunohistochemical detection of profilaggrin on sections from skin biopsies from four healthy controls and three KLICK patients. Sections were fixed in 100% ice cold acetone and endogenous peroxidase activity was blocked by Peroxidazed 1 . After reaction with Background Sniper  and incubation with primary antibodies, sections were incubated with biotinylated secondary antibodies . Thereafter the skin sections were incubated with avidin-biotin complex (Vector Laboratories) or Mach 3 Rabbit HRP polymer Detection Kit  for β5 subunit and peroxidase reactions were developed with DAB (Vector Laboratories). Filaggrin stained sections were counterstained with hematoxylin. Image analysis was performed with Leica DLMB microscope and Leica QWin software.Figure S1Immunohistochemical detection of profilaggrin in human skin. Epidermal sections from an healthy control (A) and patients with KLICK syndrome  were stained with antibodies against profilaggrin (Abcam). (C) Magnification of the cornified cell layer of patient epidermis. Bar: 50 µm.(PDF)Click here for additional data file.Figure S2mRNA analysis of POMP siRNA transfected cell lines.POMP was silenced in HeLa and HaCaT cells by siRNA transfection and cells transfected with mock siRNA and cells without transfection (+/−1 µM MG132) were used for comparison. mRNA levels of POMP, α7, β5 (A–B), BiP, ATF4 and CHOP (C–D) were analyzed by qPCR at 48 h  and 72 h  post transfection. Beta actin was used as internal control. Differences between POMP siRNA and mock siRNA transfected cells were analyzed using Student's t-test; * = p<0.05, *** = p<0.001.(PDF)Click here for additional data file.


It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with  Isogenic cell populations of both prokaryotic and eukaryotic organisms can exhibit a highly dynamic transcriptome, proteome and metabolome. In many cases cellular heterogeneity occurs in response to environmental stresses, functioning as an important mechanism for the survival of cells under adverse conditions Beauveria and Metarhizium are both comprised of anamorph entomopathogenic fungi that infect a wide range of arthropod hosts. Fungal strains for both these genera are frequently isolated from soil and have been the most widely employed as mycoinsecticides for the biological control of insect pests worldwide. In recent years, interest in the nature of the secondary metabolites responsible for their pathogenesis has increased. As a result, toxic cyclic depsipeptides, such as beauverolides in Beauveria sp. Metarhizium sp. Beauveria and Metarhizium are phylogenetically distinct by at least 150 million years The genera ex vivo insect culturing assay. As such, we have grown vegetative mycelium of a Beauveria and Metarhizium isolate in the presence of Camponotus pennsylvanicus ant brains and muscles kept alive in an insect cell culture medium. Liquid chromatography coupled with quadrupole time-of-flight mass spectrometry on the medium after prolonged contact between fungus and ant tissue shows that these two fungal isolates indeed secrete a very different array of metabolites (hereafter referred to as their secretome) on both live and dead ant material. In addition, they each exhibit a significantly different secretome when encountering different insect tissues. Furthermore, using this novel culturing approach, we discovered additional cyclic depsipeptides for both species that have not yet been described. These compounds have been reported to have roles in cellular paralysis and cytotoxic effects, which have been shown to have an antitumor, antibiotic, antifungal, immunosuppressant and anti-inflammatory effect Beauveria isolate secretes its beauverolides mainly on live tissues, while the Metarhizium isolate adopts a strategy of releasing destruxins predominantly on dead tissue.To address this hypothesis, we adopted a global metabolomics approach in combination with a newly developed Metarhizium brunneum and Beauveria bassiana. Their secondary metabolite secretion was tested when vegetative mycelium encountered C. pennsylvanicus tissues. Carpenter ants of the species C. pennsylvanicus are considered a pest in the United States since they preferably nest in dead wood and cause structural damage to many houses every year C. pennsylvanicus workers, something that is commonly being performed in insect physiology and behavioral studies ex vivo tissues stay viable , creating fungal growth conditions that can be used to simulate an ant infection and examine the reaction of entomopathogens to the different tissues it encounters. Tissue viability was checked after a week of incubation using a trypan blue staining (Sigma), which indicated that ant brain and muscle tissue cultured ex vivo were indeed still alive  on which extensive analyses, using both univariate and multivariate statistics, were performed. Principal component analysis (PCA) showed that despite the complex nature of the medium used, there is a distinguishable clustering of the four major groups of samples: medium controls, ant tissue controls, Beauveria growth and Metarhizium growth  and of those 31.5% and 32.2% were unique to either of the fungal isolates  5600 quadrupole time-of-flight mass spectrometer connected to a Shimadzu  UFLC system. Prior to data analysis the raw LC-MS peak data of all 60 samples were aligned together using the parameters described in the material and methods. Subsequently, the dataset was processed to include only monoisotopic ions between 100 and 800 m/z with retention times between 1 and 16 minutes. This resulted in a total of 15000 unique retention time/mass to charge ratio peaks  retention time/mass to charge ratio peaks for growth on live brain and muscle tissue respectively , allowing the identification of this residue. The accurate mass capabilities of the mass spectrometer used in this study easily discriminated between the isobaric 2-hydroxy-4-methylpentanoic and 2-hydroxy-4,5-epoxypentanoic side chains. The presence of