Tas2r (bitter receptor) genes in vertebrate species, we tested the hypothesis that if bitter perception exists primarily to protect animals from poisonous plant compounds, the genome of the domestic cat (Felis catus) should have lost functional bitter receptors and they should also have reduced bitter receptor function. To test functionality of cat bitter receptors, we expressed cat Tas2R receptors in cell-based assays. We found that they have at least 7 functional receptors with distinct receptive ranges, showing many similarities, along with some differences, with human bitter receptors. To provide a comparative perspective, we compared the cat repertoire of intact receptors with those of a restricted number of members of the order Carnivora, with a range of dietary habits as reported in the literature. The numbers of functional bitter receptors in the terrestrial Carnivora we examined, including omnivorous and herbivorous species, were roughly comparable to that of cats thereby providing no strong support for the hypothesis that a strict meat diet influences bitter receptor number or function. Maintenance of bitter receptor function in terrestrial obligate carnivores may be due to the presence of bitter compounds in vertebrate and invertebrate prey, to the necessary role these receptors play in non-oral perception, or to other unknown factors. We also found that the two aquatic Carnivora species examined had fewer intact bitter receptors. Further comparative studies of factors driving numbers and functions of bitter taste receptors will aid in understanding the forces shaping their repertoire.Cats are obligate carnivores and under most circumstances eat only animal products. Owing to the pseudogenization of one of two subunits of the sweet receptor gene, they are indifferent to sweeteners, presumably having no need to detect plant-based sugars in their diet. Following this reasoning and a recent report of a positive correlation between the proportion of dietary plants and the number of  Tas1r2) is a pseudogene. This loss has happened independently in several obligate carnivore species in the order Carnivora  feeding on the foliage of eucalyptus species, which are typically rather poisonous to most animal species) [What factors could maintain bitter taste receptor function in terrestrial obligate carnivores? First, it could be that even obligate carnivores such as cats are actually exposed to plant material through consumption of prey viscera that contains plant material consumed by the prey. There are two arguments against this having an important role. First, plants eaten by prey may not be bitter and highly toxic since the prey species consumed them themselves. However, some species have evolved detoxification mechanisms enabling them to consume potentially toxic plants  . Second,species) .Another possible reason for maintenance of bitter receptor number and function in cats and perhaps other carnivores is that there are also bitter compounds in many non-plant prey items in the carnivore diets (but see reference ). For exA third reason why the number of bitter taste receptors may not be strongly influenced by the amount of dietary plant material relates to the possible non-oral functions of these receptors. Bitter receptors are found in cell types other than taste on the tongue. Neither the natural ligands nor the functions of these receptors are fully known, but we suggest they may be important in maintaining bitter receptor functionality in species that might not otherwise “need” them to avoid plant-based or animal-based toxins. For instance, respiratory-expressed bitter receptors are important for innate defense against bacterial infections –35.Domestic cats have a reputation as picky eaters. Perhaps their apparently intact bitter system provides some explanation for their dietary habits. While the cell-based assays used here may not exactly recapitulate the native taste system, it allows investigators to gauge the response of the cat to bitter chemicals, including items that are poisonous, without risk to the animal. Commercial cat foods and veterinary medicines may contain bitter compounds that at least some cats find off-putting, and one way to predict their response in advance is through use of these cell-based systems.S1 Table). The coding sequences were inserted into an expression cassette with the first 45 amino acid residues of rat somatostatin receptor 3 as the signal peptide at the N-terminal and the herpes simplex virus glycoprotein D epitope at the C-terminal, following previously established methods [The coding sequences of feline Tas2r receptors were amplified from feline genome DNA using gene-specific primers , diphenidol hydrochloride (Reagent World), and limonin (LKT Laboratories), all chemicals were purchased from Sigma-Aldrich. For the catalog numbers and concentrations used in the assays, see of et al  to ensurHuman embryonic kidney 293 (Peakrapid) cells were obtained from ATCC and cultured in Opti-MEM supplemented with 5% fetal bovine serum. Cells were seeded in 96-well plate at a density of 50,000 per well. The next day, cells were transiently transfected with a Tas2r construct (0.1 μg/well) with a coupling chimeric G protein Gα16-gust44 (0.1 μg/well) using Lipofectamine 2000 (0.5 μl/well). After 24 h, cells were washed once with Hanks’ balanced salt solution (HBSS) and loaded with Fluo–4 for 1 h. After three washes with HBSS, cells were assayed for their responses to bitter compounds using a FlexStation III. Relative fluorescence units  were read every 2 s after addition of HBSS supplemented with 2× tastants. Calcium mobilization traces were recorded.Calcium mobilization was quantified as described previously . In shorwww.cbs.dtu.dk/services/TMHMM) [The protein sequences of dog and ferret Tas2rs obtained from Li and Zhang  were uses/TMHMM) . The deds/TMHMM) . A phylos/TMHMM) . All DNAS1 Dataset(TXT)Click here for additional data file.S2 Dataset(TXT)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Follistatin (FST), a single chain glycoprotein, is originally isolated from follicular fluid of ovary. Previous studies have revealed that serum FST served as a biomarker for pregnancy and ovarian mucinous tumor. However, whether FST can serve as a biomarker for diagnosis in lung adenocarcinoma of humans remains unclear.The study population consisted of 80 patients with lung adenocarcinoma, 40 patients with ovarian adenocarcinoma and 80 healthy subjects. Serum FST levels in patients and healthy subjects were measured using ELISA. The results showed that the positive ratio of serum FST levels was 51.3% (41/80), which was comparable to the sensitivity of FST in 40 patients with ovarian adenocarcinoma  using the 95th confidence interval for the healthy subject group as the cut-off value. FST expressions in lung adenocarcinoma were examined by immunohistochemical staining, we found that lung adenocarcinoma could produce FST and there was positive correlation between the level of FST expression and the differential degree of lung adenocarcinoma. Furthermore, the results showed that primary cultured lung adenocarcinoma cells could secrete FST, while cells derived from non-tumor lung tissues almost did not produce FST. In addition, the results of CCK8 assay and flow cytometry showed that using anti-FST monoclonal antibody to neutralize endogenous FST significantly augmented activin A-induced lung adenocarcinoma cells apoptosis.These data indicate that lung adenocarcinoma cells can secret FST into serum, which may be beneficial to the survival of adenocarcinoma cells by neutralizing activin A action. Thus, FST can serve as a promising biomarker for diagnosis of lung adenocarcinoma and a useful biotherapy target for lung adenocarcinoma. Follistatin (FST), a single chain glycoprotein, is originally isolated from follicular fluid of ovary, which has the effect on inhibiting the secretion of follicle-stimulating hormone (FSH) of pituitary cells The most commonly diagnosed cancers worldwide are lung cancer , breast cancer  and colorectal cancer , and the most common cause of cancer death is lung cancer  It was reported that lung tumors can produce neuroendocrine hormone, for example, some small cell lung carcinomas secrete adrenocorticotropic hormone (ACTH) The study was approved by The Ethics Committee of China-Japan Union Hospital of Jilin University, China, and written informed consents were obtained from all participants prior to study entry.Patients were diagnosed according to the new lung cancer staging system Serum samples were obtained according to the previously described methods Serum FST levels were measured in patients with lung adenocarcinoma, ovarian mucinous adenocarcinoma, lung benign disease tuberculosis and healthy subjects, respectively in triplicate, using ELISA kit  according to manufacturer’s instructions. Absorbance was then detected at 450 nm to quantify FST levels.Ten slides of each type lung adenocarcinoma in well differetiation, moderate differetiation and poor differetiation were obtained from the Department of Pathology at China-Japan Union Hospital of Jilin University for immunohistochemical staining. Immunohistochemical staining for FST was carried out as previously described 5 cells were plated into 12-well cell culture plates and cultured in Iscove’s Modified Dulbecco’s Medium (IMDM)  supplemented with 10% heat-inactivated fetal calf serum (FCS) . Cells were incubated at 37°C in a humidified, 95% air and 5% CO2 atmosphere for 24 h. Then the cells were washed twice with IMDM, and cultured in IMDM with 2.5% and 10% FCS for 24, 48 and 72 h, respectively. Finally, the supernatant of cultured cells was harvested by centrifuging at 1,500 g for 10 min at 4°C, and stored at −80°C for the measurement of FST levels.Lung adenocarcinoma tissues and non-tumor lung tissues as control were obtained from patients who underwent a surgical resection. Single cell suspension was prepared by enzyme dispersion 2 incubator Human lung adenocarcinoma cell line A549 was obtained from the American Type Culture Collection , and cultured in modified polystyrene T-75 tissue culture flasks in RPMI 1640  supplemented with 10% FCS at 37°C in a humidified 5% CO4 cells per well and incubated in the absence or presence of various concentrations of recombinant human activin A  or anti-FST antibody (1 µg/ml) in 2.5% FCS-RPMI 1640 for 24 