Chronic stress can affect skin function, and some skin diseases might be triggered or aggravated by stress. Stress can activate the central hypothalamic–pituitary–adrenocortical (HPA) axis, which causes glucocorticoid levels to increase. The skin has HPA axis elements that react to environmental stressors to regulate skin functions, such as melanogenesis. This study explores the mechanism whereby chronic stress affects skin pigmentation, focusing on the HPA axis, and investigates the role of glucocorticoids in this pathway. We exposed C57BL/6 male mice to two types of chronic stress, chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS). Mice subjected to either stress condition showed reduced melanogenesis. Interestingly, CRS and CUMS triggered reductions in the mRNA expression levels of key factors involved in the HPA axis in the skin. In mice administered corticosterone, decreased melanin synthesis and reduced expression of HPA axis elements were observed. The reduced expression of HPA axis elements and melanogenesis in the skin of stressed mice were reversed by RU486 (a glucocorticoid receptor antagonist) treatment. Glucocorticoids had no significant inhibitory effect on melanogenesis in vitro. These results suggest that, high levels of serum corticosterone induced by chronic stress can reduce the expression of elements of the skin HPA axis by glucocorticoid-dependent negative feedback. These activities can eventually result in decreased skin pigmentation. Our findings raise the possibility that chronic stress could be a risk factor for depigmentation by disrupting the cutaneous HPA axis and should prompt dermatologists to exercise more caution when using glucocorticoids for treatment. Substantial evidence suggests that chronic stress can affect the function of multiple physiological systems, including skin function During exposure to stressful events, the central hypothalamic–pituitary–adrenocortical (HPA) axis is activated. In brief, during stress, corticotropin-releasing hormone (CRH) is synthesized and released, which increases pro-opiomelanocortin (POMC) expression. POMC is converted into adrenocorticotropic hormone (ACTH) and other melanocortin peptides, such as α-MSH. ACTH then binds to the melanocortin type 2 receptor (MC2R) of the adrenal cortex and stimulates glucocorticoid synthesis and secretion into systemic circulation to exert various physiological effects. In the adrenal cortex, P450scc (Gene symbol: CYP11A1) acts as a key enzyme in glucocorticoid synthesis. Glucocorticoid binding to glucocorticoid receptors (GRs) in hypophysiotropic neurons and the anterior pituitary gland can inhibit the release of CRH and ACTH to allow for negative feedback regulation of the HPA axis The skin is the largest organ in the human body and can be regulated by the immune and neuroendocrine systems Because of the evolutionary conservation of HPA-like networks at the central and cutaneous levels, interactions may exist between these systems, and such interactions could affect the functions of each systerm Male C57BL/6 mice  were obtained from the Laboratory Animal Services Center of the Yangzhou University. Animals were maintained on a 12-h light/dark cycle at a regulated temperature (22±2°C), humidity (50±10%) and fed a standard diet and water ad libitum. Animals were acclimatized for 7 days. This study was carried out in strict accordance with the guidelines of the “Principles of Laboratory Animal Care” . This study was specifically approved by the Animal Experimentation Ethics Committee of the Chinese Pharmaceutical University  2011–0003). All efforts were made to minimize suffering.Mice were randomly divided into the following three groups: (1) control group, (2) chronic restraint stress (CRS) group, and (3) chronic unpredictable mild stress (CUMS) group. The CRS procedure was performed as described before Corticosterone was purchased from Sigma-Aldrich . Mice were randomly divided into control and corticosterone (CORT) groups. Mice received corticosterone injections , once per day for 21 days.RU486 (a glucocorticoid receptor antagonist) was purchased from Sigma-Aldrich. Mice were randomly divided into the following three groups: (1) control group, (2) CUMS group with no drug injection and the application of CUMS, and (3) CUMS+RU486 group with the application of CUMS concomitant with 100 mg/kg/day of RU486 injected subcutaneously for 21 days.On day 9, all mice received epilation with rosin and paraffin  to induce anagen of the hair cycle  [39]. MiThe B16F10 murine melanoma cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Cell culture was performed as described before 2. To detect the effects of DEX and α-MSH on melanogenesis, cells were incubated with 1 µM DEX or 50 nM α-MSH. Melanocytes at passage numbers between 3 and 6 were used for the experiments. The studies on human material were approved by Nanjing Drum Tower Hospital, Medical Ethics Committee. All participants provided their written informed consent to participate in this study. This consent procedure was approved by the Nanjing Drum Tower Hospital, Medical Ethics Committee.Normal human epidermal melanocytes were derived from young male adult foreskins (ethnic Han/aged 18–22 years) obtained at circumcision following standard protocols The body weight of all mice were recorded on days 3, 6, 9, 12, 15, 18, and 21. Serum corticosterone concentrations were measured using the IBL-AMERICA Corticosterone rat/mouse ELISA kit  according to the manufacturer's instructions. Serum samples were incubated at room temperature and then directly used for detection. The lowest detectable concentration of corticosterone that could be distinguished from the “zero calibrator” was 4.1 ng/mL.All mice were photographed with a digital camera  once every day after depilation. The grayscale (0–255) of specific area in the photographs (the region from neck to tail) were analyzed by Image J software and presented as ratios .Cells were incubated with compounds for 72 hours. After they were washed twice with ice-cold phosphate-buffered saline (PBS), cells were lysed by incubation in cell lysis buffer   at 4°C for 10 min, then the lysates were centrifuged at 14,000 rpm for 15 min. The supernatant containing protein was removed and conserved. Protein concentrations were determined by BCA Protein Assay kit  with bovine serum albumin (BSA)  as a standard. The precipitate containing total melanin was dissolved in 100 µL of 1N NaOH/10%DMSO for 2 h at 80°C. Total melanin content was estimated by absorbance at 405 nm and comparison made with total protein concentration, then calculated as a percent of the control.The protein suspension of skin tissues was obtained using a Total Protein Extraction Kit  and the protein concentrations were detected by BCA Protein Assay kit with BSA as a standard. The same amount of total protein was assayed for each Western blot. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies: POMC  and β-Actin  for 1.5 h at room temperature then washed with Tris-buffered saline, including 0.1% Tween-20, exposed to peroxidase-conjugated secondary antibodies  for 1 h at room temperature, washed. Proteins were visualized using an enhanced chemiluminescence detection system. Densitometric analysis was performed by using Quantity One . Three animals were used for each data point. Western blot assay results reported here are representative of three independent experiments. The related protein bands, cut and displayed in the same figure, are from the same membrane.-ΔΔCT method and the results were from at least three independent experiments according to the manufacturer's protocols Total RNA was extracted from cells or mouse dorsal skin using TRIZOL reagent  and total RNA concentration was quantified spectrophotometrically. First strand cDNA was synthesized with PrimeScript RT Master Mix  according to the manufacturer's instructions. The quantitative real-time PCR was performed on an iQ5 multicolor real-time PCR detection system  by using SYBR Premix Ex TaqTM2  according to the manufacturer's instructions. Primer sequences are shown in Statistical analysis was performed using GraphPadPrismVersion 5.0c (GraphPad Software). Data were analyzed by unpaired, two-tailed Student's t test or by one-way ANOVA with Tukey's post hoc test, as appropriate. P<0.05 was regarded as significant. Results are presented as mean ± SEM.Mice were photographed after epilation. Upon visual examination, all groups of mice had a similar skin color on the second day after epilation. However, on the thirteenth day, the skin color showed visible differences, and stressed mice showed lighter skin color than control mice .Experiments were performed to detect any changes in microphthalmia-associated transcription factor (MITF) or tyrosinase (TYR) mRNA expression levels in the dorsal skin. MITF is a key transcription factor for melanogenesis that effectively transactivates the TYR genes by binding to an M-box motif in the TYR promoter The expression of POMC was examined in mouse skin. POMC expression levels were reduced by both CRS (P<0.05) and CUMS (P<0.05) . SimilarAlterations of POMC expression in the skin of stressed mice may be caused by changes in UCN1 expression, thereby affecting the cutaneous HPA axis, and may lead to changes in the expression of CYP11A1. To test this possibility, experiments were performed to measure mRNA expression of UCN1 and CYP11A1, and both genes were found to be reduced in the skin of stressed mice . UCN1 exOur data indicated that CRS and CUMS did not significantly inhibit mouse body weight gain compared to the control group .Sustained activation of the central HPA axis by chronic stress can lead to elevated glucocorticoid levels Considering that chronic stress caused a marked elevation of serum corticosterone levels, further studies were conducted to investigate whether corticosterone affects the dorsal skin of mice in vivo. On the thirteenth day after epilation, mice that received corticosterone injections had a significantly lighter observable skin color than controls . In the The expression levels of key factors along the HPA axis were evaluated in the dorsal skin of mice that received chronic corticosterone injections. POMC expression was reduced after corticosterone injections . CorticoBecause chronic stress caused a marked elevation of serum corticosterone levels, further studies were conducted to test whether RU486 (a glucocorticoid receptor antagonist) could reverse the effects of CUMS on the dorsal skin of mice in vivo. On the thirteenth day after epilation, mice that received RU486 injections had a significantly darker observable skin color than CUMS mice . The CUMTo determine whether corticosterone could directly inhibit melanogenesis, NHEMs or B16F10 cells were incubated with α-MSH or DEX, a synthetic corticosteroid. In NHEMs, both α-MSH and DEX significantly increased melanin content, and α-MSH showed a stronger effect than DEX . Next, wOur data demonstrate that chronic stress can suppress cutaneous melanogenesis and the expression levels of cutaneous HPA axis elements. Moreover, chronic stress can cause the elevation of serum corticosterone levels, suggesting that increased corticosterone levels may contribute to the suppression of chronic stress. Consistent with this possibility, mice that received subcutaneous injections of corticosterone showed reduced expression of cutaneous HPA axis elements and decreased pigmentation. Additionally, the glucocorticoid receptor antagonist increased the CUMS-induced reductions of melanogenesis and cutaneous HPA axis element expression levels. These findings suggest that stress can suppress the activation of the cutaneous HPA axis through glucocorticoids and thereby cause reduced melanogenesis.In a study of human hair follicles in vitro, the glucocorticoid receptor agonist hydrocortisone could reduce follicular CRH expression In addition to the decreased expression of hormones along the cutaneous HPA axis in situ, chronic stress also leads to changes in the expression of cognate receptors for these hormones. Mice that experienced chronic stress or long-term corticosterone injections exhibited reduced MC2R mRNA expression levels in skin. ACTH binds to MC2R to promote glucocorticoid synthesis Based on our findings, glucocorticoids play an important role in the regulation of chronic stress. In humans, two key enzymes that regulate local cortisol availability are 11β-HSD1 and 11β-HSD2, which induce intracellular conversion of cortisol, and, together with GR, play a key role in regulating glucocorticoid activities human skin expresses mRNAs for three obligatory enzymes of steroid synthesis including cytochromes P450scc, P450c17 and P450c21 Clinically, glucocorticoids are used to treat many skin diseases Melanogenesis is an important function of the skin that protects the body against radiation and helps maintain homeostasis of the skin microenvironment In summary, our data demonstrate that chronic stress can suppress the expression of skin HPA axis-related genes and proteins. This restriction of the cutaneous HPA axis may be caused by negative feedback control via high glucocorticoid concentrations induced by stress. Additionally, the suppressed expression of elements of the cutaneous HPA axis is accompanied by reduced pigmentation, emphasizing that chronic stress may be a risk factor for the development of skin problems.