prominent b5, b4, a4 b3 and b2 ions allows the sequence determination of these compounds. Depending on the amino acids available in the environment, derivatives of these secondary metabolites are being formed that can have different activities and biological functions within metabolic pathways.For both residues . Thirty-Beauveria to all Metarhizium samples. Using this statistical analysis to look at tissue specificity, none of them appear to be above the set threshold of 0.8. This implies that although these important secondary metabolites are clearly heterogeneously secreted, they are overall not the main heterogeneous compounds driving PCA clustering of the different sample conditions used in this study. Other, yet unidentified compounds seem to be of more biochemical significance. We therefore hope our supplementary data sets will help researchers interested in the secondary metabolites of these two fungi to identify those compounds eventually.Analyzing the metabolite peaks obtained in this study we have identified 20 beauverolides of which 14 have not been described before  and 14 dBeauveria secreted its beauverolides mainly in the presence of live muscle tissue, Metarhizium secreted its destruxins mostly when in contact with dead brain tissue . Colonies were grown in a two-dimensional way (sandwich culture) using two perforated polycarbonate membranes Experiments were done with strain F52 of Camponotus pennsylvanicus worker’s heads using a stereomicroscope (Nikon SMZ645) holding C-W10× eyepieces and a zoom range of 0.5× to 5×. Prior to dissection, whole ants were surface sterilized in 100% ethanol on ice for 5 minutes and subsequently rinsed in PBS, again on ice. After dissection, tissues were briefly surface sterilized (5–10 seconds) in 100% ethanol and rinsed in Schneider’s media (Gibco) freshly supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories Inc.), 100 µg/mL kanamycin (Invitrogen), 100 units/mL penicillin and 100 µg streptomycin/mL (Invitrogen) Ant tissues (brains and muscles) were dissected in PBS from ex vivo ant tissues in the experimental set up was tested using Trypan blue (Sigma). Medium was removed by draining the cell culture insert and placing it in a clean well. Subsequently the tissues were washed three times using 1 ml PBS. After washing, tissues were stained with 1 ml of a 10-fold dilution of 0.4% Trypan blue (Sigma). The stain was removed by draining and the tissues were again washed 3 times in PBS. Subsequently, fresh medium was added and the tissues were examined using a dissecting scope (Nikon). When tissues failed to absorb the stain they were considered to be viable Viability of the In order to measure secreted metabolites, tissues were separated from the medium after incubation by draining the cell culture inserts. The medium was harvested, snap frozen using liquid nitrogen, and stored at −80°C upon processing. Subsequently, 100 µl of the medium was mixed with 1 volume of acetonitrile for protein precipitation prior to LC-MS/MS analysis. Samples were randomized and run using the HPLC-QTOFMS (Shimadzu Prominence UFLC XR and AB Sciex 5600 quadrupole time-of-flight mass spectrometry) platform. Samples (5 ul) were separated on a C18 Column  using a gradient elution program with aqueous acetonitrile (3–90%) at a flow rate of 250 ul/min. Positive ion electrospray ionization mass spectra were acquired over the mass range 50–1250 Da in IDA (Information Dependent Acquisition) mode with one 100 ms survey scan and up to twenty 100 ms MS/MS product ion scans per duty cycle. The survey scan LC-MS datasets were aligned together using MarkerView (AB Sciex) software and the following parameters were used to extract the peaks from the raw data: Retention times between 0.00 and 16.00 min.; Subtraction Offset of 10 scans; Subtraction Mult. Factor of 1.3; Noise Treshold of 50; Min. Spectral Peak Width of 15 ppm; Min. RT Peak Width of 3 scans; Retention Time Tolerance of 0.10 min.; Mass Tolerance of 10.0 ppm; 3 required samples; Maximum of 100000 peaks. Analyses were done looking at the monoisotopic ions with an m/z between 100 and 800 and a retention time between 1 and 16 minutes, and making use of the multivariate analysis software package SIMCA-P+13 (Umetrics).Figure S1Dead/live staining on ex vivo cultured ant tissues. Result of the trypan blue staining on ant brains (A) and ant muscle (B) kept in Schneider’s insect resembling medium, Grace’s insect resembling medium and PBS for 3 days at 28°C. Tissues failing to absorb the stain are considered viable. Tissues appear to be most viable in Schneider’s medium, hence our choice to use this medium to perform our live tissue experiments in.(TIF)Click here for additional data file.Figure S2Loading plots of representative peaks found to be significantly different across sample types. Each panel in this figure shows an example of what the loading plots across sample types of 10 representative peaks found to be significant in the univariate analyses done in this study, looked like. This figure illustrates the striking differences between and the reproducibility within sample types when comparing A) all Beauveria samples to all other samples, B) all Metarhizium samples to all other samples, C) Beauveria grown on live brain tissue versus other types of Beauveria growth, D) Beauveria grown on live muscle tissue versus other types of Beauveria growth, E) Metarhizium grown on live brain tissue versus other types of Metarhizium growth, and F) Metarhizium grown on live muscle tissue versus other types of Metarhizium growth.(TIF)Click here for additional data file.Data File S1All unique retention time/mass to charge ratio peaks that are monoisotopic and found between 100 and 800 m/z with retention times between 1 and 16 minutes.(XLSX)Click here for additional data file.Data File S2(XLSX)Click here for additional data file.Data File S3(XLSX)Click here for additional data file.Data File S4(PDF)Click here for additional data file.