h. Cell proliferation was determined by using the cell counting kit-8  and measured by microplate reader scanning at 450 nm according to the manufacturer’s instruction A549 cells were plated in triplicate in a 96-well plates at 2×105 cells per well and pre-cultured in 2.5% FCS-RPMI 1640 with or without 1 µg/ml anti-FST monoclonal antibody for 2 h, and then were incubated in the presence of 10 ng/ml activin A for 24 h. A549 cells were stained by Annexin V-Alexa Fluor-488/PI and the stained cells were analyzed by flow cytometry to determine the percentages of AnnexinV+/PI− (early apoptosis) and AnnexinV+/PI+ (late apoptosis) cells. The experiment was repeated 3 times.Annexin V-PI staining was used to evaluate A549 cells apoptosis according to the manufacturer’s instructions . Briefly, A549 cells were plated in a 12-well cell culture plates at 2×10Sensitivity for lung cancer was considered as 100×[the number of lung cancer patients with positive tumor marker (true positive)/total number of lung cancer patients ] %, specificity for lung cancer as 100×[the number of control healthy subjects with negative tumor marker (true negative)/total number of control healthy subjects ] %, The main study participants comprised 80 healthy subjects, 40 patients with stage I–III ovarian mucinous adenocarcinoma and 80 patients with stage I–III lung adenocarcinoma. Demographic and clinical characteristics of the study participants are given in By assaying serum FST levels, we found that it was significantly higher in patients with lung adenocarcinoma and ovarian mucinous adenocarcinoma than that in healthy control group P<0.01, . In contUsing the 95th confidence interval in the healthy subjects as the cut-off value, there is no difference in the positive ratio of serum FST levels between female (50%) and male (52.2%) patients with lung adenocarcinoma. In contrast, the positive ratio of serum FST levels were related to disease stages and differentiation grades of lung adenocarcinoma and ovarian mucinous adenocarcinoma .To confirm FST expression in lung adenocarcinoma, immunohistochemical staining was performed to examine FST protein. The results showed FST immunoreactivity in the glandular duct-like tissues of lung adenocarcinoma. The expression of FST in lung adenocarcinoma was correlated with the differentiation, that is to say, its expression became more pronounced as the grade became worse, i.e. from well-differentiated to poorly-differentiated lung adenocarcinoma . Using Ein vitro and FST levels in the supernatants of cultured cells were detected using ELISA. As shown in To clarify FST production in lung adenocarcinoma, cells obtained from lung adenocarcinoma tissues and non-tumor lung tissues were cultured Previous studies have reported that activin A can inhibit proliferation and induce apoptosis of lung cancer cells P<0.01).To further investigate the biological role of FST, we analyzed the effect of FST on activin A-induced lung adenocarcinoma cell apoptosis. The results showed that after lung adenocarcinoma cells were cultured only in the presence of 10 ng/ml activin A for 24 h, there was no significant apoptosis of lung adenocarcinoma cells compared with control P>0.05, . While aBased on the global cancer statistics, lung cancer was the most commonly diagnosed cancer and the leading cause of cancer death in males in 2008 globally, and among females, it was the fourth most commonly diagnosed cancer and the second leading cause of cancer death Planque and his coleagues To confirm FST production from lung adenocarcinoma, cells derived from lung adenocarcinoma and non-tumor lung tissue were cultured and the levels of FST in the supernatant were detected by ELISA. The data revealed that lung adenocarcinoma cells secreted FST in FCS-dependent and time-dependent manner, while cells from non-tumor lung tissues could not secrete FST. Lung adenocarcinoma cells secreted FST in a serum-dependent manner, which is consistent with the FST secretion of pituitary cells as described previously Activin has very extensive bioactivities, such as cell differentiation, proliferation, apoptosis and regulation of immune responses In conclusion, the data in our present studies demonstrated that the elevated FST in serum of patients with lung adenocarcinoma was directly produced by lung adenocarcinoma cells, and might promote the survival of lung adenocarcinoma cells. Thus FST may be an important biomarker for diagnosis of lung adenocarcinoma in humans and a useful target for lung adenocarcinoma biotherapy in humans.


Peripheral insulin resistance and hyperandrogenism are the primary features of polycystic ovary syndrome (PCOS). However, how insulin resistance and hyperandrogenism affect uterine function and contribute to the pathogenesis of PCOS are open questions. We treated rats with insulin alone or in combination with human chorionic gonadotropin (hCG) and showed that peripheral insulin resistance and hyperandrogenism alter uterine morphology, cell phenotype, and cell function, especially in glandular epithelial cells. These defects are associated with an aberration in the PI3K/Akt signaling pathway that is used as an indicator for the onset of insulin resistance in classical metabolic tissues. Concomitantly, increased GSK3β (Ser-9) phosphorylation and decreased ERK1/2 phosphorylation in rats treated with insulin and hCG were also observed. We also profiled the expression of glucose transporter (Glut) isoform genes in the uterus under conditions of insulin resistance and/or hyperandrogenism. Finally, we determined the expression pattern of glycolytic enzymes and intermediates during insulin resistance and hyperandrogenism in the uterus. These findings suggest that the PI3K/Akt and MAPK/ERK signaling pathways play a role in the onset of uterine insulin resistance, and they also suggest that changes in specific Glut isoform expression and alterations to glycolytic metabolism contribute to the endometrial dysfunction observed in PCOS patients. Insulin resistance is traditionally defined as insensitivity or unresponsiveness to the metabolic actions of insulin, and thus requires an increased insulin level in order to achieve a given metabolic action in its target tissues3468Insulin plays a pivotal role in glucose and lipid metabolism through the phosphorylated insulin receptor (IR), insulin receptor substrates (IRSs), and the phosphatidylinositide-3-kinase (PI3K) signaling pathwayet al. reported that the development of insulin resistance is different between tissues in obese female mice, suggesting a tissue-specific difference in insulin resistance in vivo171819206To date, the significant findings related to IR signalling defects under conditions of hyperinsulinemia and insulin resistance have been mostly obtained from metabolically relevant tissues such as adipose tissue, skeletal muscle, the liver, and the ovary14in vivoin vivo and, if so, if the insulin resistance would lead to altered glycolytic metabolism in the uterus.Rodent models of insulin and human chorionic gonadotropin (hCG) treatment222325282930Our results showed that both hCG-treated and insulin+hCG-treated rats gained body mass and ovarian weight . Increas232528232523282930in vivo2829Fasting glucose levels were significantly increased in insulin+hCG-treated rats . However222325282223Because the uterine weights of hCG-treated and insulin+hCG-treated rats increased , we wentBecause cytokeratin 8 and vimentin are well known as cellular marker proteins for the identification of epithelial and mesenchymal/stromal cells in the rodent uterusTo further assess the effect of insulin and hCG in cell mitosis and apoptosis, we examined the expression of phospho-histone H3 and cleaved caspase-3 using immunohistochemical analyses. While phospho-histone H3 was found to have a diffuse distribution in all groups , the intAberrations in the PI3K/Akt signaling pathway have been implicated in insulin resistance in several peripheral tissues1Because the immediate effect of an activated insulin signaling pathway is glucose uptake, which depends on the actions of a number of Glut proteinsHaving found that most of the Glut genes were downregulated in hCG-treated and insulin+hCG-treated rats, we examined whether such alterations might affect glycolytic metabolism by measuring the expression of glycolytic enzymes and intermediates using Western blot analysis . QuantitIn the present study, we extend previous research into the dissection of the molecular mechanism of uterine insulin resistance by investigating the altered insulin-mediated PI3K/Akt signaling pathway and glycolytic metabolism in the rat uterus. We present evidence that combined chronic treatment with insulin and hCG results not only in peripheral insulin resistance, as reported in previous studies28Because of the heterogeneous nature of PCOS622232425in vivoin vitroIGF mRNA and circulating IGF-1 protein levels are higher in PCOS patients than normal subjectsThe diverse biological effects of insulin are believed to be mediated by interactions with IRα and ligand-mediated autophosphorylation of IRβ in different tissues and cells204041IDE represents a key player in the regulation of peripheral and cellular insulin and IGF degradation, and impaired endogenous IDE activity has been implicated in several human diseases such as type 2 diabetes mellituset al.In the present study, we have analysed the expression of crucial components of the PI3K/Akt signaling pathway as they relate to the alteration of insulin signaling in metabolic tissues during the development of insulin resistanceWe attempted to determine whether inhibition of the PI3K/Akt signaling pathway results in alteration of Glut isoform expression and glycolytic metabolism. Our studies reveal that both insulin-dependent and -independent Glut isoform mRNA expression is more frequently decreased in insulin+hCG-treated rats compared to saline-treated and insulin-treated rats. These findings are in agreement with the conclusion that inadequate expression of different Glut isoforms in the uterus is associated with impaired cellular function41503in vivo effects of chronic insulin and hCG exposure on the development of uterine insulin resistance. In addition, our results demonstrate that dysregulation of the IR-mediated PI3K/Akt and MAPK/ERK signaling pathways and glycolytic metabolism in the uterus is strongly associated with insulin resistance and hyperandrogenism. We speculate that peripheral and local insulin resistance might share the same insulin/IGF receptor signaling mechanism and thus inhibit the PI3K/Akt signaling pathway that regulates uterine cell metabolism and function. There is great uncertainty regarding the relative importance of different tissues in the development of insulin resistance, and further investigation is warranted into whether the induction of uterine insulin resistance is a primary determinant for uterine abnormalities in PCOS patients.In summary, the results of this study highlight for the first time the hCG was from NV Organon . Human recombinant insulin (Humulin NPH) was from Eli Lilly . 