Mycobacterium tuberculosis. A comprehensive histopathological analysis revealed that C3HeB/FeJ mice develop three morphologically distinct lesion types in the lung that differ with respect to cellular composition, degree of immunopathology and control of bacterial replication. Mice displaying predominantly the fulminant necrotizing alveolitis lesion type had significantly higher pulmonary bacterial loads and displayed rapid and severe immunopathology characterized by increased mortality, highlighting the pathological role of an uncontrolled granulocytic response in the lung. Using a highly sensitive novel fluorescent acid-fast stain, we were able to visualize the spatial distribution and location of bacteria within each lesion type. Animal models that better reflect the heterogeneity of lesion types found in humans will permit more realistic modeling of drug penetration into solid caseous necrotic lesions and drug efficacy testing against metabolically distinct bacterial subpopulations. A more thorough understanding of the pathological progression of disease in C3HeB/FeJ mice could facilitate modulation of the immune response to produce the desired pathology, increasing the utility of this animal model.Cost-effective animal models that accurately reflect the pathological progression of pulmonary tuberculosis are needed to screen and evaluate novel tuberculosis drugs and drug regimens. Pulmonary disease in humans is characterized by a number of heterogeneous lesion types that reflect differences in cellular composition and organization, extent of encapsulation, and degree of caseous necrosis. C3HeB/FeJ mice have been increasingly used to model tuberculosis infection because they produce hypoxic, well-defined granulomas exhibiting caseous necrosis following aerosol infection with  Summary: C3HeB/FeJ mice develop three morphologically distinct lesion types, which differ with respect to bacterial load, cellular composition and degree of immunopathology following low-dose aerosol infection with Mycobacterium tuberculosis. Mycobacterium tuberculosis, with the vast majority of these individuals serving as a latently infected asymptomatic reservoir  computed tomography (CT), it has recently become apparent that individual pulmonary lesions can change substantially over time  is a pulmonary (lung) disease spread by aerosol transmission. Around 2-billion people worldwide are infected with ResultsHere, the authors investigate the histopathological response to aerosol infection in the C3HeB/FeJ mouse model, which develops caseous necrotic pulmonary granulomas, a type of lung lesion often seen in humans with TB. A comprehensive pathological evaluation reveals that three morphologically distinct lesion types emerge following infection, in contrast to the more commonly used mouse strains, which only present with a single lesion type. Notably, the bacterial load varies between lesion types and this variability reflects differences in the immunological control of bacterial replication. Finally, the authors identify a single lesion type that is responsible for the significant early mortality observed in a subpopulation of C3HeB/FeJ mice following infection.Implications and future directionsCost-effective small animal models that represent the full spectrum of human TB are needed to augment drug development efforts, evaluate novel multi-drug combination regimens and prevent the emergence of drug resistance. Although the C3HeB/FeJ mouse model has shown promise, variability in pulmonary colony-forming unit (CFU) counts and early mortality have complicated its usage. The realization that three distinct lesion types with different bacterial loads can emerge following aerosol infection of C3HeB/FeJ mice will allow researchers to understand and properly interpret the variability of pulmonary bacterial counts typically observed when using C3HeB/FeJ mice as a model for TB. Moreover, a more complete understanding of the pathological process that underlies lesion development in the lungs will increase the reproducibility, and therefore the utility, of this animal model. Immunomodulatory and chemotherapeutic strategies are presently being investigated to reduce early mortalities while preserving the caseous necrotic lesions that make this animal model relevant to human TB.The ultimate goal of TB drug development programs is to shorten the duration of therapy needed to cure disease, prevent the emergence of drug resistance and phenotypic drug tolerance, and target difficult-to-treat persistent subpopulations of bacilli. Small animal models of TB infection are an important component of drug screening efforts and represent a cost-effective means of accelerating preclinical drug development. In order to obtain results that have predictive utility for human clinical trials, small animal models should replicate the pathophysiological conditions believed to exist within human pulmonary TB lesions. Important parameters include: (1) the production of caseous necrosis accompanied by collagen encapsulation, which can influence drug penetration and the nutrient supply available to bacilli ; (2) hyp bacilli ; and (3) bacilli . Althougrculosis , and therculosis  and mighrculosis , which cefficacy . Mouse sefficacy .TRANSLAM. tuberculosis, and therefore represent an attractive model in which to study TB drug activity. In support of this, previous work from our laboratory demonstrated that clofazimine (CFZ) had significant activity in BALB/c mice and that this activity was highly attenuated in C3HeB/FeJ mice  generate hypoxic, caseous necrotic pulmonary lesions with abundant intracellular and extracellular bacteria following LDA infection with FeJ mice . The attOwing to the crucial role of the pathological response in the lungs in TB, a more comprehensive understanding of the histopathological response to aerosol infection in C3HeB/FeJ mice was warranted. In these experiments, we characterized the pathological progression of lesion development over time following an LDA infection and examined the cellular composition and distribution of cell types in the resulting granulomas. We identified three distinct lesion types that arose following LDA infection. Importantly, the heterogeneity of lesion types represented differing levels of host control of bacterial replication and of the progressive inflammatory response as evidenced by the number of bacteria and morphometric analysis of lesion size over time. The exceptionally large number of bacilli characteristic of C3HeB/FeJ mice coupled with the highly sensitive SYBR Gold staining methodology allowed us to easily visualize the location of the bacilli, which is normally quite challenging in most animal models  and in hM. tuberculosis Erdman, bacterial numbers within the lung rapidly increased to more than 7 log10 CFU by 30 days post-infection (10 CFU increase between 30 and 90 days.Following LDA infection of C3HeB/FeJ mice using 50-75 colony-forming units (CFU) of nfection A. The inIn a series of experiments, we measured mouse survival over time. A consistent pattern emerged whereby significant mortality was observed between 28-45 days after LDA; however, mice that survived beyond 45 days generally survived at least 14 weeks with only minimal mortality observed in the intervening time B. Of impWe next performed a comprehensive pathological examination of the pulmonary lesions that arose following LDA infection in the C3HeB/FeJ mice. Interestingly, upon histological examination we were able to identify three distinct lesion types by 5 weeks following aerosol infection that differed by cellular composition and organization. For descriptive efficiency, we arbitrarily identified these lesions as Type I, Type II or Type III.Type I lesions A most clType II lesions resembled a rapidly progressive, granulocytic pneumonia composed almost entirely of neutrophils. By 20 days following LDA, small cellular aggregates composed of activated and epithelioid macrophages and aggregates of neutrophils that had completely impacted the alveoli were evident E. By dayType III lesions in C3HeB/FeJ mice were cellular, inflammatory lesions that were indistinguishable from pulmonary lesions typically observed in BALB/c  and C57BWe next examined the mice that had to be euthanized prior to experimental endpoints to adhere to Colorado State University's Institutional Animal Care and Use Committee (IACUC) guidelines . This group of mice exhibited significant morbidity in multiple experiments within a highly reproducible window (28-45 days after infection). Morphometric analysis of the total lesion area occupied by each of the three lesion types in comparison to the total lung area was performed on hematoxylin and eosin (H&E)-stained whole lung sections to quantify disease severity. Analysis of the early-mortality mice (mice humanely euthanized between days 30 and 41 in this particular experiment) indicated that 100% of these animals had predominantly Type II lesions composed of fulminant neutrophilia A that oc10 CFU more bacteria than the mice that survived 8 weeks, even though the infection in the surviving mice had progressed approximately 3 weeks longer.Pulmonary bacterial load was assessed by plating serial dilutions of whole-lung homogenates on 7H11 agar. Seventeen mice displaying signs of morbidity substantial enough to warrant humane euthanasia  were selected for CFU determination. All of these mice were euthanized between 30-41 days post-infection in this experiment. Fourteen surviving mice were euthanized 8 weeks post-infection B. The ean=10), 35 (n=15) and 45 (n=14) days post-infection. Early-mortality mice (n=17) were euthanized between 30-41 days post-infection based upon IACUC morbidity guidelines. Pulmonary bacterial load inversely correlated with mouse body weight, which proved to be a reliable indicator of disease severity in conjunction with physical parameters  and behavioral characteristics . Initial deviations in individual animal body weights could be observed by 20 days following LDA, and were predictive of disease severity and early mortality (data not shown).We next compared pulmonary bacterial load with the terminal body weight at the time of euthanasia for individual mice C. Mice wThe progression of pulmonary disease in C3HeB/FeJ mice following LDA infection was characterized by collecting mice for histological examination at 5-day intervals through 85 days of infection .Fig. 5.M. tuberculosis infection occurred at 15 days following infection and pulmonary lesion burden increased with each subsequent time point through 45 days of infection, with a particularly abrupt transition between 35 and 40 days of