HIV exposed seronegative (HESN) women represent the population most in need of a prophylactic antiviral strategy. Mucosal cationic polypeptides can potentially be regulated for this purpose and we here aimed to determine their endogenous expression and HIV neutralizing activity in genital secretions of women at risk of HIV infection.Cervicovaginal secretions (CVS) of Kenyan women in HIV-serodiscordant relationships  and low-risk controls (n = 72) were assessed for the cationic polypeptides HNP1–3, LL-37 and SLPI by ELISA and for HIV neutralizing activity by a PBMC-based assay using an HIV primary isolate. Median levels of HNP1–3 and LL-37 in CVS were similar across study groups. Neither HSV-2 serostatus, nor presence of bacterial vaginosis, correlated with levels of HNP1–3 or LL-37 in the HESN women. However, an association with their partner's viral load was observed. High viral load  correlated with higher levels of HNP1–3 and LL-37 . SLPI was most abundant in the low-risk group and did not correlate with male partner's viral load in the HESN women. HIV neutralizing activity was found in CVS of all study groups. In experimental studies, selective depletion of cationic polypeptides from CVS rendered the remaining CVS fraction non-neutralizing, whereas the cationic polypeptide fraction retained the activity. Furthermore, recombinant HNP1–3 and LL-37 could induce neutralizing activity when added to CVS lacking intrinsic activity.These findings show that CVS from HESN, low-risk, and HIV seropositive women contain HIV neutralizing activity. Although several innate immune proteins, including HNP1–3 and LL-37, contribute to this activity these molecules can also have inflammatory properties. This balance is influenced by hormonal and environmental factors and in the present HIV serodiscordant couple cohort study we show that a partner's viral load is associated with levels of such molecules. Mucosal surfaces of the female genital tract are portals for sexual transmission of microbes and viruses, and cationic antimicrobial polypeptides are the principal effector molecules of mucosal innate immunity. Together with other innate immune cells and molecules, as well as physical and chemical barriers , these antimicrobials normally contribute to an efficient barrier against low dose HIV exposure during sexual intercourse with an HIV infected partner in vitro and are present at detectable levels in CVS , LL-37 and secretory leukocyte protease inhibitor (SLPI) have been shown to possess HIV inhibitory activity Each of these synergistically acting molecules can have multifaceted roles influenced by the immunologic, hormonal, and microbial status of the genital mucosal surface of sexually active individuals. In a previous study, we showed that cationic polypeptides were required for anti-HIV activity of human vaginal fluid from healthy women at low risk of sexual HIV exposure During the years 2007–2010, HIV serodiscordant (n = 469) couples were enrolled in a prospective cohort study in Nairobi, Kenya and followed for up to 2 years with visits every 3 months in vitro experiments.For the present study, CVS were collected from these women at the time of study enrollment and a questionnaire was administered by a nurse-counselor that included sociodemographic data and sexual/reproductive history. A physical examination, including a genital examination, was performed. CVS were collected by rotating a cotton swab 360° in the outer part of the endocervix and by rotating a different swab across the vaginal wall. Both swabs were placed in the same tube containing 5 ml sterile phosphate buffered saline (PBS), which was immediately placed on ice. The samples were then transported from the clinic to the laboratory on ice and centrifuged at 800 g for 10 minutes at 4°C. Supernatants were separated from the cell pellet, aliquoted into 4 cryovials, each containing 1 ml fluid, and stored at −80°C. CVS from healthy Swedish low-risk HIV seronegative women were collected and stored according to the same procedure and were used as internal controls for the For cationic polypeptide depletion experiments, a more concentrated CVS sample was required. HIV seronegative women, without any clinical or laboratory signs of STIs  or BV, were recruited from the Maternal and Child Health Clinic in Pumwani, Nairobi. One cotton swab soaked in mucus from the cervico-vaginal tract was placed in 0.5 ml of saline and placed on ice. The vials were transported to the laboratory where the samples were stored in −80°C.® HIV–1/2 Rapid Test, Abbott Laboratories, Abbott Park, IL; Bioline™ Recombigen HIV Test, Standard Diagnostic Inc.). Positive or indeterminate results were confirmed with HIV-1 enzyme-linked immunosorbent assay (ELISA) using the Vironostika® HIV Uni-Form II Ag/Ab ELISA kit  in the University of Nairobi's Obstetrics/Gynecology laboratory. Plasma HIV-1 RNA viral load in HIV-seropositive partners was quantified using the Gen-Probe Transcription Mediated Amplification (TMA) assay HIV rapid testing was conducted in the Study Clinic using two commercial kits (DetermineProstate-specific antigen (PSA) levels in CVS were measured by an external accredited laboratory  using the ARCHITECT Total PSA assay , a chemiluminescent microparticle immunoassay. The PSA levels in the CVS are reported as measured in the original 5 ml dilution. A level above 1 µg PSA/mL CVS were defined as a positive result Commercial ELISA kits were used, according to the manufacturers protocols, to quantify the immune molecules of interest at the following dilutions: the α-defensins HNP-1–3 (1∶500), LL-37 (1∶10) , and SLPI (1∶100-1∶1000) . The CVS levels of the factors are reported as measured in the original 5 ml dilution.50 depending on PBMC donor variability, three viral dilutions were used in each assay. Duplicate wells of 75 µl of each virus dilution and 75 µl of each sample fraction (undiluted) were incubated for 1 h at 37°C followed by addition of a mixture of 1×105 PHA-P-stimulated PBMC from two donors. After 24 h incubation at 37°C, the cells were centrifuged; unbound virus was washed away, and 200 µl of fresh medium were added to each well. On day 3, 120 µl of medium was discarded and replaced with new medium, after which day 6 supernatants were collected for analysis of virus production with a p24 antigen ELISA . Neutralization was defined as a ≥67% reduction of p24 antigen in the supernatant as compared with p24 antigen content when the virus isolate was incubated in the presence of a standard pool of Swedish HIV seronegative samples.HIV neutralization assays were performed according to a predefined protocol and neutralization cut off Carboxymethyl (CM) weak cation exchange resin (Bio-Rad) was used to deplete cationic polypeptides from vaginal fluid Recombinant SLPI , LL37 and HNP1–3  were added in serial dilutions to IgA-depleted CVS samples from low-risk HIV seronegative women who were selected based on lack of intrinsic neutralizing capacity. The recombinant peptides were incubated with the CVS samples for 1 hr. Following this pre-incubation, the samples were tested in the HIV neutralization assay as described above except for the use of another HIV isolate of the same HIV subtype .Statistical comparisons of the immune parameters between groups were performed