3,3-diaminobenzidine tetrahydrochloride (DAB) was from Sigma-Aldrich . The avidin-biotinylated-peroxidase complex detection system (ABC kit) was from Vector Laboratories Inc. . The primary antibodies used for Western blot and immunohistochemical analyses in the present study, their dilution, and sources are listed in Seventy-day-old female Sprague Dawley rats were obtained from the Laboratory Animal Centre of Harbin Medical University . Animals were kept in groups with free access to food and water and a controlled temperature of 22 ± 2 °C with a 12 h light/dark cycle. All rats used in this study needed to have normal cycles prior to treatment, and these were confirmed by examination of vaginal smears under a light microscope for two sequential estrous cycles (about 8–10 days). All animal experiments were performed according to the National Institute of Health guidelines on the care and use of animals and were approved by the Animal Care and Use Committee of the Heilongjiang University of Chinese Medicine, China.th day and the 22nd day to induce hyperinsulinemia and peripheral insulin resistance (n = 21); (C) 3.0 IU/day hCG to induce hyperandrogenism (n = 21); or (D) insulin plus hCG to induce a combination of hyperinsulinemia and hyperandrogenism (n = 27). Animals were treated with twice-daily subcutaneous injections and decapitated on the 23rd day. The treated animals have been shown to suffer no hypoglycemic episodes2223The doses and treatment protocols for insulin and hCG are described in detail in the original papers2223Estrous cycles were monitored daily by vaginal lavage according to a standard protocolOGTT was performed in live rats. One day after all treatments finished, animals were given oral doses of D-glucose (3 g/kg body weight) after a 10-h fast. Blood samples were collected from the tail vein, and glucose levels were measured using a glucometer monitor  before (0 min) and at 30, 60, 90, 120 and 180 min after the doseA detailed explanation of the Western blot analysis protocol has been published elsewhereImmunohistochemistry and immunofluorescence were performed according to previously described methods55After the sections were incubated with primary antibodies in TBST containing 5% nonfat milk overnight at 4 °C in a humidified chamber, a secondary antibody was applied at room temperature for 1 h. After the sections were washed with TBST, they were re-suspended in mounting medium containing DAPI  and examined under an Axiovert 200 confocal microscope  equipped with a laser-scanning confocal imaging LSM 510 META system (Carl Zeiss) and photomicrographed.−ΔΔCT method, and the efficiency of each reaction as determined via linear regressionQuantitative real-time RT-PCR (qRT-PCR) was performed with a Roche Light Cycler 480 sequence detection system . The PCR parameters were set according to the manufacturer’s protocols, and amplifications were performed with a SYBR green qPCR master mix . Single-stranded cDNA was synthesized from each sample (2 μg) with M-MLV reverse transcriptase  and 40 U RNase inhibitor . cDNA (1 μl) was added to a reaction master mix (10 μl) containing 2× SYBR green qPCR reaction mix (Thermo Scientific) and gene-specific primers (5 μM each of forward and reverse primers). For each sample, duplicate reactions were conducted in 96-well plates. All primers  were desSerum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E2), progesterone (P4), total testosterone (T), androstenedione (A4), sex hormone-binding globulin (SHBG), and insulin were determined using enzyme-linked immunosorbent assay (ELISA) kits . Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated to assess changes in insulin sensitivity58U-test for skewed data. For the OGTT studies, data were analyzed using one-way ANOVA repeated measures followed by Tukey’s post hoc test. A p-value less than 0.05 was considered statistically significant.Results are presented as the means ± SEM. Statistical analyses were performed using SPSS version 21.0 statistical software for Windows . No statistical method was used to pre-determine animal size. The normal distribution of the data was tested with the Shapiro-Wilk test. Differences between groups were analysed by one-way ANOVA followed by Tukey’s post hoc test for normally distributed data or the Kruskal–Wallis test followed by the Mann–Whitney How to cite this article: Zhang, Y. et al. Molecular characterization of insulin resistance and glycolytic metabolism in the rat uterus. Sci. Rep. 6, 30679; doi: 10.1038/srep30679 (2016).


Upon activation, platelets secrete more than 300 active substances from their intracellular granules. Platelet dense granule components, such as ADP and polyphosphates, contribute to haemostasis and coagulation, but also play a role in cancer metastasis. α-Granules contain multiple cytokines, mitogens, pro- and anti-inflammatory factors and other bioactive molecules that are essential regulators in the complex microenvironment of the growing thrombus but also contribute to a number of disease processes. Our understanding of the molecular mechanisms of secretion and the genetic regulation of granule biogenesis still remains incomplete. In this review we summarise our current understanding of the roles of platelet secretion in health and disease, and discuss some of the hypotheses that may explain how platelets may control the release of its many secreted components in a context-specific manner, to allow platelets to play multiple roles in health and disease. As well as serving as adhesion sites for coagulation factors , platelets themselves are an important source of those factors  signalling downstream from soluble agonists forming at the site of thrombus. Those soluble agonists, and in particular ADP released from dense granules of activated platelets, precipitate a number of positive feedback cascades leading to rapid activation of large numbers of platelets. Ultimately, an orchestrated ‘effort’ of many other secreted mediators and cells results in restoration of vessel integrity. Arguably, secretion is the most far reaching result of platelet activation, and as such can also account for functions of platelets beyond the immediate environment of the thrombus. Therefore platelet secretion is at the heart of the control of vascular integrity, in health and disease, and is the focus of this review.33.13H]-serotonin occurred more rapidly than PF-4 from α-granules or β-hexosaminidase from lysosomes, regardless of the agonist used to stimulate platelets Dense granules contain a range of small molecules 2 act back on circulating platelets and contribute to the positive feedback signalling that sustains platelet aggregation. The central role of the ADP–P2Y12 receptor axis in haemostasis is particularly well established. Platelet adhesion on vWF, inside-out activation of integrin αIIbβ3 and P-selectin expression are all defective in 12−/−P2Y mice JinxUnc13D mice) that show a complete absence of platelet dense granule secretion were generated. Those animals were also found to have significantly reduced aggregation and other markers of platelet activation such as α-granule and lysosome secretion and integrin activation 12 signalling in haemostasis and thrombosis, possibly even in different disease scenarios. The role of ADP emerges as more important and complex than initially thought and may account for some distinguishing features of thrombosis versus haemostasis. ADP/P2Y12 mediated signalling also enhances signalling towards procoagulant activity and thrombin activation Small molecules released from dense granules together with synthesised thromboxane A3.1.112 inhibition significantly decreasing the size of the ‘shell’ and not affecting the ‘core’. They also show that permeability of the ‘core’ to plasma-borne molecules is limited, suggesting that efflux of platelet granule contents in that tightly packed region could be similarly limited Despite many different agonists shown to drive platelet activation in vitro to completion, it is now known that in vivo platelet activation is non-homogeneous, possibly attributable to variations in shear under physiological conditions 12 positive feedback cascade, blocking the P2Y12 receptors and thus limiting platelet activation and aggregation and the risks of pathological thrombosis, particularly in the management of coronary heart disease 12 axis in haemostasis means that abnormal bleeding often occurs in patients treated with potent P2Y12 inhibitors such as clopidogrel, prasugrel or ticagrelor 12 signalling may help in discovering more selective platelet inhibitors that could specifically limit thrombosis without causing bleeding.The most effective anti-thrombotic treatments currently used specifically target the ADP/P2Y3.1.26) kinase required for synthesis of polyP showed that a 3-fold reduction in polyP levels led to widespread platelet function and coagulation defects, again suggesting a role in haemostasis Dense granule secretion is also important in coagulation. Released calcium for instance is required at several steps of the coagulation cascade and for activation of the prothrombinase pathway. Recently more insight was gained into the role of another dense granule cargo molecule, polyphosphate (polyP). PolyP is a highly anionic linear polymer that is synthesised from ATP and secreted by platelets after activation 3.2IIbβ3 and one third or more GPVI receptors reside in α-granules and are trafficked to the surface of the platelet following platelet activation, further amplifying platelet aggregation A number of α-granule cargoes, such as vWF and fibrinogen, act to propagate activation and aggregation of platelets at the site of injury. In addition, an estimated one third to a