infection. After 45 days, the pathology in the lungs of C3HeB/FeJ mice as measured by total lesion burden scoring began to stabilize. The 35- to 40-day time period coincided with the 28- to 45-day window of mortality observed in the survival experiments  between 28-45 days following aerosol infection. All of the mice that succumbed during this time had predominantly Type II lesions and extensive pulmonary consolidation occupying the majority of viable lung parenchyma. Thus, the characteristic window at 28-45 days after infection seemed to represent the amount of time necessary to generate and recruit enough neutrophils into the lung to compromise respiratory function. The Type II lesions observed in C3HeB/FeJ mice closely resembled polymorphonuclear alveolitis occasionally observed in humans with TB, as described by Type III lesions in C3HeB/FeJ mice were very similar to lesions found in BALB/c mice following aerosol infection. These lesions were composed predominantly of epithelioid and foamy macrophages with large numbers of lymphocytes present in cellular aggregates and interspersed throughout the lesion. Bacteria within these lesions were few in number and primarily located intracellularly within epithelioid and, at later stages, foamy macrophages. Individual cellular necrosis was evident primarily at the later stages of infection, and was predominantly the result of the disintegration of foamy macrophages. Caseous necrosis was never observed associated with this lesion type.Cavitary lesions play a crucial role in human TB because the bacilli within these lesions are extremely resistant to drug therapy and the host immunological response, and their presence correlates with a poor clinical prognosis . The intM. tuberculosis that initially produced only Type III lesions did not exhibit caseous necrosis, even 20 weeks following aerosol infection (data not shown).An important observation of comparing all three lesion types was that the number of lymphocytes was inversely proportional to the bacterial burden. As such, these three lesion types represented differing levels of host immunological control within the lung following infection. Type III lesions contained abundant lymphocytes and controlled bacterial replication more efficiently, maintained bacilli within intact foamy macrophages for the longest period of time, and progressed at a rate slower than the other two lesion types. These observations are consistent with initiation of a strong adaptive immune response capable of controlling bacterial replication and limiting host immunopathology. In contrast, Type II lesions contained few if any identifiable lymphocytes, failed to control bacterial replication, and rapidly progressed to a state in which consolidation of viable lung parenchyma negatively impacted animal survival. This lesion type reflected a failure of the host to initiate a robust protective immune response within the lung. Severe pulmonary neutrophilia might represent an ineffective and immunopathological compensatory action in the absence of strong adaptive immunity, facilitating uncontrolled bacterial replication and ultimately favoring the bacteria. Type I granulomas represented an intermediate level of host adaptive immunity, where rapidly progressive neutrophilic lung consolidation was held in check by the host pro-fibrotic response; however, the host response was unable to reverse the pathological progression once the granuloma became established. Lymphocytes were clearly visible within the peripheral margins of Type I lesions; however, the number of such cells was much less than that seen in Type III lesions. Although the number of bacteria continued to increase over time, the rate of increase was slower than that seen in Type II lesions. It is important to understand that, although each of the described lesion types differed in pathological severity, level of host control of bacterial replication, and damaging neutrophilic infiltration, all three lesion types continued to increase in size and degree of inflammation over time. The rate of increase was most rapid for Type II lesions and slowest for Type III lesions. Once established, these lesion types did not seem to interconvert over time. C3HeB/FeJ mice that were infected with a less virulent strain of M. tuberculosis bacilli  of human TB. Neutrophils represent the predominant cell type found in bronchoalveolar lavage and in sputum of individuals with active TB, and often contain large numbers of actively replicating bacilli . The ideAnother inbred mouse strain (CBA/J mice) that exhibits caseous necrotic pulmonary lesions following aerosol infection also displays a correlation between rapid weight loss, clinically observable signs of morbidity, and extensive neutrophilic infiltration of lung parenchyma . InteresM. tuberculosis might not generate the same level of mortality, and could therefore result in larger experimental variation. However, even after elimination of the majority of Type II lesions, the variability of the surviving C3HeB/FeJ mouse population was still somewhat higher than that typically observed in BALB/c mice, which only present with Type III lesions. To maintain the statistical power of experiments, extra mice are now routinely added to experimental and control groups  important for assessing drug activity and the pharmacokinetic profile, while still maintaining experimental variation within useful limits to discern statistical differences between drugs.M. tuberculosis), and (4) histological verification of the pulmonary pathology at the start of treatment.Based upon results presented in this paper as well as other experiments performed in our laboratory, important caveats for optimal utilization of the C3HeB/FeJ mouse model include: (1) collection of all lung lobes to mitigate the variability in the distribution of lesion types, (2) use of larger numbers of mice per group to increase statistical power of the experiment, (3) allowing sufficient time for the caseous necrotic pathology to develop . Mice were housed in a bio-safety level III animal facility and maintained with sterile bedding, water and mouse chow. Specific pathogen-free status was verified by testing sentinel mice housed within the colony.M. tuberculosis Erdman strain (TMCC 107) was used for aerosol infections of mice, and the inocula were prepared as previously described . The number of viable organisms was determined by plating serial dilutions of whole lungs homogenized in 4 ml of PBS on Middlebrook 7H11 agar plates supplemented with OADC . Colonies were counted after at least 21 days of incubation at 37°C as previously described .Whole lungs were collected at necropsy and fixed in 4% paraformaldehyde in phosphate buffered saline (PBS). Tissue sections were embedded in paraffin and sectioned to 5 μm thickness. Subsequent tissue sections were mounted on glass slides, deparaffinized and stained either with: H&E, Masson's Trichrome collagen stain or SYBR Gold fluorescent stain  as descr2O and slides were subsequently stained with 4′,6-diamidino-2-phenylindole  at 200 ng/ml final concentration for 10 min and washed again with water. Slides were mounted with Prolong Gold antifade mounting media .At each sacrifice, whole lung lobes were infused with 4% paraformaldehyde , preserved for 48 h in paraformaldehyde, then washed and kept in 70% ethanol until being processed for histopathological assessment. Paraffin sections (5 μm) were stained with SYBR Gold fluorescent dye  as previously described . BrieflyCollagen was stained in the mouse tissue sections using the Masson's Trichrome Kit  using the manufacturer's procedure. Briefly, tissue sections were dewaxed in xylene and rehydrated through a graded alcohol series. The sections were briefly rinsed in water and heated at 56°C for 1 h with Bouin's Fluid. Slides were washed then stained with Weigert's hematoxylin. Slides were rinsed and stained with Biebrich Scarlet-Acid Fuchsin for 15 min, after which they were rinsed. Slides were stained with Phosphomolybdic/Phosphotungstic acid for 5 min and placed directly into Aniline Blue stain for 10 min, then rinsed. Slides were placed in 1% acetic acid for 3 min and dehydrated by a graded alcohol series including xylene. Slides were hard-mounted using Surgipath MM24 Mounting Media .n=3 mice/time point). Slides were examined by an American College of Veterinary Pathologists board-certified veterinary pathologist blinded to the treatment groups. The relative lesion burden for each lung lobe from individual mice was determined by scoring (0-5 points) using the following scale: 0=no lesions, 1=focal lesion, 2=multiple focal lesions, 3=one or more focal severe lesions, 4=multiple focal lesions that are extensive and coalesce, and 5=extensive lesions that occupy the majority of the lung lobe.H&E-stained histological sections were prepared from whole lungs of C3HeB/FeJ mice at 5-day intervals  to analyze scanned images of representative H&E-stained slides obtained using a ScanScope XT slide scanner  at 400×. The data were expressed as the ratio of the lesion area to the lung area for individual animals.t-test. Differences were considered signiﬁcant at the 95% level of conﬁdence.The viable CFU counts were converted to logarithms, which were then evaluated using Student's