by using the Mann-Whitney U test, and calculations were performed by using the GraphPad Prism 5 software  and STATA version 11.2/IC . A p-value of <0.05 was considered statistically significant.Owing to lack of sufficient volume of some CVS samples, not all 300 samples were assessed for all parameters in the following sections. However, the number of samples assessed in each case is presented in the table and figure legends.Because a partner's viral load of 10,000 or greater has been shown as an epidemiologic determinant for higher transmission risk in this geographical setting In the present study, CVS samples were assessed from a total of 296 study participants: HIV seropositive  and HIV seronegative  women representing the serodiscordant couple cohort as well as HIV seronegative  women living with a seroconcordant HIV-negative male partner. Demographic data and sexual risk-taking profiles of the study subjects are outlined in The most abundant peptide among the ones investigated  in all three study groups was HNP1–3, with comparable median levels for the HIV-positive, HESN and low-risk groups  . Also, lWe examined several mucosal factors that could potentially influence the levels of cationic polypeptides. First, in the HESN group, partner's viral load  was compared to levels of the individual peptides . HESN woWe next assessed the association of HSV-2 serostatus and presence of BV with levels of the peptides in all study groups. HIV positive women who were co-infected with HSV-2 had significantly higher levels of LL-37 than those who were HSV-2 seronegative . Among the HESNs, lower levels of SLPI were observed for the HSV-2 seropositive as compared with the HSV-2 seronegative women  . For HESTo evaluate the influence of seminal contamination in the CVS, samples were assessed for presence of PSA (>1 µg/ml) and then compared for levels of cationic polypeptides. PSA was found in 33% of the CVS samples representing the low-risk group followed by 12% in the HIV positive group and 10% in the HESN group . AssessiAs previously shown Samples from the study groups were then divided based on the presence/absence of neutralization capacity and within each study group and comparison was made between neutralizing and non-neutralizing samples in terms of their levels of each peptide . No statIn this set of experiments, we explored the HIV-neutralizing activity of the cationic polypeptide components of CVS from HIV seronegative women. A total of 14 CVS samples were selected for the presence of intrinsic HIV neutralizing activity and pooled into three groups to reach sufficient volume for the following experiments . First, Next we studied whether adding individual recombinant cationic polypeptides to CVS could induce HIV neutralizing activity. Theoretically, the added peptides would act synergistically with other innate immune factors naturally present in CVS. For these experiments, CVS from HIV seronegative women lacking intrinsic HIV neutralizing activity were selected and the HIV neutralizing assay was adapted to establish the ratio between peptide+CVS versus medium+CVS. SLPI as well as recombinant HNP1–3 and LL-37 were evaluated in three independent experiments. As a result, both HNP1–3 and LL-37 induced a two to six-fold increase of HIV inhibiting activity when assessed at about 10–50 times the physiological concentrations, whereas the effect of SLPI was only marginal . At physin vitro, and because they are detectable in cervicovaginal lavage from healthy women (reviewed in in vitro by selectively depleting the cationic polypeptides from CVS, which we demonstrated significantly reduced the functional activity. In another set of experiments, human CVS lacking intrinsic HIV neutralizing activity gained this function following addition of recombinant HNP1–3 or LL-37, but only marginally from SLPI.We document here the presence of cationic polypeptides and intrinsic HIV neutralizing activity in CVS of Kenyan women who are HIV infected, HESN, and HIV-uninfected low risk. The three cationic polypeptides HNP1–3, LL-37 and SLPI were selected for assessment because they were previously shown to be important mediators of HIV neutralization Detailed demographic data were collected from the study participants. Despite being thoroughly counseled on sexual risk behavior, signs of recent unprotected sexual intercourse as assessed by measurement of PSA were documented in 10% of the HESN women and in 12% of the HIV seropositive women. The corresponding figure in the low risk women was 33%. Furthermore, none of the HIV infected partners of the HESN women were on ARV treatment at enrollment; about two-thirds of the partners had a plasma viral load above 10,000 HIV RNA copies per ml, a threshold shown to correlate with a high risk of HIV transmission in this setting Levels of the cationic polypeptides were also related to study group, HSV-2 serostatus and presence of BV. Levels of both HNP1–3 and LL-37 were similar when compared between study groups, whereas SLPI levels were lowest in the HIV positive group and highest in the low risk group. SLPI can be degraded by endogenous proteases and by proteolytic activity of pathogenic agents and, although not assessed here, HIV positive individuals differ in many aspects with regard to the genital mucosal environment that could account for the findings. HIV infected individuals who were HSV-2 seropositive had higher levels of LL-37 than their HSV-2-seronegative counterparts within the same study group. In HESNs, levels of SLPI were lower for HSV-2 seropositive than HSV-2 seronegative individuals. Active HSV-2 infection and bacterial STIs have previously been shown to affect levels of cationic polypeptides, including increased levels of LL-37 and HNP1–3 in Kenyan high risk women HIV inhibitory activities of mucosal fluids of low-risk HIV-seronegative individuals have been reported and assigned to a number of different innate molecules in addition to the ones assessed in the present study in vitro they have been associated with an increased rate of HIV acquisition in a cohort of high-risk sex workers Additional interactions and synergistic effects of the cationic polypeptides may occur at the mucosal tissue level if the virus penetrates beyond the cervical secretion. Indeed, molecules that perform antimicrobial activity in human secretions may have a counteractive effect in the genital mucosa and contribute to inflammation and recruitment of immune cells that in turn could be infected Several important confounders must be considered in the context of HESN individuals and assessment of mucosal immune responses. Direct effects of sexual intercourse and secondary immune effects of inflammation were here partly controlled for by assessment of PSA and excluding subjects with ongoing STIs. However, hormonal factors and other inflammatory conditions influence the local mucosal environment Considerable attention has been directed to developing microbicides for topical use to prevent sexual HIV transmission, with promising results from recent clinical trials