half of total α3.2.1−/−Tsp1 mice, but activation as measured by PS exposure was significantly reduced. Similarly, they also showed that in the whole blood perfusion model, −/−Tsp1 or −/−CD36 thrombi disintegrated faster, but the thrombus size was the same between genotypes. In vivo both −/−CD36 and −/−Tsp1 mice showed longer time to occlusion and increased embolization in thrombosis assays. The Tsp1/CD36 axis may therefore be another contributor to the complex haemostasis/thrombosis balance.In addition, a recent study identified the thrombospondin (TSP1)/CD36 axis as another pathway that may help differentiate physiological haemostasis from thrombosis. TSP1 is one of the most highly expressed proteins in platelet α-granules and unlike vWF or fibrinogen is present in plasma in very low concentrations. As shown by Kuijpers et al., knockdown of the TSP1/CD36 axis in mouse platelets leads to a defect in thrombus stabilisation without affecting primary haemostasis 3.2.2Factors V, XI and XIII are all stored in α-granules, as is the thrombin precursor, prothrombin. Platelet derived Factor V is a potent procoagulant, thanks to higher resistance to activated Protein C-mediated inactivation than its plasma counterpart and targeted release at the site of injury Importantly, platelet α granules also contain numerous anti-coagulation cargoes. Tissue factor pathway inhibitor (TFPI), protein S, protease nexin-2 (amyloid β-A4 protein), plasmin and its inactive precursor plasminogen can all limit progression of coagulation by inhibiting or cleaving activated clotting factors or initiating fibrinolysis 4Since the primary function of platelets is haemostasis, or prevention of bleeding, it is no surprise that many patients presenting with abnormal bleeding are found to have defects in their platelet function and secretion. There are two classes of heritable bleeding disorders associated with poor platelet secretion: (i) those that result from defective platelet granule formation, leading to low numbers of platelet granules and cargo and (ii) those that arise from defects in secretory machinery, where platelets may have normal numbers of granules, but have defects in their ability to secrete them. Both of these classes of secretion deficiency have helped us to understand the role and mechanisms of platelet secretion on different levels.4.1Platelets derive from megakaryocytes (MKs) — large polyploid progenitor cells that reside primarily in the bone marrow. The synthesis and packaging of granules are thought to occur mainly at the early megakaryocyte stage 4.1.1Since dense granules are lysosome-related organelles, the best described dense granule formation defects are associated with systemic lysosome-like organelle biogenesis defects Hermansky–Pudlak Syndrome (HPS) was first described in 1976 Another dense granule storage defect with much poorer prognosis than HPS is Chediak–Higashi syndrome (CHS) Isolated dense storage pool disorders not associated with systemic defects in lysosomal-related organelles are quite common and can result in mild to severe secretion defects with or without affecting secondary aggregation, and thus presenting with varying degrees of bleeding diathesis 4.1.2NBEAL2 (neurobeachin-like 2) gene in a large number of GPS cases NBEAL2 belongs to the same gene family as LYST responsible for CHS, and appears to be directly implicated in α-granule biogenesis in MKs. −/−Nbeal2 mice show defects in primary thrombus formation and are protected from inflammatory brain infarction following focal cerebral ischaemia α-Granules are the storage site for many proteins including those synthesised in MK or endocytosed from plasma, with multiple functions in haemostasis and other processes described later in this article. Mechanisms of α-granule formation are less well understood. Defects of protein packaging and α-granule biogenesis in MK result in a set of heterogeneous disorders collectively known as Grey Platelet Syndrome, resulting in a usually mild to moderate bleeding disorder which can however on occasions be life-threatening 4.2Relatively recently another family of congenital disorders was linked with its genetic correlates to help us understand how secretion in platelets is regulated. Familial haemophagocytic lymphohistiocytosis (FHL) patients present with an abnormal bleeding diathesis without any morphological changes to platelet granules. Unlike storage pool disorders that rarely affect both α- and dense granules, some FHL subtypes affect all granule types, suggesting a common ‘core’ mechanism to regulate secretion. In addition, systemic complications beyond platelets suggest a common mechanism of secretion in other haematopoietic cells.Soluble NSF Attachment Protein Receptor (SNARE) family of proteins, with other mammalian cells. In the simplest model, the universal SNARE-mediated fusion involves transport of the vesicle to the target membrane and ‘priming’ it for release, followed by calcium-mediated conformational change in the SNARE complex that leads to the completion of membrane fusion, and eventually to the release of granule contents Indeed, platelets share their secretory machinery, centred on the In FHL subtypes 3, 4 and 5, deficiency of MUNC13-4 5The roles of platelets are not limited to initial aggregation and plug formation. The cascade of events leading to vessel repair is summarised in 5.1After the stable fibrin and platelet rich clot forms to stop the immediate bleeding, the further steps of the repair process begin. Close bidirectional cooperation between the haemostatic and immune systems is required to ensure restoration of normal tissue function following the injury.In the arterial circulation, the high shear environment leads to wash out of any chemoattractant molecules from the site of the injury. Therefore platelets stably adherent to the ECM are required for ‘capturing’ circulating immune cells and recruiting them to the thrombus. Platelet α-granules mediate inflammatory responses both by expressing adhesion receptors that facilitate interactions with endothelial cells and leukocytes, and by secreting a wide range of chemokines. P-selectin translocates to the cell surface from α-granules on adherent platelets and can recruit circulatory monocytes, neutrophils and lymphocytes, inducing inflammatory responses in those cells Inflammatory cell infiltration of the arterial subendothelium, promoted by platelet cytokines, also contributes to atherosclerotic plaque formation −/−ApoE mice In addition to their essential roles in physiological responses to injury, platelet–immune cell interactions are also likely to be very important in the pathophysiology of atherosclerotic plaque formation. Modulation of platelet secretion could therefore help reduce plaque growth. For example, inhibition of platelet derived CXCR4–CCL5 heterodimer formation was shown to attenuate monocyte recruitment to inflamed vessel wall in 5.2Escherichia coli or Salmonella species Trauma to tissue or introduction of foreign objects into the vessel is the most direct way by which bacterial and viral pathogens can invade the body. Therefore it stands to reason that antimicrobial and haemostatic responses are intimately linked. Platelets are the most numerous cells first to arrive at the site of injury, and the role of platelets in combating pathogens is increasingly apparent. It has been known for many years that platelets localize and adhere to sites of bacterial lesions in the circulation, for example in endocarditis 5.3The final steps of the haemostatic response to external injury, or rupture of atherosclerotic plaque in the arterial circulation, lead to restoration of the integrity of the vascular wall. This process involves orchestrated proliferation and migration of smooth muscle cells (SMCs), fibroblasts and endothelial cells. Platelets' wealth of growth factors and chemokines is known to contribute to regulation of those processes. Platelet derived growth factor (PDGF) in particular is instrumental in regulating SMC proliferation and migration in both the arterial and venous circulation + bone marrow derived progenitor cell recruitment to the injury site and their differentiation into endothelial progenitor cells + progenitor cells into mature foam cells is of particular importance in atherosclerotic plaque formation Another important mediator in healing and remodelling is platelet-derived SDF-1α which mediates CD34In addition to angiogenic factors, platelets also store and secrete a number of tumour necrosis factor (TNF)-α-related apoptosis regulators such as CD95, Apo2-L and Apo3-L which can induce inflammatory responses and apoptosis in other circulating cells, as well as anti-apoptotic molecules In summary, the increasing understanding of the role of platelet secretion in wound healing and tissue regeneration led to the development of multiple applications, especially in trauma management. Autologous platelet-rich plasma gels and growth factor concentrates are used in a number of clinical scenarios, from healing of acute skin wounds and diabetic ulcers to regeneration of tendon, ligament and nerve tissue 6Platelets' wealth of potent bioactive molecules is essential for the regulation of the complex physiological processes outlined so far. However, their ability to influence other cells means that they are also central to the pathophysiology of disease. Platelet secretion can cause disease beyond the obvious thrombotic scenarios, such as atherosclerosis, or heart attacks and strokes resulting from occlusive thrombus formation in circulation. Multiple roles in conditions from cancer progression and metastasis, to chronic inflammatory conditions, sepsis and acute lung injury have also been proposed.6.1Early evidence for platelet function in cancer progression was originally derived from studies in a mouse model of severe thrombocytopenia Platelet α-granules contain a number of growth factors that are important in physiological wound healing and angiogenesis including VEGF, PDGF, epidermal growth factor (EGF), and transforming growth factor beta (TGFβ) JinxUnc13d mice, that lack expression of Munc13-4, were protected from lung metastases and suggested that lack of ATP secretion from platelet dense granules was responsible for that protection 2 receptors on endothelial cells contributes another mechanism that increases endothelial cell permeability and facilitates