Motivation: Many intracellular signaling processes are mediated by interactions involving peptide recognition modules such as SH3 domains. These domains bind to small, linear protein sequence motifs which can be identified using high-throughput experimental screens such as phage display. Binding motif patterns can then be used to computationally predict protein interactions mediated by these domains. While many protein–protein interaction prediction methods exist, most do not work with peptide recognition module mediated interactions or do not consider many of the known constraints governing physiologically relevant interactions between two proteins.Results: A novel method for predicting physiologically relevant SH3 domain-peptide mediated protein–protein interactions in S. cerevisae using phage display data is presented. Like some previous similar methods, this method uses position weight matrix models of protein linear motif preference for individual SH3 domains to scan the proteome for potential hits and then filters these hits using a range of evidence sources related to sequence-based and cellular constraints on protein interactions. The novelty of this approach is the large number of evidence sources used and the method of combination of sequence based and protein pair based evidence sources. By combining different peptide and protein features using multiple Bayesian models we are able to predict high confidence interactions with an overall accuracy of 0.97.Availability and implementation: Domain-Motif Mediated Interaction Prediction (DoMo-Pred) command line tool and all relevant datasets are available under GNU LGPL license for download from http://www.baderlab.org/Software/DoMo-Pred. The DoMo-Pred command line tool is implemented using Python 2.7 and C ++.Contact:gary.bader@utoronto.caSupplementary information:Supplementary data are available at Bioinformatics online. Protein–protein interactions (PPIs) are physical associations between protein pairs in a specific biological context. Their knowledge provides important insights into the functioning of a cell. Previously, experimental detection of PPIs was limited to labor intensive techniques such as co-immunoprecipitation or affinity chromatography (Multiple kinds of protein–protein interactions exist. We focus on interactions involving peptide recognition modules (PRMs), in particular Src homology three (SH3), which are important in many cellular signaling processes. These domains bind to small, linear sequence motifs (peptides) within proteins . SH3 domS. cerevisiae. A straightforward approach is to construct position weight matrices (PWMs) from phage peptides and scan the whole proteome for potential binding sites in target proteins using some threshold score .Position weight matrices (PWMs) are statistical models for representing sequence motifs. They are real valued S. cerevisiae often lie within the disordered regions of a protein (ip is the disorder score of the ith significant amino acid (either one for disordered or zero for ordered) and N is the number of significant amino acids in the binding site.PRMs bind to small peptide stretches containing a specific motif. Specifically interactions between proteins having SH3 domains and their targets are often mediated by proline rich peptide sequences containing PXXP, [R/K]xxPxxP, PxxPx[R/K] motifs. Proline disrupts the secondary structure of a protein by inhibiting the formation of helices and sheets . Also, s protein . DISOPREip is the surface accessibility score of ith significant amino acid and N is the number of significant amino acids in the binding site.Sequences present on a protein’s surface are more accessible to binding by SH3 domains than those that are buried inside a protein structure. The degree of solvent-accessible surface area of amino acid residues in a sequence indicates its level of exposure and is measured in terms of relative solvent accessibility (RSA)  are ideected by  is used ijc is the normalized contact area of the ith aligned domain and peptide residues of the jth base model. Alignment gaps in contact residues will negatively impact the average contact area as only the aligned residues are used for scoring . N is the number of aligned contact residues.Known 3-D structures of SH3 domains complexed with peptides can be used to assess the binding potential of a query SH3 domain and peptide by reducing residue-residue contacts in 3-D structures to a binary 2-D contact matrix . Six yeaa and b are the query proteins and C, P, F are the cellular component, biological process and molecular function ontologies.Physical PPIs require proteins to be in close proximity to each other i.e. they should co-localize in the same cellular compartment. Also, interacting proteins are more likely to be part of same biological process or have the same function. The Gene Ontology (GO) contains a hierarchy of controlled terms describing cellular location, biological process and molecular function of proteins . The funSupplementary Figure S1. Correlation coefficients from 86 gene expression profiles from GeneMANIA EX=1−ijp is the probability of seeing motif i on one protein and motif j on other protein in the experimentally verified PPI set, ip is the probability of seeing motif i and jp is the probability of seeing motif j in the same set.Sequence signature based PPI prediction methods are based on the notion that protein domains are correlated with specific functions. For instance, it has been shown that functionally related proteins have similar domain composition or they belong to the same ‘domain club’ .The objective of a Bayesian PPI prediction model is to estimate the probability that a given protein pair interacts, conditioned on the biological evidence in support of that interaction. A naïve Bayesian model simplifies this problem by assuming independence between different types of biological evidence. While modeling the PRM mediated PPI prediction problem a set of observations are made on domain-peptides while others are made on full-length proteins. Assuming that peptide and protein features are independent of each other, two separate naïve Bayes models  theorem  (see sup(P1) and an equal number of randomly selected negative interactions (N1). The protein classifier is trained on a high confidence set of 5215 pairwise yeast PPIs from the iRefIndex database (P2) and an equal number of randomly selected negative interactions (N2) .The goal is to construct a generalized model which can predict high confidence, in vivo yeast SH3 domain–peptide physical interactions. To achieve this, both peptide and protein classifiers are trained on their respective positive and negative datasets. The peptide classifier is trained on a high confidence set of 628 SH3 domain–peptide interactions in yeast from the MINT database Supplementary Fig. S2). Biological process (BP), cellular component (CC) and sequence signature (SS) outperform molecular function (MF) and gene expression (EX) in the protein feature set. Proteins could have the same molecular function but still belong to different processes and this could be one of the reasons behind molecular function feature’s weak performance. Gene expression data alone is not as powerful as others in discriminating positives from negatives  and accuracy (ACC). Peptide and protein classifiers with all features outperformed other models on at least one of statistical measure .Highly correlated features can negatively affect the performance of a naïve Bayesian classifier. Maximal information coefficient (MIC) is used to quantify the correlation between different features. DR and SA in the peptide feature set and CC and BP in the protein feature set are correlated with MICs of 0.72 and 0.5 respectively. The effect of correlation on classifier performance is measured by comparing different models without one of the correlated features. Further, to identify the feature subset which maximizes the performance of both classifiers, all possible combinations of features are compared using different statistical measures, such as area under ROC curve (AUROC), area under precision-recall curve (AUPRC), Brier score (BRIER), Mpep and protein Mpro naïve Bayesian classifiers. The majority of interactions in the P1 dataset are from two peptide array experiments . These PWMs are then used to predict SH3 domain–peptide interactions using the combined classifier. 534 unique PPIs (1481 binding sites) are predicted as positives for the stringent P-value PWM threshold of 1e-05 with no missing features (Supplementary Table S3). Approximately 55%  of these interactions are known at the PPI level (iRefIndex and MINT) and at least 172 (464 binding sites) out of 295 PPIs are known SH3 domain mediated interactions at the peptide level (with Supplementary Table S4). For example, some interacting partners of the MYO3 SH3 domain are found to be enriched in PI3K/AKT signaling. AKT is known to regulate actin organization and cell motility during endocytosis (Supplementary Table S5). These examples support our results and suggest that our predicted interactions are biologically relevant.30 PWMs representing multiple binding specificities of 25 SH3 domains in yeast are constructed using phage display data from nt  features, including presence in a disordered region of the protein, surface accessibility, conservation across different yeast species, and structural contact with the SH3 domain, as well as protein features such as cellular proximity, shared biological process, similar molecular function, correlated gene expression and sequence signature. Two separate Bayesian models are used to combine peptide and protein features. Their respective posterior probabilities are further combined using Bayes rule for predicting high confidence interactions. The combination of peptide and protein models achieved a higher accuracy of 0.97 compared to individual models on a curated benchmark dataset from


Peripheral nerve injury results in dramatic upregulation in pituitary adenylate cyclase activating polypeptide (PACAP) expression in adult rat dorsal root ganglia and spinal motor neurons mirroring that described for the neurotrophin brain derived neurotrophic factor (BDNF). Thus, we posited that injury-associated alterations in BDNF expression regulate the changes in PACAP expression observed in the injured neurons. The role of endogenous BDNF in induction and/or maintenance of PACAP mRNA expression in injured adult rat motor and sensory neurons was examined by intrathecally infusing or intraperitoneally injecting BDNF-specific antibodies or control IgGs immediately at the time of L4-L6 spinal nerve injury, or in a delayed fashion one week later for 3 days followed by analysis of impact on PACAP expression. PACAP mRNA in injured lumbar sensory and motor neurons was detected using in situ hybridization, allowing quantification of relative changes between experimental groups, with ATF-3 immunofluorescence serving to identify the injured subpopulation of motor neurons. Both the incidence and level of PACAP mRNA expression were dramatically reduced in injured sensory and motor neurons in response to immediate intrathecal anti-BDNF treatment. In contrast, neither intraperitoneal injections nor delayed intrathecal infusions of anti-BDNF had any discernible impact on PACAP expression. This impact on PACAP expression in response to BDNF immunoneutralization in DRG was confirmed using qRT-PCR or by using BDNF selective siRNAs to reduce neuronal BDNF expression. Collectively, our findings support that endogenous injury-associated BDNF expression is critically involved in induction, but not maintenance, of injury-associated PACAP expression in sensory and motor neurons. The peptidergic phenotype in sensory and motor neurons is dramatically changed in response to nerve injury and is implicated in the neurons’ capability to survive and regenerate. However, the signals inducing this phenotypic switch, to a regenerative state, are relatively unknown. We and others have