The development of second generation sequencing technology has resulted in the rapid production of large volumes of sequence data for relatively little cost, thereby substantially increasing the quantity of data available for phylogenetic studies. Despite these technological advances, assembling longer sequences, such as that of entire mitochondrial genomes, has not been straightforward. Existing studies have been limited to using only incomplete or nominally intra-specific datasets resulting in a bottleneck between mitogenome amplification and downstream high-throughput sequencing. Here we assess the effectiveness of a wide range of targeted long-range PCR strategies, encapsulating single and dual fragment primer design approaches to provide full mitogenomic coverage within the Araneae (Spiders). Despite extensive rounds of optimisation, full mitochondrial genome PCR amplifications were stochastic in most taxa, although 454 Roche sequencing confirmed the successful amplification of 10 mitochondrial genomes out of the 33 trialled species. The low success rates of amplification using long-Range PCR highlights the difficulties in consistently obtaining genomic amplifications using currently available DNA polymerases optimised for large genomic amplifications and suggests that there may be opportunities for the use of alternative amplification methods. Mitochondrial DNA markers are the cornerstones of contemporary molecular systematics and contribute greatly to our understanding of organellar evolution de novo construction of phylogenetic hypotheses for taxa without sequenced transcriptomes As more sequence data becomes available for elucidating the tree of life, large-scale sequencing efforts and interrogation of expressed sequence tag (EST) libraries or sequenced transcriptomes The number of informative phylogenetic characters within the mitochondrial genome has been appreciated for some time Whilst the potential benefits of analysing whole mitochondrial genomes  in furthering our understanding of the mechanisms behind the evolution of organellar DNA are clear, all of the data produced to date have been of comparatively low volume. The lack of empirical data means that we still cannot accurately assess the utility of full mitogenomic sequences as a tool for resolving complex phylogenies in many taxa Taq polymerases. The PCR approach was chosen as it circumvents the need for (often unavailable) large starting biomass associated with the direct pelleting of mitochondria. Whilst mitochondrial genomes can be readily amplified in large numbers of small fragments, this is both costly and labour intensive. To this end, we adopted both single and dual fragment amplification approaches across the phylogenetic breadth of the order to assess the feasibility of both conserved and directed approaches for expedient whole mitochondrial DNA amplification.The Araneae are among the oldest and most diverse group of terrestrial organisms Selenops annulatus, Deinopis sp. and Cithaeron praedonius had been stored in 70% ethanol and were incorporated in order to maximise taxonomic coverage across the order. No specific permits were required for the described field studies and no specific permissions were required for these locations or activities. No locations were privately owned or protected and the field studies did not involve protected or endangered species. All four legs were removed from the left side of the thorax prior to DNA extraction. The rest of the body was stored in 100% ethanol for vouchering and subsequent identification purposes. Whole genomic DNA was extracted from a single femur of each species using the Qiagen DNeasy Blood & Tissue Kits (Qiagen).Spiders were obtained from across the United Kingdom and Gambia by the authors and members of the British Arachnological Society (BAS) and either ethanol preserved  or freshly frozen (stored at −80°C) directly from living individuals . Sampless rRNA), were amplified for 33 taxa. Both amplifications were performed in 25 µl reactions using the primer combinations of CHELF1 (5′- TACTCTACTAATCATAAAGACATTGG) and CHELR2 (5′-GGATGGCCAAAAAATCAAAATAAATG) (COI) 5′- CGCCTGTTTAACAAAAACAT) and LR-J-12887 (5′- CCGGTCTGAACTCAGATCACGT) (16s) 2, 0.4 µM of each primer, 0.625 units ThermoPrime Taq DNA polymerase (Thermo Scientific) and 1 µl template DNA and thermocycling was performed using a DNA engine (Tetrad 2) Peltier Thermal Cycler (BIORAD). Cycling conditions were 60 seconds at 94°C, 5 cycles of 60 seconds at 94°C, 90 seconds at 45°C and 90 seconds at 72°C followed by 35 cycles of 60 seconds at 94°C, 90 seconds at 50°C and 90 seconds at 72°C U shrimp alkaline phosphatase (Promega) to dephosphorylate residual deoxynucleotides and 0.5 U Exonuclease I (Promega) to degrade excess primers In order to identify an efficient approach for the amplification of whole mitochondrial genomes we adopted two long-range PCR strategies to amplify the complete mitogenome in one or two large fragments s anchoring regions, chromatographs were checked and sequences were manually edited where necessary, using CodonCode Aligner , prior to alignment using Clustal W s ribosomal subunit using the Primer 3 software bp), primer Tm (57.0–70.0°C) and GC content (40–60%) which followed consensus recommendations from the Taq manufacturers (Primer sequences available on DRYAD entry doi:10.5061/dryad.8dd3n). For the single fragment protocol, primers were designed within the COI sequences, with the light strand primer situated downstream of the heavy strand primer, thus taking advantage of the circular nature of the genome. For the dual fragment protocols, two sets of primers were designed for each taxon, to bridge the gaps between the COI and 16s regions , NEB LongAmp (NEB), GeneAmp XL (Applied Biosystems) & Expand Long Range PCR (Roche), using the protocols and cycling conditions recommended by the manufacturers. All DNA polymerases were initially tested on five phylogenetically disparate samples prior to using the most successful ones on the remaining samples. Successfully amplified PCR products were electrophoresed and subsequently purified using Qiagen QiaQuick Gel extraction kits (Qiagen) or Origene Rapid PCR Purification System (Origene), dependent on fragment size. Following long-range PCR amplification, primers were assigned to groups based on amplification success  and subsequently tested for significant differences in primer length, GC content and TMeta menardi, Xysticus audax, Psalmopoeus cambridgei, a Gorgyrella sp., Eupalaestrus campestratus, a Linyphiidae sp., Zelotes apricorum, Malthonica silvestris, Araneus diadematus, Pisaura mirabilis & Dysdera erythrina) were fragmented using a Covaris DNA shearer (Covaris) at 10% duty cycle, intensity: 4 with 200 cycles per burst for 65 seconds. Following quantification using a Qubit 2.0 fluorometer (Invitrogen), MID barcoded adapted sequencing libraries were constructed using the NEBNext DNA sample prep master mix set in accordance with manufacturer’s instructions (New England Biolabs), pooled in equimolar concentrations and sequenced at low putative coverage on 1/16th of a 454 Roche GSFLX Titanium platform . Please note that the sequencing step here was intended as a quality control measure and not intended to sequence to a depth required for full mitochondrial genome assembly . The 454 Roche sequences were assembled using GS De Novo Assembler software (Roche). Following trials with Roche’s GS De Novo software, MIRA (B. Chevreux) and CLC Genomics Workbench , no substantial