tumour cell extravasation, additional to the well-established pro-angiogenic functions of α-granule releasate.In addition, Schumacher et al. recently showed that 6.2Due to their ability to initiate and sustain inflammatory responses platelets are often one of the main culprits and reasons for mortality in a series of other conditions. Therefore in some cases, platelet inhibition or depletion correlates with better outcomes. The exception to that is sepsis, where a role for platelets in exuberating the disease was proposed. On the one hand, platelet accumulation in vital organs can cause inflammatory responses by platelet-mediated immune cell recruitment The platelet P-selectin/PSGL interaction mediating platelet–neutrophil interaction in infection, is also a proposed mechanism for monocyte recruitment accompanying transplanted graft rejection Via their pro-inflammatory potential, platelets are also linked to inflammatory bowel disease (elevated levels of RANTES are found in Crohn's disease patients) 7As outlined in this review, the roles of platelets extend beyond initial platelet adhesion and aggregation at the site of injury. Platelets are not ‘merely’ contributing to processes such as coagulation, inflammation, and antimicrobial responses or wound healing, they are arguably central and essential in the majority of those processes. As outlined, platelet secretion of 300 + molecules from intracellular stores is likely to be a major driver of those interactions  1 or PAR4 agonist resulted in the preferential release of VEGF or endostatin,  In 2012, the Whiteheart group performed a systematic quantification of granule secretion using micro-ELISA arrays for 28 distinct α-granule cargo molecules in response to 4 different agonists 8Platelet secretion is undoubtedly of pivotal importance for regulating not only canonical platelet functions, but also mediating their impact on other cells, and understanding platelet secretion may have consequences reaching far beyond the ‘platelet field’. Yet platelets remain less well studied in terms of mechanisms governing their function, relative to other secretory cells such as neuroendocrine or immune cells. In this review we have shown examples of the ways in which platelet secretion is essential for sustaining haemostasis and ensuring an adequate response to injury, but also how the same mechanisms may mediate pathological responses. This means that any approaches targeting platelets or platelet secretion have to be carefully tailored for each specific application. Dissecting mechanisms of differential secretion of different cargoes may be helpful in designing therapies that can avoid currently common bleeding side effects while limiting pathological inflammation, tissue hypertrophy or cancer cell metastasis.None.


Consistent with a neuroergonomics approach, task performance can be facilitated by non-invasive neuromodulation techniques, such as anodal transcranial direct current stimulation-atDCS  and were required to perform with their non-dominant left hand a grooved pegboard test (GPT) over four training blocks and a retest 2 weeks later. Three priming tDCS conditions were investigated on the right primary motor cortex (M1): (1) Sham: Sham ctDCS (15 min) 10 min before Sham Online atDCS (20 min); (2) Online atDCS: Sham ctDCS (15 min) 10 min before Online atDCS ; (3) ctDCS priming: ctDCS  10 min before Online atDCS . Transcranial magnetic stimulation (TMS) parameters  were assessed before and up to 60 min after the tDCS conditions. The results indicated that although both Online atDCS conditions improved GPT performance  over Sham after the four training blocks, only the priming ctDCS/Online atDCS condition further enhanced GPT performance 2 weeks later. These latter findings were explained in relation to homeostatic metaplastic mechanisms based on the Bienenstock-Cooper-Munro (BCM) theory that postulates a “sliding threshold” for bidirectional synaptic plasticity (Karabanov et al., The recent study of Christova et al.  aimed toAn important tDCS parameter that requires further investigation is the influence of the time delay between priming and test tDCS application on homeostatic metaplasticity and its effects on motor performance/learning (Karabanov et al., Since the design of the Christova et al.  study coIn conclusion, priming tDCS protocols are promising ways to optimize tDCS facilitatory effects on motor performance/learning, which has relevance from a neuroergonomic standpoint. Thus, future studies are necessary to determine the optimal polarity and timing of tDCS applications to modulate neuroplasticity and enhance performance in clinical, sports, and real-world settings.All authors listed, have made substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NMDA receptors (NMDAR) are glutamate-gated calcium channels that play pivotal roles in fundamental aspects of neuronal function. Dysregulated receptor function contributes to many disorders. Recruitment by NMDARs of calcium-dependent enzyme nNOS via PSD95 is seen as a key contributor to neuronal dysfunction. nNOS adaptor protein (NOS1AP), originally described as a competitor of PSD95:nNOS interaction, is regarded an inhibitor of NMDAR-driven nNOS function. In conditions of NMDAR hyperactivity such as excitotoxicity, one expects NOS1AP to be neuroprotective. Conditions of NMDAR hypoactivity, as thought to occur in schizophrenia, might be exacerbated by NOS1AP. Indeed GWAS have implicated NOS1AP and nNOS in schizophrenia. Several studies now indicate NOS1AP can mediate rather than inhibit NMDAR/nNOS-dependent responses, including excitotoxic signaling. Yet the concept of NOS1AP as an inhibitor of nNOS predominates in studies of human disease genetics. Here we review the experimental evidence to evaluate this apparent controversy, consider whether the known functions of NOS1AP might defend neurons against NMDAR dysregulation and highlight specific areas for future investigation to shed light on the functions of this adaptor protein. To address this controversy, here we review how NOS1AP might contribute to NMDAR signaling pathways, taking into account the structural motifs critical for interactions between these three proteins. We discuss previously proposed models and develop for consideration an additional model of nNOS/PSD95/NOS1AP function that is more consistent with experimental findings.Candidate downstream mediators of nNOS include p38MAPK and JNKs. Both are involved in disorders including neurodegeneration, cerebral ischemia and chronic pain .Subsequent structural studies clearly showed the core PDZ domain of nNOS is is sufficient to interact with NOS1AP. Furthermore, C-terminal residues of NOS1AP are required for interaction  Two-State Model  × V did not inhibit nNOS:PSD95 interaction and YAGQWGESV peptide co-precipitated in a ternary complex with nNOS and PSD95-PDZ2  5 Figure , and docinhibitor of nNOS function (Jaffrey et al., mediator of nNOS signaling and contributor to NMDAR/nNOS-dependent regulation of neuronal functions (Fang et al., functional inhibition of nNOS has actually been reported (Jaffrey et al., not directly inhibit the enzyme activity of nNOS (Jaffrey et al., NOS1AP was regarded as an The potential of nNOS:NOS1AP interaction as a candidate drug target for neurological and cardiovascular disorders (Li et al., Pre-conditioning should also be considered. While p38MAPK mediates excitotoxicity, paradoxically it also facilitates the resistance to toxic insults preceded by prior sub-toxic stimuli, in brain and tissues such as heart and liver (Hausenloy and Yellon, Clearly more work is required to understand the impact of NOS1AP on NMDAR-driven nNOS signaling pathways, both at the molecular level as well as in a range of translational models for those diseases and conditions urgently needing new therapeutic approaches, including neurodegenerative diseases, stroke, chronic pain, as well as depression and other psychiatric conditions. Only then can we determine whether we should seek to boost endogenous functions of NOS1AP or to inhibit its function to achieve desirable therapeutic outcomes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chlorine solutions (CS), mostly containing sodium hypochlorite (SH), have been widely used for hand hygiene (HH) in the West African countries affected by the Ebola outbreak due to unavailability of alcohol-based handrub solutions and soap, easiness of use and “fear” factors leading to  sense of safety given by using an easily available disinfectant. However, no HH guidelines recommend the use of CS and concerns have been raised about skin tolerability among users.We conducted a systematic review to investigate whether the use of CS causes skin side effects when used for HH.Contact Dermatitis journal and the reference lists of relevant articles were also screened separately.PubMed and EMBASE were searched on 26/09/14 with no time, age, human, language or geographical restrictions. Out of 3241 hits, 14 articles about skin side effects were included; 10 case reports, 3 surveys and one comparative study. Only one case report was related to the use of SH 4-6% for HH by a veterinary surgeon who developed allergic contact dermatitis (CD) with a positive patch test to SH at dilutions 100 times lower than 4-6 %. In 5 case reports, CD with a variable severity was reported following use of SH for disinfection (2 papers) and for domestic cleaning (3papers); these cases had positive patch tests for SH; conversely, their experimental control groups showed either no reaction or very low-intensity skin reactions. Two papers reported severe dermatitis related to environmental cleaning with SH, and 2 others reported unusual systemic allergic reactions with accidental exposure and bathing the foot. One comparative study showed that SH was the most irritating product even at low concentrations when compared to alcohol, chlorhexidine and iodine. Finally, one survey among 706 nurses showed that 33.5% of cases of allergic or irritant CD had a history of chlorine exposure; 2 other surveys among cleaners showed higher prevalence of hand dermatitis with SH exposure.Overall, only one report described allergic CD related to use of high-concentration CS for HH. Very low-quality evidence shows that SH used for other purposes might cause skin irritations even at low concentrations following bare skin exposure.None declared.