previously shown that one of these peptidergic changes is an injury-induced expression of the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) in both sensory and motor neurons. These changes in PACAP expression parallel changes that we have observed in brain derived neurotrophic factor (BDNF) in the injured neurons over the same time frame. The aim of this study was to examine our hypothesis that injury-induced changes in BDNF expression regulate induction of injury-associated PACAP expression in sensory and motor neurons.1, is detectable in spinal cord dorsal and ventral horn neurons but not in DRG neurons, suggesting a paracrine role for primary sensory neurons In sensory and motor neurons PACAP expression is markedly upregulated in response to various types of nerve damage (see below) where it serves roles in modulation of nociception Neurotrophins are potent modulators of neuropeptide expression in sensory neurons with three members having been studied in this capacity – nerve growth factor (NGF), neurotrophin 3 (NT-3) and BDNF All animal procedures were conducted under the approval of the Animal Research Ethics Board at the University of Saskatchewan (protocol number: 19920164) and in accordance with the Canadian Council on Animal Care. All efforts were made to minimize suffering and animals were given buprenorphine  subcutaneously pre- and postoperatively.Male Wistar rats (∼250–300 g) were anaesthetized with sodium pentobarbital , and all animals underwent unilateral sciatic spinal nerve transection, whereby the right sciatic nerve was transected, at its origin from the L4, L5 and L6 spinal nerves, and a 5 mm segment resected to prevent regeneration. Animals were divided into two groups –those receiving continous 3 day intrathecal infusion or those receiving intraperitoneal injections (i.p.) of anti-BDNF or control IgG. In a previous study we confirmed that the antibody employed is highly selective for BDNF and does not recognize other neurotrophins, nor cytochrome C a protein of similar molecular weight and isoelectric point Intrathecal infusion group: Animals were given continuous intrathecal infusions of sheep anti-BDNF  or control IgG  either immediately  or 7 days after injury  at 1.5 µg/µl/hour for 3 days  or sheep control IgG (n = 3),  were used to assess the impact of infusing/injecting IgGs into the animal. Three days after introduction of the pump or injections, animals were anaesthetised and perfused (see below).In vivo reduction of neuronal BDNF expression by small interfering RNAs (siRNA), was employed as an additional method by which to interfere with endogenous BDNF as in To deliver the siRNAs, a laminectomy was performed at the lumbar sacral junction and a sterile indwelling catheter inserted into the subarachnoid space such that the tip of the catheter lay along the midline at the level of the L5 DRG. siRNA (20 µg - 15 µl siRNA from the 100 µM stock) was combined with 2 µl oligofectamine (Invitrogen) and incubated for 15 minutes at room temperature prior to intrathecal injection over a 5 minute span, followed by 3 µl of sterile PBS to flush the catheter. This first injection was performed to help deplete existing stores of BDNF protein that could be released upon injury. Three days later the same animals were subjected to a second injection of BDNF siRNA (n = 2) or control non-targeting siRNA (n = 2) which was administered via the indwelling catheter immediately followed by unilateral transection of the L4, 5, 6 spinal nerves. Fluorescently labeled control scrambled siRNA – siGLO (n = 2) were also delivered intrathecally in an identical manner to BDNF siRNA and non-targeting control siRNA to visualize and ascertain the cellular uptake within the DRG. An additional 4 control animals received unilateral transection of the L4, 5, 6 spinal nerves alone. Three days later animals were perfused and DRGs (L5) removed and processed for in situ hybridization to detect PACAP mRNA and assess the impact of BDNF silencing on PACAP expression.The efficacy of BDNF siRNA treatment in reducing BDNF mRNA and protein expression in DRG neurons was assessed, using BDNF in situ hybridization and immunohistochemistry respectively.On the day of dissection, animals were deeply anaesthetized and perfused via the aorta with cold phosphate buffered saline , followed by 4% paraformaldehyde (PF) in phosphate buffer . Dissected tissues  were postfixed (1–1.5 hours) and cryoprotected in 20% sucrose. Control and experimental tissues were embedded in the same cryomolds, covered in OCT and frozen in cooled isopentane prior to storing at −80°C until processing.Sections were analysed and photographed with an Olympus BX-60 microscope connected to an Olympus DP-50 digital camera. Brightness and contrast were adjusted with the software Adobe Photoshop 5.0.Injured neurons in spinal cord sections were identified using an antibody against activating transcription factor 3 (ATF-3), a specific marker induced in injured DRG and motor neurons DRG and spinal cord sections were, rinsed in PBS, and incubated with biotinylated-anti-sheep IgG  for 1 hour at room temperature. Sheep IgG distribution was visualized in target tissues using a standard avidin/biotin/peroxidase procedure as per manufacturer’s instruction .BDNF immunohistochemistry was performed essentially as decribed in Geremia et al., –260 – M61175, rat BDNF (2) complementary bases 626–673 – X16713 were synthesised and purified . All cDNA regions used were checked against the Genbank database ; no greater than 60% homology were found to sequences other than the cognate transcript. Labeling of probe (at a concentration of 3.1 ng probe/µl), with 35S–dATP  and terminal transferase enzyme  was performed in a terminal transferase buffer, containing sodium cacaodylate 500 mM, CoCl2 (pH 7.2) 10 mM, mercaptoethanol 1 mM, for 1.5–2 hours at 37°C. The reaction was stalled by adding 500 µl, 0.1 M Tris HCl (pH 8.0), after which probe was purified through a NENSORB-20 column , and dithiothreitol added to a final concentration of 10 mM. The activity was measured to approximately 0.27–0.28×109 cpm/ml, and the labeled probe was stored at 4°C.Oligodeoxyribonucleotide (OligoDNA) probes complementary to and selective for PACAP mRNA -nucleotides 700–747 2O (5 minutes), and dehydrated in increasing ethanol concentrations . Sections were hybridized with radiolabeled probe at a concentration of 107 cpm/ml in a hybridization solution consisting of 50% formamide, 4×saline sodium citrate , 1×Denhardts solution , 0.02% Ficoll and 0.02% polyvinylpyrrolidone), 10% dextran sulphate, 0.5 mg/ml salmon sperm DNA, 1% sarcosyl and 0.2 M DTT. Hybridization, with approximately 100 µl hybridization solution/slide, was conducted over night at 43°C in air sealed, humidified chambers to prevent evaporation. Following hybridization, the slides were washed in 1×SSC , dipped twice in distilled water, dehydrated in ascending ethanols and air dried. Slides were dipped in Kodak NTB2 photoemulsion (diluted 1∶1 in distilled water), to generate autoradiograms. After 4.5–10 weeks exposure the slides were developed in Kodak D19 (3–5 minutes), rinsed in water, fixed in Kodak rapid fix (5 minutes) and rinsed in water (20 minutes). For darkfield viewing and photography slides were left unstained, whereas slides for brightfield examination were counterstained with 0.5 % toluidine blue , and mounted with Permount . The specificity of hybridization signal for the individual probes was determined by hybridization of adjacent 6 micron sections with labeled probe with the addition of either 1000-fold excess corresponding unlabeled probe which abolished the signal, or 1000-fold excess of a dissimilar unlabeled probe of the same length and similar G-C content which left the signal unchanged from that observed with labelled probe alone.All steps prior to hybridization were performed under RNase free conditions, and all dilutions were performed in autoclaved double distilled water. The slides were air-dried and postfixed in 4% PF (20 minutes), washed in PBS (3×5 minutes), treated with proteinase K at 37°C , rinsed in PBS (5 minutes), fixed in 4% PF (5 minutes), rinsed in PBS (2×5 minutes), rinsed in diethyl pyrocarbonate (0.1%) -HTo evaluate the impact of immunoneutralizing or reducing endogenous BDNF expression on PACAP mRNA expression in axotomised DRG neurons, slides from all animals were first analysed qualitatively to ensure that consistent alterations in expression between experimental groups were observed. All slides examined had both experimental and control tissues mounted on the same slide, thus avoiding bias induced by slide to slide variability in hybridization efficiency and allowing for an accurate determination of relative changes between experimental groups. Qualitative analysis was performed where the relative changes in hybridization signal between experimental groups mounted on the same slide were noted. Representative slides were selected for quantitative analysis based on the criterion that all DRG sections on that slide contained similar numbers of neurons. Photomontages of DRGs for all experimental groups mounted on the same slide were prepared and individual neurons with a visible nucleus were identified and numbered. All neurons within individual DRGs on the same slide were assessed (using a 63X objective) as being labeled, i.e. having levels of hybridization signal above background or not, thus yielding information on incidence of expression. In this manner approximately 820 neurons per experimental group were quantified for immediate infusion of anti-BDNF and control IgG groups and approximately 495 neurons per experimental group were analysed for i.p. injections.Only immediate intrathecal infusion of anti-BDNF (but not i.p. injections) had a discernible impact on the incidence and the degree of expression per individual neuron, the latter assessed qualitatively. Therefore only intrathecally infused animals were subjected to additional computer-assisted image analysis. Further, assessment of impact of treatment on PACAP expression in STEP I revealed neither discernible changes in the level of PACAP hybridization signal between injury alone and control IgG-infused injured tissue, nor any effect on incidence of expression between these two control groups. Thus, only control IgG- and BDNF IgG-infused groups were examined quantitatively using computer-assisted image analysis. An additional two animals from each of the immediately infused experimental groups were selected for this form of analysis bringing the total number of animals quantitatively assessed to 4 for each experimental group. In addition, quantification