differences in number of contigs or length were present using any of the approaches. Therefore, in accordance with the proven functionality of the 454 Roche software, all sequences were assembled using the GS De Novo program. Following assembly, the resulting contigs where compared to existing Araneae mitochondrial DNA sequences within GenBank via BLAST Amplicons from eleven purified samples .Of the 33 species used, 27 and 24 taxa were successfully amplified for the COI and the 16families   or NEB LongAmp  Taq polymerases. The probability of obtaining successful amplifications of complete mitochondrial genomes utilising either the 1 or 2 fragment amplification protocol was not demonstrably different (based upon presence/absence of bands), suggesting that the limiting factor in the amplification of the genomes was not necessarily the size of the target fragment. In some two-fragment amplifications  the COI to 16s region amplified consistently more often than the reverse, highlighting potential inhibition to amplification within the target sequence. However, of the initial 33 samples trialled, from which a subset of 22 (based on sequence availability and prior PCR amplification success) were used for long-range PCR, only 11 mitochondrial genomes could be amplified robustly and with a guaranteed level of reproducibility . The NEB LongAmp polymerase (NEB) amplified genomes in two fragments with greater reproducibility whilst Clontech Advantage 2 (Takara Biotech) amplified single fragment genomes with greater consistency. Statistical analysis of the primer properties  yielded no significant differences between any combination of primers that amplified repeatedly, intermittently, or those that consistently failed to amplify mitochondrial genome fragments.Initially, five specimens, covering a broad taxon range, were used to test the performance of the fication . FollowiMalthonica silvestris) were unable to be recovered by the MID identifying software. De novo read assembly was unable to construct complete mitochondrial genomes but provided an average of an estimated 65% sequence assembly .The sequencing of the 11 amplicon libraries provided a total of 27,892 tagged reads with an average length of approximately 278 bases . Reads fIn light of the likelihood of tRNA gene rearrangements and because only 6 mitochondrial genomes were available on Genbank at the time of analysis, a highly dissimilar BLAST search was performed on the nucleotide collection database. Of the 10 samples, all contigs created through short-read assembly had maximum identification values ranging from 78–100% Max ID for spider mitochondrial DNA, either through an unrestricted blast search or with searches focused on Araneae accessions. Nucleotide sequences for all reads were deposited in the GenBank short-read archive (SRA051390.1).s) across our taxon range, but were unable to identify or develop degenerate primers for any other potential anchoring region within the spider mitochondrial genome following sliding window analyses of all regions most commonly used in spider phylogenetics. Sliding window analysis was performed on all 13 protein coding genes and both ribosomal RNAs (rRNAs) of the six previously published spider mitochondrial genomes using the Drosophila Polymorphism database, SNP Graphics (http://dpdb.uab.es/dpdb/diversity.asp) (sliding window plots available on DRYAD entry doi:10.5061/dryad.8dd3n). This most likely highlights the high level of mitochondrial genetic diversity that can be found within the spiders along with the absence of universal primers available for no more than a handful of protein coding genes, resulting in few reference sequences available for comparison for large portions of the Araneae mitogenome We were able to amplify most of the target genes . The arachnid mitochondrial genome, as for most animal mitochondrial genomes, is a small extra-chromosomal genome comprising 37 genes including 22 coding for transfer RNAs (tRNAs), 13 coding for proteins and 2 coding for ribosomal RNAs (rRNAs) s) of the dual fragment approach amplified successfully more often than the shorter 16s-COI counterpart, the G/C content of both fragments is comparable  with no significant differences between the highest and lowest values of the comprising genes within each fragment. However the lesser-amplified small fragment (16s-COI) does contain the control region, also known as the A+T rich region due to its low G/C content, which has been known to inhibit DNA polymerases in insects and platyhelminths Nevertheless, even following an extensive campaign of PCR strategies and optimization, using a variety of long range s regions, using a variety of commercially available DNA polymerases optimised for large genomic amplifications. However, we have also highlighted the difficulty in obtaining amplifications with a guaranteed level of reproducibility, using multiple currently available polymerases and thus highlighted potential limitations to the feasibility of mitogenomic studies featuring large numbers of independently amplified taxa using single, or dual fragment approaches. Recent studies utilising mitogenomics to resolve phylogenetic incongruences have avoided this issue by focusing on the production of datasets comprising either incomplete genome sequences Our results provide evidence for the successful amplification of several whole mitochondrial genomes, in one or two long fragments overlapping the COI and/or 16, JQ406614–JQ406637 and SRA051390.1.DNA sequences: Genbank accessions JQ412439 - JQ412463Primer sequences, Contigs & Sliding window analysis: DRYAD entry doi:10.5061/dryad.8dd3n.


Poxviridae.A wildlife hospital and rehabilitation center in northwestern United States received several big brown bats with necrosuppurative osteomyelitis in multiple joints. Wing and joint tissues were positive by PCR for poxvirus. Thin-section electron microscopy showed poxvirus particles within A-type inclusions. Phylogenetic comparison supports establishment of a new genus of  Bats have several physiologic, cellular, and natural history characteristics that may make them particularly suited to their role as reservoir hosts (Chordopoxviridae is a subfamily of Poxviridae that contains large double-stranded DNA viruses that replicate in the cellular cytoplasm and are known to infect a wide range of vertebrates. Many of these viruses cause zoonotic disease in humans. Although poxviruses are known to have incorporated host genes into their genomes to subvert the host immune system (–Eptesicus fuscus) were brought to a wildlife hospital and rehabilitation center  during late spring or summer because they could not fly. All but 1 of the bats had >1 visibly swollen and occasionally contused joints involving the long bones of the legs and wings; 1 had contusions of the oral commissures. During 2009–2011, six  adult big brown bats  for further investigation. Histologic examination showed severe fibrino-suppurative and necrotizing tenosynovitis and osteoarthritis that involved the long bones and occasionally facial flat bones and joints with occasional localized vasculitis. No bacterial or fungal agents were seen by light microscopy of specimens stained with hematoxylin and eosin, Giemsa, Warthin-Starry, Brown and Brenn, or Gomori methenamine