Cytotoxic T lymphocyte (CTL) responses against the HIV Gag protein are associated with lowering viremia; however, immune control is undermined by viral escape mutations. The rapid viral mutation rate is a key factor, but recombination may also contribute. We hypothesized that CTL responses drive the outgrowth of unique intra-patient HIV-recombinants (URFs) and examined gag sequences from a Kenyan sex worker cohort. We determined whether patients with HLA variants associated with effective CTL responses  were more likely to carry URFs and, if so, examined whether they progressed more rapidly than patients with beneficial HLA-variants who did not carry URFs. Women with beneficial HLA-variants (12/52) were more likely to carry URFs than those without beneficial HLA variants (3/61) . Beneficial HLA variants were primarily found in slow/standard progressors in the URF group, whereas they predominated in long-term non-progressors/survivors in the remaining cohort (p = 0.0377). The URFs may sometimes spread and become circulating recombinant forms (CRFs) of HIV and local CRF fragments were over-represented in the URF sequences (p < 0.0001). Collectively, our results suggest that CTL-responses associated with beneficial HLA variants likely drive the outgrowth of URFs that might reduce the positive effect of these CTL responses on disease progression. Millions of patients are already receiving expensive, life-long anti-HIV treatment, and millions more will require treatment because of the continued spread of HIV-1, particularly in resource-poor settings worldwide (www.unaids.org). The solution to this epidemic will require the development of a preventive vaccine and, potentially, therapeutic vaccinesThe immune system never successfully clears HIV-1 during the course of natural infection; however, in a few patients, the virus can be contained for decades through cytotoxic T lymphocyte (CTL) responses234All CTL responses can select for mutations within or flanking CD8 epitopes, which allows HIV-1 to escape CTL recognition and prevents infected cells from being killed. These mutations may affect HLA binding, T cell receptor contact sites and/or antigen processing278910In vitro studies have demonstrated that at least 2.8 crossovers occur per replication cycle per genome−5 mutations per nucleotide per cycle)141518Escape mutations may occur relatively slowly and one-by-one in individual viruses over time or they may spread rapidly en-bloc through the HIV-1 population by recombination. Recombination occurs when the viral reverse transcriptase (RT) protein switches between the two HIV-1 RNA genomes and generates a recombinant DNA strand during transcription. Most phylogenetic analyses of HIV-1 do not accommodate recombination because it violates the assumption of phylogeny inference of descent from a common ancestor161919212224By contrast, recombination between two different HIV subtypes is easy to identify and the effect of recombination between subtypes is evident on the epidemiological scale. Most circulating HIV-1 strains belong to the main (M) group, which is divided into 10 homogeneous subtypes , two sub-subtypes  and several circulating recombinant forms (CRFs) of two or more subtypes (n > 60). Although the number of CRFs far exceeds the number of pure subtypes/sub-subtypes, the majority of HIV-infected people worldwide remain infected by a pure HIV subtypes/sub-subtypes, although CRFs may predominate in specific regions  or CRF versus URF), HLA profile and clinical progression rate in HIV-infected female commercial sex workers (CSWs) from the Pumwani slum in Nairobi, Kenya, where several HIV-1 subtypes and CRFs co-circulate2728Approximately 1200 bases spanning the HIV-1 p17-p24 region from peripheral blood mononuclear cell (PBMC) DNA samples from each of the 113 patients from Pumwani were PCR amplified and sequenced using population sequencing to optimize primers followed by single genome amplification of the PBMC DNA and sequencing. The sequences were subtyped and each section of the recombinant sequences was annotated with breakpoints using jpHMMThe following HIV-1 subtypes were prevalent in our cohort: HIV-A1, 61% (69/113); HIV-C, 6% (7/113); and HIV-D, 18% (20/113) . These r27To identify women in whom selection had favoured the outgrowth of URFs, we developed an algorithm to distinguish between URFs and any CRFs that had infected the women by chance. This analysis was based on an examination of subtype-section specific phylogenetic trees constructed from alignments of sequences from this cohort and from the HIV database  both recombinant sequence-sections were derived from HHSs; 2) one section was derived from an HHS and the other section was derived from a CRF; or 3) the recombinant sequence had a unique breakpoint pattern. Based on this analysis, 15/17 women (88%) were likely to carry a URF and 2/17 carried the same CRF (12%) . This CRNext, we examined the composition of each URF and found that 6/15 (40%) of the URFs contained sections of two HHSs and that 9/15 (60%) contained one section of a local CRF and one section of an HHS . The latThis overrepresentation of CRF sections in the URF group is highly unlikely to occur by chance given the frequency of CRFs in the cohort (2/98 (2%) versus 9/15 (60%), Fisher’s exact test, p < 0.0001; odds ratio = 9.8), Thus, our results indicate that local CRFs may have a specific survival advantage in the URF group.To test our hypothesis that unique HIV-1 recombinants are more likely to occur in hosts with CTL responses that are more efficient than average, we compared the frequency of HLA variants associated with delayed disease progression 23536Women with beneficial HLA variants (12/52) were over 5 times more likely to carry URFs than those without beneficial HLA variants (3/61) . However, Fisher’s exact test is a test of equality of proportion and, with the proportions in question being based upon URF classifications derived from Bayes Rule, no account has been taken of the uncertainty in those classifications. Although strict classification followed by Fisher’s exact test has the virtues of simplicity and ease of interpretation, information has been discarded; therefore the analyses were improved by fitting binary generalised linear models (GLMs) to the log(odds) of the Bayesian posterior probabilities. Odds ratios quoted for these results refer to a change in Bayesian log(odds) of unity, which corresponds to a posterior probability of 0.75; odds-ratios corresponding to a posterior probability of 0.9 can be approximated by squaring the quoted odds ratio, and cubing it would correspond to a posterior probability of 0.95. This more meticulous approach confirmed that women with beneficial HLA variants were more likely to carry URFs than those without . Likewise we observed a significantly higher frequency of beneficial HLA variants in the URF group  compared with the group with HHSs/CRFs  .These results suggest that the efficient CTL responses associated with beneficial HLA variants selects for the outgrowth of HIV-1 recombinants.Next, we investigated whether the relationship between beneficial HLA variants and disease progression in the group of women with URFs was similar to that in the group of women with either HHSs or CRFs. We used the biannual CD4-counts to classify the patients into groups according to clinical progression rate and examined the distribution of beneficial, detrimental and neutral HLA variants in each group (LTNPs (HIV-infected for >7 years and stable CD4 counts >400/μl), long-term survivors (HIV-infected for >7 years and stable CD4 counts >200/μl and <400/μl), slow progressors (HIV-infected for 5–7 years and unstable CD4 counts ≥200/μl and <400/μl), standard progressors (HIV-infected for 3–5 years and CD4 counts >200/μl) and rapid progressors (HIV-infected for <3 years and CD4 counts <200/μl)) .For both the URF and the HHSs/CRF patient groups, we generated two disease progression (DP) groups; one with long-term control of HIV infection (LTNPs and long-term survivors = DP1) and one with slow/standard disease progression (slow progressors and standard progressors = DP2); rapid progressors (n = 2) were not included because these were found only in the HHS/CRF patient group. We grouped standard progressors and slow progressors because standard progressors often had CD4 counts close to or above 400/μl, consistent withIn the group of women with HHSs/CRFs, we compared the frequency of beneficial, neutral and detrimental HLA variants in DP1 and DP2 by fitting a binary GLM to the HLA variant using neutral as the reference category. We found that beneficial HLA variants predominated in DP1 , whereas no difference was observed between the frequencies of neutral and detrimental HLA variants (p = 0.3858) . Thus, iNext, we tested whether the presence of either URFs or HHSs/CRFs affected disease progression in patients with beneficial HLA variants, i.e., if the patient would be likely to belong to DP1 or DP2. By regressing on the Bayesian log(odds) we found that patients with URFs were marginally more likely to be in DP2 and that patients with HHSs/CRFs were slightly more likely to be in DP1 . Second,Collectively, these findings suggest that beneficial HLA variants might have less beneficial effects on disease progression in women with URFs than in women with either HHSs or CRFs.Here, we compared the HLA variant distribution and clinical progression rate in groups of HIV-infected women with or without unique HIV-1 recombinants. Women with URFs had significantly higher frequencies of HLA variants, which were associated with delayed disease progression. In addition, the presence of URFs appeared to