of PACAP mRNA expression in tissues from animals receiving delayed intrathecal infusions was also performed .The proportion of labeled neurons, as well as the level of expression in each neuron and the distribution of labeling in relation to neuronal size, i.e. in small vs medium-large diameter neurons were determined. For each DRG analysed montages were made, neurons with a visible nucleus identified and each individual neuron was analysed using computer assisted image analysis. This form of analysis was performed for animals receiving immediate intrathecal infusions - 2 rats infused with anti-BDNF and 2 rats infused with control IgG with ∼225 neuronal profiles/L5 DRG section being examined for a total of ∼450 neurons per experimental group. It was also performed on animals receiving delayed intrathecal antibody infusions - 3 rats infused with anti-BDNF and 3 rats infused with control IgG with ∼300 neuronal profiles/L5 DRG section were examined. In short, cross sectional areas and the percentage of the cytoplasmic area covered by silver grains were measured for all the identified neurons in all the ganglia, using the public domain NIH Image program . In DRG, neurons were readily distinguished from glial cells  based on histological criteria in the toluidine blue stained sections with neurons exhibiting lightly stained nuclei and a stained cell soma, in contrast to the glial cells which appear as darkly stained, more oblong nuclei and are significantly smaller in size Analyses of DRG neurons were further subdivided into small (diameter <35 µm), and medium-large (diameter ≥35 µm) For spinal cord ventral horn neurons, a similar approach was used. While spinal nerve lesion injures virtually all the DRG neurons, the injured motor neurons need to be identified in an adjacent section by the presence of nuclear ATF-3 immunoreactivity, which is an injury marker. Only motor neurons identified as injured were quantified. Montages were prepared from adjacent sections (hybridized for detection of PACAP mRNA and ATF-3 immunohistochemistry), and all neurons showing nuclear ATF-3 immunoreactivity were analysed to determine the density of PACAP mRNA hybridization signal over the injured motor neuron.Neurons were considered labeled for PACAP mRNA if they contained more than 4 or 6 times background labeling of silver grains for ventral horn and DRG neurons, respectively. This threshold corresponds to a level of silver grains that must be present to confidently identify the neuron as positively labeled when examined under 63 X oil immersion as in STEP I of the analysis. Background levels of hybridization signal were determined by averaging the silver grain density over 5–10 defined areas of the neuropil devoid of positively labeled cell bodies for each section being analysed.Differences in the proportions of PACAP mRNA expressing neurons between the control IgG treated and anti-BDNF treated DRGs, were analysed using the chi-square test, with p≤0.05 being considered significant, whereas differential induction of PACAP mRNA in injured motor neurons were analysed using the Mann Whitney U test and p≤0.05 was considered significant.′- GCCTACGCCCTTTACTACCC -3′5 and ′- GAAGATGCCGTCCGAGTG -3′5 with a qRT-PCR machine . The levels of housekeeping gene GAPDH (amplified by primers ′- GGTGCTGAGTATGTCGTGGAGTC-3′5 and ′- GTGGATGCAGGGATGATGTTCT-3′5) were used as internal control to verify the qRT-PCR reaction. A non-template control was included in each set of reactions.Total RNA was isolated from the L4 and L5 DRGs using the RNeasy Kit . The quality of RNA was analyzed by electrophoresis using the Agilent 2100 bioanalyzer . The RNA was converted to cDNA using the QuantiTect Rev. Transcription Kit . The PACAP cDNA was amplified by primers Statistical analyses were performed with Prism . Differences between experimental group means were assessed by one-way analysis of variance (ANOVA) with post hoc Tukey's analysis. Differences were considered significant at p≤0.05.To determine whether intrathecally infused antibodies effectively penetrate the surrounding DRGs and corresponding regions of the spinal cord, these tissues were processed immunohistochemically to detect sheep IgGs. DRG sections from antibody infused (control IgG or anti-BDNF) animals revealed immunoreactivity in the parenchyma as well as the surrounding connective tissue in lumbar DRG contralateral and ipsilateral to axotomy , whereasWe wanted to determine whether endogenous BDNF is involved in the regulation of PACAP mRNA expression after nerve injury. Thus, the impact of immediate intrathecal infusion of sheep anti-BDNF or sheep IgG, for 3 days at the time of sciatic spinal nerve transection, on PACAP mRNA expression was investigated. The 3 day infusion/injury period was chosen since BDNF expression is elevated in ∼80% of the injured sensory neurons at this timepoint Immunoneutralization of endogenous BDNF by immediate intrathecal infusion of anti-BDNF had a dramatic impact on the induction of PACAP expression in 3 day injured sensory neurons, resulting in decreased incidence and level of PACAP mRNA expression  with onlUninjured lumbar motor neurons show negligible levels of PACAP mRNA expression. L4-6 spinal nerve injury however, induced, PACAP mRNA expression in lumbar motor neurons, in agreement with what we have previously shown Delayed immunoneutralization of endogenous BDNF by intrathecal infusion of BDNF antibodies for 3 days beginning at 1 week after injury (10 day injury period) did not have any discernible effect on injury-induced PACAP mRNA expression in DRG or spinal cord motor neurons . PACAP mqRT-PCR served as an alternate way to quantitatively assess fold change in mRNA expression between experimental and control conditions, but does so without information about impact on select neuronal subpopulations. We performed these additional experiments in triplicate using immediate or delayed intrathecal anti-BDNF delivery to immunoneutralize endogenous BDNF actions and assess the impact of this treatment on PACAP mRNA expression in injured DRG. in vivo reduction of neuronal BDNF expression using BDNF selective siRNAs was adapted from Baker-Herman et al. (2004) Finally, In contrast to anti-BDNF that was intrathecally delivered immediately following injury, i.p. injections of anti-BDNF also delivered immediately following injury did not discernibly alter either the incidence nor the level of PACAP hybridization signal observed over injured sensory neurons when examined 3 days later. After i.p. injection of anti-BDNF, 67% of injured DRG neurons expressed detectable PACAP mRNA hybridization signal as compared the 65% of the sensory neurons that expressed PACAP following injury and i.p. injection of control IgG (data not shown). Once again no discernible difference in expression was found between animals receiving injury alone or injury and control IgG injection. Since the systemic injections of anti-BDNF after nerve transection did not have any obvious effect on PACAP mRNA expression, further quantifications of these tissues were not performed.We and others have shown that the neuropeptide PACAP is markedly upregulated in sensory and motor neurons, in response to nerve transection, peripheral inflammation and compression injury BDNF is normally expressed in small DRG sensory neurons that express the nerve growth factor receptor trkA We found PACAP to be expressed in roughly one fifth of intact DRG neurons, predominantly small sized neurons, consistent with previous findings An alternate approach of interfering with BDNF signaling, by siBDNF injection, led to a similar, though not as robust, reduction of injury-induced PACAP expression. This further supports a role for endogenous BDNF in modulation of PACAP expression. Two major differences between this form of BDNF interference and anti-BDNF infusion may factor into this less robust response. First, the BDNF siRNA is likely only interfering with neuronal sources of BDNF since the fluorescently tagged siRNAs were only detected in neurons and not perineuronal or other glial cells . This implies that BDNF is not necessary for maintaining an increased PACAP expression once it is induced.We have previously examined the possibility that immunoneutralization of BDNF, by BDNF antagonizing antibodies, might lead to a non-selective effect on gene expression, downregulating expression of all genes. This theory was negated by the findings that infusion of anti-BDNF after injury had no obvious effect on the injury-induced expression of neuropeptide Y We have also recently found that anti-BDNF treatment impairs the intrinsic ability of 3 day injured sensory neurons to extend neurites when assayed in vitro following the in vivo manipulations In spinal cord motor neurons the impact of interfering with the BDNF axis was similar to what was found for DRG neurons. The incidence of injury-induced PACAP mRNA expression in the injured motor neurons (3 days after injury) was also reduced by immediate anti-BDNF treatment at the time of injury, decreasing from ∼55% to only 35% of the neurons expressing PACAP mRNA after anti-BDNF infusion. Delayed antibody infusion on the other hand had no discernible effect on PACAP mRNA expression in the spinal cord. Hence, BDNF seems to be important for the induction but not the maintenance of PACAP mRNA expression after nerve injury in motor neurons, as well as in sensory neurons. Even though the infused antibody was not readily detectable at the level of the lumbar enlargement, the route of how anti-BDNF is able to affect and mitigate PACAP expression in spinal cord ventral horn neurons after intrathecal infusion may be a result of retrograde axonal transport of small amounts of the infused antibody via the ventral root to the motor neurons. Specific receptor mediated retrograde transport of antibodies has been shown in motor neurons The lack of a measureable effect of i.p. anti-BDNF injections on PACAP mRNA expression in injured sensory neurons might be due to the systemic dose not being sufficient to neutralize the endogenous BDNF implicated in this response In conclusion, the findings in this study indicate that the endogenous injury-associated BDNF upregulation is involved in the positive regulation of PACAP expression in sensory and motor neurons in response to the pathology. However, while BDNF is critical for the induction, it is not essential for the maintenance of injury-induced PACAP expression in these neurons. The relevance of the injury-associated upregulation in PACAP expression while not being fully elucidated at this point, provides a novel target in manipulation of regenerative states.