silver stains or in a Wright-Giemsa–stained cytologic preparation of a joint aspirate. Cultures for aerobic and anaerobic bacteria, and cultures of the joint from 1 bat for mycoplasma showed negative results.Thin-section electron microscopy of synovial tissue extracted from a wax histoblock showed poxvirus particles in inflammatory cells , panel Al-glutamine, and penicillin/streptomycin. Growth medium from a T25 flask containing green monkey kidney epithelial cells (BSC40) was removed and the virus mixture added. The flask was incubated for 1 hour at 56°C, after which 6 mL of RPMI 1640 medium was added to each flask. Cells and supernatant were harvested after 95% of the monolayer was infected. Material was taken from the wing and joint of an affected bat for real-time PCR testing and cell culture isolation. Wing and joint material was positive by real-time PCR for a poxvirus with low genomic G + C content  was briefly applied to the grid and blotted. The grid contents were visualized by using a Tecnai BioTwin electron microscope  operating at 120 kV. Digital images were captured by using a 2K × 2K camera (Advanced Microscopy Techniques). Poxvirus particles were identified in cell culture supernatant , panel Bwww.illumina.com/technology/sequencing_technology.ilmn). DNA sequences from 7 open reading frames  were extracted on the basis of sequence similarity. Data were deposited in GenBank under accession nos. KC181855–KC181861. Open reading frames were translated into amino acid sequences and aligned by using the ClustalW alignment option in Geneious version 6.0.5 (www.geneious.com/). The tree search was conducted by using MrBayes in Geneious 6.0.5 under default settings and Amsacta moorei (red hairy caterpillar) entomopoxvirus (Moyer) was used as the outgroup with a burn in of 10%.Genome sequencing produced data that were used to construct a phylogenetic tree . Virus D,Historically, osteomyelitis with arthritis has been reported in smallpox patients (osteomyelitis variolosa) and in smallpox vaccine recipients (vaccinia osteomyelitis), but did not occur frequently (–Chordopoxvirus; its nearest neighbor is Cotia virus, an as yet unclassified chordopoxvirus first isolated from sentinel suckling mice in a state reserve in Cotia County, São Paulo State, Brazil, in 1961 (Results of at least 3 investigations that involved detection of viral DNA detection in bat guano have been published; 2 involved bats from North America and 1 involved bats in China (Chiropoxvirus. Further efforts should be undertaken to determine whether Cotia virus should be included in this genus. The bat-derived virus requires a new species designation for which we propose Eptesipox virus because of its isolation from an Eptesicus fuscus bat specimen.Although the 2 viruses are nearest neighbors, levels of shared nucleotide and amino acid identity between them suggest they should likely be considered separate genera (


We show that Cranial Neural Crest (CNC)-derived cartilage precursors migrate correctly into the pharyngeal arches, but fail to differentiate into chondrocytes. This phenotype is specific for cartilage precursors, since other neural crest-derived cell lineages, including glia, neurons and chromatophores, are formed normally in ring1b mutants. Our results therefore reveal a critical and specific role for Ring1b in promoting the differentiation of cranial neural crest cells into chondrocytes. The molecular mechanisms underlying the pathogenesis of craniofacial abnormalities, which are among the most common genetic birth defects in humans, remain poorly understood. The zebrafish ring1b mutant provides a molecular model for investigating these mechanisms and may lead to the discovery of new treatments or preventions of craniofacial abnormalities.Polycomb group (PcG) genes are chromatin modifiers that mediate epigenetic silencing of target genes. PcG-mediated epigenetic silencing is implicated in embryonic development, stem cell plasticity, cell fate maintenance, cellular differentiation and cancer. However, analysis of the roles of PcG proteins in maintaining differentiation programs during vertebrate embryogenesis has been hampered due to the early embryonic lethality of several PcG knock-outs in the mouse. Here, we show that zebrafish Ring1b/Rnf2, the single E3 ubiquitin ligase in the Polycomb Repressive Complex 1, critically regulates the developmental program of craniofacial cell lineages. Zebrafish  Craniofacial abnormalities are amongst the most common genetic birth defects and result from defects in cranial neural crest patterning and morphogenesis. However, the molecular mechanisms regulating cranial neural crest differentiation are not completely understood. Most of the cartilage and bones of the skull in vertebrate embryos are formed by cranial neural crest cells (CNCs), a subpopulation of the neural crest (NC), a vertebrate-specific, ectoderm-derived, multipotent cell population that is induced at the beginning of neurulation at the border between epidermal and neural ectoderm During cartilage development, the most anteriorly-located CNC-derived cartilage precursors will mainly form skeletal elements that encase the brain (neurocranium), whereas more posteriorly-located NC cells give rise to the ventral viscerocranium. Viscerocranial precursors migrate ventrally as three streams to populate the pharyngeal arches, embryonic structures that will form several tissues of the head-neck region in higher vertebrates. The mandibular and hyoid arches form the jaw and its supportive elements, respectively, whereas the five posterior  arches give rise to the cartilaginous elements of the gills.Cranial bones are also derived from CNC cells and ossification occurs through two distinct processes: endochondral and dermal ossification. In endochondral ossification, a skeletal template comprising of replacement cartilage is formed first, which is then replaced by bone. In dermal ossification, bones form directly from osteogenic condensations. Both processes are essential for cranial bone development.Skeletogenesis is a complex process guided by specific genetic and epigenetic programs. The Polycomb group (PcG) proteins are transcriptional repressors that mediate epigenetic silencing of target genes and thereby regulate differentiation programs. PcG proteins are key regulators of cell proliferation, cell-fate maintenance during embryonic development; and in adults, they are implicated in tissue homeostasis, cellular differentiation and cancer ring1b knock-outs The study of PcG proteins in maintaining differentiation programs during vertebrate embryonic development is hampered by the early lethality of mice defective for the core proteins of PRC2 and PRC1 (Ring1b). We have recently generated Ring1b-deficient zebrafish that survive early development -mediated targeted gene inactivation ring1b mutants The ring1b mutants were first observed at around 40 hours post fertilization (hpf), and consisted of a reduction in the amount of tissue at the level of the anterior pharyngeal arches. In contrast to WT embryos at 72 hpf  as well as in a single domain in which the vagal nerve (gX) and the posteriolateral line ganglia (gP) are located , iridophores (iridescent) and xanthophores (yellow). While melanophores are found in a wide variety of vertebrates, iridophores and xanthophores are not iblings.  