decrease the beneficial clinical effect of these HLA variants.URFs could potentially be generated and selected in most CSWs in our cohort because recombination occurs as a result of the inherent properties of RT, the CSWs likely share the same risk factors, and the risk of HIV superinfection for patients with any given HLA variant is similar to that of initial infection during the first 6 months and subsequently decreases by approximately half2418The significant overrepresentation of patients with beneficial HLA variants in the URF group suggests that the effective CTL responses in these women selects for the outgrowth of the URFs. Because the URFs eventually dominate the viral population, the URFs must be more fit than each of the parent viruses. This fitness gain may result from the selection of HIV-1 with en bloc combinations of advantageous mutations, and recombination may partly explain why HIV-1 containment fails in approximately 70% of patients with beneficial HLA variants within 16 years of infectiongag recombination in this group because the CSWs in our cohort had a very high number of daily customers (an average of 4 men per day) and because the epidemic in Kenya is caused by several co-circulating and relatively prevalent HIV-1 subtypes and CRFs272829URFs were observed in 23% of women with beneficial HLA variants. This frequency likely represents a near maximum estimate of the frequency of inter-subtype et al., who found that 41% of nearly full-length recombinant HIV-1 sequences from Kenya only contained the recombinant sequence in pol and/or envDespite the limitations of our study , we still obtained statistically significant results. However, we likely underestimated the overall URF frequency because some patients with homogeneous HIV Gag regions may have viral variants with recombined regions outside Gag. Such recombinants were described by Dowling Nevertheless, Gag is likely a key target for CTL-driven selection for recombination because only CTL responses against Gag are associated with viral containment and lower mean viral loadsAlthough CSW infection with a second HIV-1 (HHS or CRF) may be hampered by factors such as anti-HIV immune responses and target cell depletion in the genital tract, it is unclear why most CSWs are not infected with multiple HHSs/CRFs given their daily exposure to numerous clients. One possible explanation is that any superinfecting HIV-1 has to be more fit initially than the established HHS/CRF, which is already adapted to survive in the specific host, and this situation is rarely the case.The overrepresentation of CRF-sections in HIV recombinants in the URF group suggests that local CRFs may have a specific survival advantage in women with beneficial HLA variants, possibly because the CRFs arise from URFs generated in this patient group. Alternatively, or additionally, a prior successful recombination could be an indicator of subsequent recombination success possibly because mutations in the CRF already compensate for inter-subtype sequence incompatibilities. The potential local CRF fitness advantage and/or inherent CRF recombination propensity further amplifies HIV diversification in individuals with beneficial HLA variants.in vitro.Beneficial HLA variants were frequently found in LTNPs and long-term survivors in the HHS/CRF group, whereas beneficial HLA variants were concentrated in slow progressors and standard progressors in the URF group. Thus, the normally protective HLA variants appear to have less of a beneficial effect on disease progression in women carrying URFs. The fact that we predominately observed URFs in women with beneficial HLA variants who had been infected for a short duration and who were progressing faster refutes a key potential source of confounding, namely, that URFs would be overrepresented in HIV-infected women with beneficial HLA variants simply because they are more likely to be superinfected because they survive longer than those without beneficial HLA-variants. Our results are supported by previous reports demonstrating that superinfection and/or dual infection with two HIV strains lead to faster CD4+ T cell declineThese results suggest that a balance exists between CTL responses and viral adaptation in most patients with protective HLA variants and that the delayed disease progression in these patients is a result of this unique virus-host equilibrium. However, this beneficial balance may be disrupted if the CTL response results in the outgrowth of highly adapted URFs, and our results suggest that this process limits the protective effects of the immune response. Recombination and outgrowth of unique recombinants could likewise undermine successful intra-host immune containment of other human and animal retroviruses e.g., human T-lymphotropic virus type 1 (HTLV-1)Our results suggest that individuals with protective HLA variants who engage in any high-risk behaviour  constitute a hub for HIV-1 diversification that could lead to the outgrowth of unique forms of recombinant HIV-1 that have the potential to spread in the population. If these new HIV-1 recombinants do spread, then they will contribute to viral diversification locally and globally and may lead to faster progression to AIDS and/or to more rapid CD4 decline than the homogenous subtypes from which they originated, as observed in, e.g., Guinea-Bissau and Brazil47These results suggest that monitoring the effect of HIV-vaccination on HIV-1 diversification in infected vaccinees may be necessary because a vaccine that elicits effective CTL responses, but that does not prevent infection, may increase the formation of URFs and thereby increase viral diversification and adaptation to the vaccinated population.We obtained DNA samples extracted from peripheral blood mononuclear cells (PBMC) from 113 HIV-1 infected female CSWs from the Pumwani Sex Worker CohortPBMC DNA was extracted using QIAamp DNA Mini Kits  and 5–10 μl of each sample was available for this study. HLA class I genotyping was performed using multiplex PCR .HLA variants were assigned as beneficial, detrimental, and neutral with regard to disease progression based on previously published reports235364950235363738HXB2 numbering) and annealed to regions conserved amongst the subtypes. Because of limited sample availability, we initially performed bulk DNA amplification to optimize the primers and to identify the patients with mixed sequence reads. Subsequently, we used the PBMC DNA samples to performed single-genome PCR amplification of 5–10 genomes per patient, subject to DNA availability. Forward and reverse sequence reads were assembled and proofread using Pregap4 and Gap4KR781618 - KR782177).We amplified ~1200 bases spanning the HIV-1 p17–p24 region using a hot-start nested polymerase chain reaction (PCR) . The primers were GAG1 (round 1 (R1)) 728–751, GAG3 (R1) 1941–1916, GAG2 (R2) 763-788 and GAG4 (R2) 1911–1884  because we needed the longest alignments possible to ensure adequate power in our phylogenetic analyses. Briefly, we constructed separate alignments for each subtype; these alignments consisted of 1) the subtype sections of the recombinant sequences, 2) matching sections of the HHS sequences from our cohort, and, 3) matching sections of the most similar sequences in the HIV database (www.hiv.lanl.gov), as determined by BLASTnWe used Bayesian inferential techniques MrBayes to buildWe assumed that an inter-subtype recombinant HIV would not recombine again to become an HHS. From this assumption follows that the parent node of any HHS sequence must also be an HHS sequence. Therefore, any tree in which a recombinant sequence has an HHS sister sequence supports the hypothesis that the recombinant sequence is a URF because it is derived from an HHS parent node , which wThus, for each tree sampled from the posterior tree distribution derived from a subtype-section alignment  we examined whether the tree supported the URF hypothesis or the CRF hypothesis for each patient. For every patient infected with a recombinant, we identified the common ancestor of the patient’s subtype-section sequences in the tree. If the sequences from that patient were monophyletic , then we selected the sequences’ common ancestor’s parent node to assess the recombinant form. If the patient’s sequences were paraphyletic , then we selected the common ancestor of the patient’s subtype-section sequences. If the selected node had an HHS from another patient amongst its descendants, then we interpreted the tree as evidence that an HHS had infected the patient and that this HHS had subsequently recombined in the patient to form a URF . ConversTo ensure that rooting effects did not confound the analysis, we rooted each tree sampled from the posterior distribution on a random sequence from a random HHS-infected patient and excluded patients for that tree whose sequences were too close to the root, i.e., when the selected node had more than four different patient/virus combinations as its descendants. For each patient, we calculated the probability that their sequences in this subtype-section alignment were part of a URF (URF-probability) by dividing the number of trees supporting the hypothesis that the patient was infected with a URF  by the nWe implemented this algorithm in a Python script using the ete2 tree manipulation moduleEquality of proportion was tested with Fisher’s exact test. Where we incorporated the uncertainty of the Bayesian sequence analysis, binary GLMs were fitted using the posterior Bayesian log(odds) as an explanatory variable; these values were calculated from the highest Bayesian posterior probabilities of the subtype-sections of a given recombinant HIV-1 .