Transcranial direct current stimulation (tDCS) has been shown to have antidepressant efficacy in patients experiencing a major depressive episode, but little is known about the underlying neurophysiology. The purpose of our study was to investigate the acute effects of tDCS on cortical activity using electroencephalography (EEG) in patients with an affective disorder. Eighteen patients diagnosed with an affective disorder and experiencing a depressive episode participated in a sham-controlled study of tDCS, each receiving a session of active (2 mA for 20 minutes) and sham tDCS to the left dorsolateral prefrontal cortex (DLPFC). The effects of tDCS on EEG activity were assessed after each session using event-related potentials (ERP) and measurement of spectral activity during a visual working memory (VWM) task. We observed task and intervention dependent effects on both ERPs and task-related alpha and theta activity, where active compared to sham stimulation resulted in a significant reduction in the N2 amplitude and reduced theta activity over frontal areas during memory retrieval. In summary a single session of anodal tDCS stimulation to the left DLPFC during a major depressive episode resulted in modulated brain activity evident in task-related EEG. Effects on the N2 and frontal theta activity likely reflect modulated activity in the medial frontal cortex and hence indicate that the after-effects of tDCS extend beyond the direct focal effects to the left DLPFC. Transcranial direct current stimulation (tDCS) has recently emerged as a promising intervention for affective disorders. Clinical improvements following tDCS manifest as reductions in depressive symptoms et al.et al.et al.Electroencephalography (EEG) is an obvious candidate for studying changes in cortical activity following tDCS et al.Whilst these studies sought to primarily elucidate the basic neurophysiological impact of tDCS, the findings suggest a role for EEG as a marker or predictor of response in the therapeutic setting The widespread effects of tDCS on cortical functioning and disturbances in prefrontal regions can be effectively assessed using a VWM paradigm The objectives of our study were to investigate the acute after-effects of tDCS on cortical EEG activity during a VWM task in patients with an affective disorder during a depressive episode. EEG data were acquired from participants enrolled in a double-blind, crossover study testing effects of a single session of active versus sham tDCS. We studied EEG activity during a VWM task, focusing on traditional evoked potentials as well as event-related spectral changes. Based on previous results on tDCS in healthy subjects, we expected that tDCS would improve working memory and modulate the corresponding activity of prefrontal cortical regions through the propagation of stimulation-induced activity through brain networks. Additionally, we hypothesised that assessing task-related EEG activity in a VWM paradigm would allow characterisation of cognitive sub-functions and quantitative neurophysiological markers affected by tDCS in patients experiencing depression.www.clinicaltrials.gov: NCT00763230) Eighteen participants were invited to participate in our study prior to entry into a clinical trial investigating tDCS stimulation for the treatment of depression at the Black Dog Institute, Sydney, Australia  The study protocol was a double-blind sham-controlled crossover design where participants were randomised to one of two arms of the study in which they received either tDCS followed by sham (Group 1) or sham followed by active tDCS (Group 2) in a ratio of 1∶1 . Hence, Participants were seated in a light and sound attenuated room and completed a VWM paradigm http://cerco.ups-tlse.fr/~barbeau/, accessed November 2005), which do not lend themselves easily to verbal naming. Images and locations were randomised to ensure an even presentation of true and false outcomes. By manipulating the ratio of target pictures to background fillers, memory load can be parameterised from easy  to medium  and hard  whilst keeping the visual load and visual scan path constant. Participants undertook 12 trials of each level of difficulty with a total of 36 trials. The order of trials was counterbalanced across subjects.The stimuli consisted of a 5×5 grid on which pictures and filler items were presented. Picture stimuli consisted of abstract, multi-coloured designs obtained from an online database  and custom electrode caps  arranged according to the international 10–20 system. All data were referenced against an electrode centred on the midline between Fz and Cz and sampled at 5 kHz. Electrodes impedances were set below 5 kΩ. The electrooculogram and two electrocardiogram channels were also recorded. The delay between tDCS and the EEG acquisition – due to placement of the cap – was approximately 60 minutes.Task-related EEG data were characterized by conventional ERP analysis to investigate time-locked changes in scalp voltage, and by time-frequency decomposition to assess changes in oscillatory activity that reflect event-related synchronization (ERS) and desynchronization (ERD) of a cortical population EEG data were analysed using Brain Vision Analyzer software (version 2.0.3). Data was segmented, down sampled to 2 kHz and filtered using a Butterworth zero-phase high-pass filter (cut-off frequency 0.5 Hz). To remove artefacts, EEG data was decomposed using independent components analysis (ICA) using the infomax algorithm. Components containing eye, heart or muscle artefacts were rejected and the remaining component were back-transformed to obtain artefact-corrected EEG data. Cleaned EEG data was re-referenced to the average reference and filtered using a low-pass 45-Hz Butterworth zero-phase filter.ERP analysis was used to assess differences between active and sham tDCS conditions on cognitive manipulations relating to memory load. EEG data were aligned to the stimulus onset of the encoding (t = 6 s), maintenance (t = 12 s) and retrieval phase (t = 18 s), and averaged across trials. Grand averages over 14 participants were subsequently calculated and ERP components were labelled as P1, N2, P3 and slow wave corresponding to the time intervals of 90–160, 160–250, 250–400 and 400–800 ms, respectively. The average amplitudes (voltage deviations) over these time intervals were computed and analysed for statistical significance.Wavelet decompositions were used to characterise the event-related changes in spectral power during the VWM task. Event-related synchronisation and desynchronisation (ERS/ERD) in identified frequency bands were subsequently computed for statistical comparison across conditions intervention (active or sham tDCS) and memory load . For the behavioural data, the percentage correct responses, i.e. hits and correct rejections, reaction times, and d-prime were compared across conditions to assess intervention effects on cognitive performance. All ERP and ERS/ERD components were identically analysed using a 2×3 ANOVA with statistical significance set at p<0.05. The ANOVA was repeated for multiple EEG channels, task conditions and time intervals and significance threshold was adjusted for multiple comparisons using a false discovery (FDR) approach that accounts for the number of conditions and includes a correction for their correlations A 2×3 repeated-measures ANOVA was conducted on all dependent variables  with the within-subject factors memory load, consistent with the task design. Reaction time  = 75.6, p<0.0005)) and accuracy decreased  = 22.9, p<0.0005) with increasing memory load  = 19.68, p<0.0005). There were no significant main effects of intervention (active versus sham tDCS) on response accuracy, reaction times or d-prime (p>0.7). No significant interaction effects were observed.Behavioural data showed significant effects of ion time  increaseory load . In addiory load  decreaseEvent-related potentials showed waveforms consistent with visually evoked activity. intervention and memory load on the N2 component during the retrieval interval (18–19 s) was found in channel FCz  = 6.87, p = 0.004). Post-hoc t-tests revealed a significant reduction in N2 amplitude after active tDCS compared to sham stimulation in the medium load condition (t(13) = 4.01, p = 0.001; intervention or interaction effects were found for the event-related potentials.A significant interaction effect of Visual inspection of changes in the spectral content of the EEG across a broad range of frequencies revealed that task-related changes manifest in the theta (4–8 Hz) and alpha (8–12 Hz) frequency bands . Spectraintervention and memory load was observed for alpha power in parietal channel Pz during the maintenance period (t (13) = 2.63, p = 0.021) in the condition of high memory load. In addition to the effect on theta activity over parietal cortex, a main effect of intervention was observed for theta power in frontal channel FCz during the retrieval period (t (13) = 2.19, p = 0.048) in theta power for the medium task load. No other significant intervention or interaction effects were found for ERS/ERD in the theta or alpha band.Statistical analysis of ERS/ERD data revealed robust tDCS intervention effects on power in both the alpha and theta frequency ranges in frontal and parietal channels . In partAfter the current study all participants continued with tDCS treatment in a clinical trial to study the antidepressant efficacy over multiple tDCS sessions Despite its emerging therapeutic utility the effects of tDCS on cortical activity in patients with an affective disorder remains largely undetermined. Here we examined acute effects of a single session of anodal tDCS to the left DLPFC on task-related EEG activity using a sham-controlled crossover design. We assessed behavioural correlates, ERPs and spectral changes during a VWM task in these patients after receiving tDCS. Respectively we observed significant effects of tDCS on cortical EEG activity in both electrophysiological measures (ERPs and ERS/ERD) during the retrieval period of the VWM task, but found no significant effects on behavioural measures. A distinct finding was that active tDCS resulted in a reduction of the event-related N2 component over prefrontal brain areas during medium memory load. In addition, we observed a significant reduction in frontal theta activity following active compared to sham stimulation and increased occipito-parietal alpha desynchronisation after active tDCS during the retention phase.et al.et al.To our knowledge, this is the first sham-controlled EEG study investigating the effects of tDCS stimulation in a group of patients with an affective disorder. Our results corroborate previous findings from a case study which recorded EEG after anodal tDCS in a patient with major depressive disorder showing reduction in alpha and theta power The behavioural effects of tDCS in the literature are mixed and in our study we found no behavioural effects of tDCS on the VWM task. Previous studies have found improved task performance This exploratory study sought to elucidate the effects of tDCS on EEG measures in view of investigating potential biomarkers for use in a clinical setting. However, in the current study only 18 patients were recruited and four patients were excluded due to high levels of EEG artefacts or inadequate task performance. We chose a within subjects design to optimise the power, given the sample size. The within subjects design is an efficient use of participants time and allowed detection of differences across our different variables with sufficient statistical power to draw inference about the treatment effects. Our study was a satellite study to a clinical trial assessing the efficacy of tDCS in depression The current findings may also help to uncover potential physiological mechanisms underlying the clinical improvements following tDCS. The observed changes in theta and N2 were found in channel FCz over the medial frontal cortex. The modulation of these EEG components most likely reflects indirect effects of brain stimulations rather than direct effects on the left DLPFC. That is, the frontal theta rhythm in working memory has been associated with the medial frontal cortex In sum, the present study shows the suitability of EEG to detect the cortical after-effects of tDCS in patients experiencing depression see also . The obsText S1Correlation between EEG measures and subsequent improvement in clinical trial.(DOC)Click here for additional data file.