During liblings. . In cont spindly . It has  mutants . Yellow  mutants . In addi mutants  although mutants .ring1b mutants. Thus, the arrest in cartilage formation appears to be a specific developmental defect in ring1b CNC cells.Together, these results allow us to conclude that migration and differentiation of non-ectomesenchymal NC derivatives is only modestly affected by the loss of Ring1b. Glia, neurons and the three chromatophore lineages were formed in ring1b mutants, we investigated whether CNC cells that will form the prospective viscerocranium migrated correctly into the pharyngeal arches.Because craniofacial cartilage development was impaired in dlx2a that is expressed in CNC cells and hand2, whose expression is confined to a ventral subset of dlx2a-positive cells in post-migratory CNC cells dlx2a-expressing CNC cells had migrated into the pharyngeal arches in both WT and ring1b embryos , hyoid arch (2) and the posterior branchial arches (3–7) .We next investigated whether differentiation of post-migratory CNC cells into chondrocytes was impaired. We stained WT and es (3–7) ’, B, B’.l arches ’, albeitextended ’. Sox9b  missing ’. Notablsox10 and dlx2a that remained expressed in post-migratory CNC cells. Both genes were expressed in the two anterior pharyngeal arches of ring1b mutants, whereas trabecular expression was completely absent , the major collagen in cartilage and marker of differentiating chondrocytes in zebrafish Col2a1 expression is controlled by sox9a/bcol2a1 was strongly expressed in the trabeculae cranii, otic vesicle and to a lesser extent in the mandibular and hyoid arch , an extracellular matrix component known to be directly regulated by runx2Col10a1 expression is specific to developing dermal bones at 72 hpf. Indeed, strong staining was observed in the cleithrum, operculum and parasphenoid of WT embryos  Craniofacial birth abnormalities result from defects in cranial neural crest patterning and morphogenesis. A better understanding of the molecular mechanisms that regulate chondrogenesis will facilitate development of therapies and/or prevention. The rapid development of the jaw cartilages in the zebrafish, its transparency and amenability to genetic manipulations makes it an excellent model for studying the genetic basis of craniofacial development. In addition, zebrafish mutants isolated in forward and reverse genetic screens have enabled the identification of critical genes involved in cartilage development ring1b cartilage phenotype is quite rare as only few mutants showing a total lack of pharyngeal elements and neurocranium have been identified. Remarkably, the ring1b mutant is the only one among those with cartilage defects that is missing the cleithrum, the pectoral girdle element that is not of Neural Crest origin. Not surprisingly, the sox9a/sox9b double mutant displayed severe craniofacial abnormalities. Zebrafish mutants in Trap230/Med12, a transcriptional co-activator for Sox9, were characterized by disrupted cartilage development runx2b and runx3 are critically involved in chondrogenesis: runx2b morphants lack the entire pharyngeal skeleton whereas only rudimentary trabeculae are formed in runx3 morphants The dramatic lim-absent (lia)/fgf3 mutant, posterior arch formation is abrogated Finally, in the ring1b mutants. We demonstrated previously that the Fgf-target genes dusp6, pea3 and spry4 were expressed at near normal levels in the pharyngeal arches of 32 hpf ring1b mutants Our data indicate that Ring1b acts genetically upstream of Sox9 and Runx2, as their expression in (pre)chondrocytes is markedly decreased in Chondrogenesis involves a cascade of events that requires the concerted action of an intricate gene regulatory network. Although recent studies have provided insight into the molecular mechanisms underlying chondrogenesis, much remains unknown. For instance, the role of (epigenetic) chromatin modifications such as chromatin remodeling, DNA (de) methylation and histone modifications has largely been overlooked. Our results illustrate the critical role of epigenetic control in cartilage differentiation and highlight for the first time an essential and specific role for Polycomb genes in craniofacial development.ring1b cartilage phenotype affects specific groups of cells and at a specific developmental point, namely the late stages of differentiation of the cranial neural crest cells into cartilage and muscle of the head. Our results indicate that epigenetic regulation of transcription in specific cell populations is very important for late differentiation events.Despite the numerous molecular targets of Polycomb-mediated repression, the ring1b mutants consists of a block in terminal differentiation of chondrocytes. We propose that this is the result of deregulated expression of developmental programs rather than individual genes. This is in line with the proposed role of Ring1b to control differentiation programs in ES cells and of the Polycomb complex to regulate programs rather than individual genes.The cartilage phenotype in ring1b mutants, they provide an attractive platform to interrogate the function of (human) candidate genes for skeletogenesis, dissect possible genetic interactions, as well as screening of compounds to identify regulators of cartilage development.Since no cartilage is formed in Danio rerio) were maintained as previously described Ring1b founder fish were out-crossed to AB and TL genetic backgrounds and genotyped as described ring1b/rnf2 that led to the insertion of 4 base pairs (bp) within the ZFN target site was named ibl30/ibl30rnf2  and the mutation that caused deletion of 14 bp at the ZFN target site was named ibl31/ibl31rnf2  (www.zfin.org).Zebrafish . The posterior hypaxial muscle (phm), had delaminated from the somites in both WT and ring1b mutants at 48 hpf . At 56 hpf, the sternohyoideus (sh) is formed in both WT and ring1b mutants and the pectoral fin muscle is prominently visible . During later development, the sh and phm elongated and attached to the cleithrum, a bone of the fin girdle in WT embryos, . Elongation of these muscles was completely abrogated in ring1b mutants . Cranial musculature is almost completely absent in ring1b mutants (H). Abbreviations: am: anterior mandibularis; ah: adductor hyomandibulae; ao: adductor opercule; do: dilator operculi; hh: hyohyoideus; ih: interhyoideus; ima: intermandibularis anterioris; imp: intermandibularis posterioris, lap: levator arcus palatine; phm: posterior hypaxial muscle; pfm: pectoral fin muscle; sh: sternohyoideus.(TIF)Click here for additional data file.Figure S2Persistence of apoptotic cells in ring1b mutants. Ventral views of WT and ring1b embryos at 48 hpf stained with Acridine Orange. No apoptotic cells are detected in the pharyngeal arch region of WT embryos (A) whereas few AO-positive apoptotic cells have persisted in the ring1b mutants (B) Arrows in (B) indicate apoptotic clusters in the prospective jaw region and anteriorly to the otic vesicle. Figure also shows images of live WT and ring1b embryos depicting the fin and pharyngeal cartilages in WT  and their absence in the ring1b mutants .(TIF)Click here for additional data file.Methods S1(DOCX)Click here for additional data file.