Differential gene expression profiles were classified by post-analyses of microarray data, followed by western blot analyses to confirm the microarray data of selected genes. In total, 243 genes were differentially expressed (125 upregulated and 118 downregulated) between the PCOS and non-PCOS granulosa cells. These genes are involved in reproductive system development, amino acid metabolism and cellular development and proliferation. Comparative analysis revealed genes involved in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) signaling pathways. Western blot analyses confirmed that mitogen-activated protein kinase kinase kinase 4 and phospho-ERK1/2 were decreased in PCOS granulosa cells. This study identified candidate genes involved in MAPK/ERK signaling pathways that may influence the function of granulosa cells in PCOS.Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. Although its aetiology and pathogenesis remain unclear, recent studies suggest that the dysfunction of granulosa cells may partly be responsible. This study aimed to use cDNA microarray technology to compare granulosa cell gene expression profiles in women with and without PCOS to identify genes that may be aetiologically implicated in the pathogenesis of PCOS. The study cohort included 12 women undergoing  Polycystic ovary syndrome (PCOS) is a common heterogeneous endocrinopathy characterised by hyperandrogenemia (HA), arrested ovarian follicle development, menstrual irregularity and polycystic ovaries that results in anovulation and infertility in women of reproductive age1245679Granulosa cells are important in ovarian folliculogenesis as they provide a suitable microenvironment for follicular development and oocyte maturation. In response to pituitary gonadotropin secretion, granulosa cells upregulate the expression of a variety of genes that encode the components of steroidogenic pathways involved in estrogen biosynthesis1213141516in vitro fertilization (IVF) have not yet been reportedDNA microarray technology is a suitable modality to examine changes in global gene expression among granulosa cells in PCOS and may aid in the identification of novel candidate genes that contribute to the aetiology of PCOS. Previous studies comparing gene expression arrays in theca cells, ovarian cells, oocytes, cumulus cells and granulosa cells between women with PCOS and without PCOS have identified several candidate pathways underlying differential gene expression among various cell types181920212223Therefore, we compared the gene expression profiles of non-PCOS and PCOS granulosa cells from preovulatory follicles using Affymetrix® microarray chips to assess global changes in granulosa cell gene expression profiles and alterations in genetic networks, signal transduction, transcription/translation and/or metabolic pathways critical to PCOS granulosa cell phenotypes that may impact oocyte maturation and follicular development.2 to minimize the effects of obesity and fasting blood glucose . The study protocol was approved by the Research Ethics Committee of the National Taiwan University Hospital  and conducted in accordance with approved institutional guidelines. All participants provided signed informed consent.All study participants were women undergoing IVF at the Center for Reproduction at the National Taiwan University Hospital . A diagnosis of PCOS was based on the Rotterdam diagnostic criteriaAfter overnight fasting, venous blood samples were collected in the early follicular phase from all subjects, allowed to clot for 30 min and then centrifuged at 2500 rpm for approximately 10 min to obtain serum. Serum total testosterone levels [detectable range (DR): 0.03–14.4 ng/mL] were measured using radioimmunoassay 2526During ovarian stimulation, the dose of gonadotropin  was adjusted for each individual patient according to FSH levels, serum E2 concentrations and growth and number of follicles. Follicular fluid was aspirated and pooled from small antral follicles (4–8 mm in diameter) visible on ultrasonography during the retrieval of transvaginal oocytes for IVF. The granulosa cells were isolated and prepared from follicular aspirates after the removal of oocytes. The samples were then centrifuged at 400 × g for 10 min to separate the fluid from cells. The cell pellets were resuspended in Dulbecco’s phosphate-buffered saline , layered on 50% Ficoll–Paque™ density gradient media  and then centrifuged at 400 × for 30 min to remove maximum red blood cells. The granulosa cells at the interface were collected and washed with DPBS. Total RNA was extracted from individual patient samples using the RNeasy® Mini Kit  according to the manufacturer’s instructions and then treated with deoxyribonuclease. The concentration of isolated RNA was determined using a NanoDrop® ND-100 spectrophotometer . The RNA extracts were stored at −80 °C until all samples were collected. The quality and integrity of RNA samples were analyzed using the Agilent 2100 Bioanalyzer .Affymetrix® Gene Chip Human Genome Gene 1.0 ST microarray chips  were hybridized at the National Health Research Institutes Microarray Core Facility . Briefly, total RNA was reverse transcribed into double-stranded cDNA using an oligohttp://www.ingenuity.com) to identify gene networks. Gene networks were used to identify biologic functions, molecular processes, disorders and pathways of genes identified by Limma data analysis.Data were normalized using quantile normalization methodsTotal protein was extracted from an additional three non-PCOS granulosa cells and three PCOS granulosa cells, and 20 μg of protein per lane was loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes and blocked for 1 h with 5% nonfat milk in Tris-buffered saline with Tween-20 (Gibco). The membranes were incubated overnight at 4 °C with one of the following primary antibodies: phosphorylated extracellular signal-regulated kinase , ERK (diluted 1:1000), mitogen-activated protein kinase kinase kinase 4 , or glyceraldehyde 3-phosphate dehydrogenase  . Next, PVDF membranes were incubated for 60 min at room temperature with secondary antibodies  and then immune complexes were detected by chemiluminescence using a western blotting analysis system . Relative protein levels per sample were then normalized to GAPDH signals.U test, Student’s t-test and paired t-test of variance were used where appropriate for statistical analyses. Data from at least three independent measurements are presented as means ± standard deviation of the mean (SEM). The SYSTAT statistical graphics software package  was used for analysis, and a probability (P) value of <0.05 was considered statistically significant.Clinical features of interest were selected as dependent variables. The Mann–Whitney The granulosa cells were isolated from six women without PCOS and six women with PCOS undergoing IVF. The clinical and biochemical characteristics of study participants are listed in All 12 samples were analyzed to select genes that were significantly upregulated or downregulated. A total of 243 genes were differentially expressed in the granulosa cells from the non-PCOS group compared with those from the PCOS group . DiffereThe functional differences between the two groups were used to construct a differential gene–protein interaction network of 243 differentially expressed genes identified in granulosa cells. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis suggested that these genes were typically involved in MAPK signaling, VEGF signaling, androgen and estrogen metabolism and drug metabolism . Of a toIn this study, we coupled the KEGG and IPA data to identify statistically significant differences in MAPK/ERK signaling between the non-PCOS and PCOS granulosa cells. To further validate whether there were differences at the protein level, we compared MAP3K4 expression in granulosa cells obtained from three other independent patients from each group by western blot analysis and observed a 30% reduction in MAP3K4 expression in the PCOS granulosa cells . The mos31The purpose of this study was to use DNA microarray data to identify differentially expressed genes in the granulosa cells of women with and without PCOS to provide novel information for the further study of associated disease mechanisms and also to bring about advancement in disease treatment. These microarray profiles indicated that the levels of activated MAP3K4 and p-ERK1/2 were reduced in the PCOS granulosa cells, which are potentially correlated with reproduction. These findings provide further evidence that the MAPK signaling pathway plays an important role in the pathogenesis of PCOS. Furthermore, these differentially expressed genes were also involved in androgen and estrogen metabolism, drug metabolism and VEGF signaling.2 and glucose AC level of 70–100 mg/dL were included to minimize the effects of obesity and insulin resistance.A limitation to this genetic study of PCOS was the relatively small number of subjects, which was due to the limited clinical access to patient tissues. PCOS is a genetically complex heterogeneous endocrinopathy affecting 5%–10% of reproductive age women. The strength of this study was that PCOS was assessed only among women of reproductive age  who were undergoing IVF. It was particularly difficult to collect sufficiently large cohorts of such patients because our recruitment of women with PCOS was based on the Rotterdam diagnostic criteria and only those with a normal BMI of 19–24 kg/mWe used microarray analysis, IPA and KEGG data to identify several important pathways, biological function networks and groups of significant candidate PCOS genes. We also compared pathways to identify common, unique and unions of pathway participants by calculating and comparing the significance of various potential genes between our cases and controls. Although differences in gene expression profiles were fairly small, the significance fulfils the criteria of the IPA software for further comprehensive research of these unique genes.Among the 10 most highly differentially expressed genes detected in this study, some were previously shown to be associated with PCOS, including aldehyde oxidase 1, DEAH (Asp-Glu-Ala-His) box polypeptide 16, chromosome 1 open reading frame 54 and F-box protein 519213336Our results also indicated that a group of genes that were upregulated or downregulated in the PCOS granulosa cells were involved in the reproductive system development and function. These genes included the transcription regulators, T-cell acute lymphocytic leukemia 1 and zinc finger and BTB domain containing 17; the transporters, neuroglobin and alpha-2-glycoprotein 1and the zinc-binding proteins, PLA2G4A, SRD5A2, TK1, GDF9, myeloperoxidase, transmembrane receptor lymphocyte-activation gene 3 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1. The PLA2G4A enzyme is stimulated by the preovulatory LH/hCG surge in granulosa cells of ovulatory follicles4045474951In conclusion, we identified several genes that were differentially expressed between non-PCOS and PCOS granulosa cells. The present data show for the first time that expression of the novel player MAP3K4 was decreased in the PCOS granulosa cells and participated in the MAPK/ERK signal transduction pathway. These differentially expressed genes are involved in various biological functions, particularly reproduction and development, and provide a mechanism through which granulosa cells may contribute to the pathogenesis of PCOS. These results will help to further clarify the molecular basis and the functional significance of several novel genes involved in the pathogenesis of PCOS.How to cite this article: Lan, C.-W. et al. Functional microarray analysis of differentially expressed genes in granulosa cells from women with polycystic ovary syndrome related to MAPK/ERK signaling. Sci. Rep.5, 14994; doi: 10.1038/srep14994 (2015).


Capnocytophaga canimorsus is a bacterium from the normal oral flora of dogs and cats that causes rare generalized infections in humans. In an attempt to determine whether infections could be caused by a subset of strains and to identify pathogenicity factors, we sequenced the genomes of three strains isolated from human infections. Capnocytophaga canimorsus is a Gram-negative commensal bacterium from the normal canine oral flora that causes life-threatening septicemia in patients who have been in contact with dogs or cats , were isolated from human patients (blood samples) who developed septicemia (C. canimorsus strain 5 (Cc5)   and to crameters . Genome rameters . The gen’s mouth . Also, 1CDOJ00000000 (Cc2), CDOK00000000 (Cc11), and CDOE00000000 (Cc12). The versions described in this paper are the initial versions.These whole-genome shotgun projects have been deposited in ENA under the accession numbers