Mitochondrial dysfunction is a crucial mechanism by which cisplatin, a potent chemotherapeutic agent, causes nephrotoxicity where mitochondrial electron transport complexes are shifted mostly toward imbalanced reactive oxygen species versus energy production. In the present study, the protective role of tempol, a membrane-permeable superoxide dismutase mimetic agent, was evaluated on mitochondrial dysfunction and the subsequent damage induced by cisplatin nephrotoxicity in mice.Nephrotoxicity was assessed 72 h after a single i.p. injection of cisplatin (25 mg/kg) with or without oral administration of tempol (100 mg/kg/day). Serum creatinine and urea as well as glucosuria and proteinuria were evaluated. Both kidneys were isolated for estimation of oxidative stress markers, adenosine triphosphate (ATP) content and caspase-3 activity. Moreover, mitochondrial oxidative phosphorylation capacity, complexes I–IV activities and mitochondrial nitric oxide synthase (mNOS) protein expression were measured along with histological examinations of renal tubular damage and mitochondrial ultrastructural changes. Tempol was effective against cisplatin-induced elevation of serum creatinine and urea as well as glucosuria and proteinuria. Moreover, pretreatment with tempol notably inhibited cisplatin-induced oxidative stress and disruption of mitochondrial function by restoring mitochondrial oxidative phosphorylation, complexes I and III activities, mNOS protein expression and ATP content. Tempol also provided significant protection against apoptosis, tubular damage and mitochondrial ultrastructural changes. Interestingly, tempol did not interfere with the cytotoxic effect of cisplatin against the growth of solid Ehrlich carcinoma.This study highlights the potential role of tempol in inhibiting cisplatin-induced nephrotoxicity without affecting its antitumor activity via amelioration of oxidative stress and mitochondrial dysfunction. Cisplatin is an effective chemotherapeutic agent that is widely used against several types of solid tumors. However, its clinical use is limited by its potent nephrotoxicity Cisplatin nephrotoxicity has been recognized as a complex multifactorial process that includes oxidative stress, mitochondrial dysfunction and alteration of signal transduction pathways involved in apoptosis Mitochondrial injury seems to play an important role in cisplatin-induced nephrotoxicity. Several functional and structural alterations of the mitochondria have been observed in cell cultures and in vivo animal models of cisplatin nephrotoxicity Oxidative stress and mitochondrial damage have been proposed as important factors that are involved in the activation of apoptotic pathway and cisplatin-induced cell death in vitro Tempol (4-hydroxy tempo) is a membrane-permeable radical scavenger which has SOD and catalase activities. Tempol has been reported experimentally to ameliorate oxidative stress-mediated renal dysfunction and glomerular injury Tempol, as an antioxidant, has been previously demonstrated to prevent injury induced by cisplatin using established renal epithelial cell line, LLC-PK1 Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health  and was approved by the Ethics Committee for Animal Experimentation at Faculty of Pharmacy, Cairo University (Permit Number: PT 983).Male Swiss albino mice weighing 21–24 g were obtained from the animal facility of Faculty of Pharmacy, Cairo University, Egypt. Animals were housed under controlled environmental conditions at constant temperature (25±2°C) and a 12/12 h light/dark cycle. Mice were allowed standard chow diet and water ad libitum. The investigation complies with the Tempol, cisplatin, fine chemicals and reagents, unless otherwise specified, were obtained from Sigma-Aldrich Chemical Co. .For selection of cisplatin dose, two doses have been examined; 12 mg/kg One kidney was used for mitochondrial separation while the other was homogenized in ice-cold saline to prepare 10% homogenate for determination of adenosine triphosphate (ATP) content as well as caspase-3 activity.Serum creatinine and urea levels were estimated using commercial kits purchased from Stanbio Chemicals (USA) and Quimica Clinica Aplicada (Spain), respectively. Procedures were performed according to the manufacturer’s instructions. Results were expressed as mg/dl.Urinary glucose and protein levels were estimated using commercial kits purchased from Biodiagnostic, Egypt. Procedures were performed according to the manufacturer’s instructions. Urinary creatinine was also evaluated as previously mentioned for estimation of urinary glucose/creatinine ratio and protein/creatinine ratio to compensate for variations in urine concentration in the used samples.Mitochondrial isolation was performed as mentioned in a previous study Mitochondrial and postmitochondrial lipid peroxidation products were estimated by determination of the level of thiobarbituric acid reactive substances (TBARS) that was measured according to the assay of Buege and Aust Mitochondrial and postmitochondrial GSH contents were determined using the method of Beutler et al. Mitochondrial and postmitochondrial SOD activities were assessed according to the method of Marklund 2O2) per min at 25°C.Mitochondrial and postmitochondrial catalase activities were assessed according to the method of Abei Mitochondrial phosphate utilization was assayed following the method of Hinkle A part of mitochondrial pellet was resuspended in lysis buffer and quantified for protein levels using a commercial assay kit . An aliquot of 20 µg protein from each sample was separated on 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane  using a semidry transfer apparatus . Membranes were blocked with 5% nonfat milk in Tris- buffered saline with 0.05% Tween-20 (TBST) at 4°C overnight. The membranes were then washed with TBST and incubated with a 1∶2,000 dilution of anti-iNOS antibodies  for 1 h at room temperature with constant shaking. The filters were washed and subsequently probed with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin . Chemiluminescence detection was performed with the Amersham detection kit according to the manufacturer’s protocols and exposed to X-ray film. The amount of NOS protein was quantified by densitometric analysis of the autoradiograms using a scanning laser densitometer . Results were expressed as arbitrary units after normalization for beta-actin (β-actin) protein expression.+ and glucose-6-phosphate dehydrogenase. Results were expressed as µmol/g wet tissue.ATP content was estimated according to the method of Lowry et al. + and the results were expressed as nmol NADH oxidized/min/mg protein. Complex II (succinate dehydrogenase) was assayed using phenazine methosulfate as an artificial electron acceptor from succinate and recording the increase in absorbance of ferrocytochrome-c where complex II activity was calculated as nmol succinate oxidized/min/mg protein. For estimating the activity of complex III (ubiquinol-cytochrome c oxidoreductase), the method depends on the oxidation of decylubiquinol to the corresponding quinone with the concurrent reduction of ferricytochrome-c to ferrocytochrome-c and its activity was computed as the initial reaction rate per mg protein. Finally, the activity of complex IV (cytochrome-c oxidase) was assayed using the method of Storrie and Madden Complexes I–III activities were estimated according to the method of Sharman and Bondy Caspase-3 activity was estimated using caspase-3 colorimetric assay kit . Results were expressed as nmol p-nitroanilide (pNA)/h/mg protein.For light microscopic examination, the right kidney was separated, rinsed in ice-cold saline and immediately fixed in 10% formalin for 24 h. Specimens were processed for paraffin embedding and 5 µm sections were prepared. Sections were stained with haematoxylin and eosin (H&E) and examined microscopically (magnification x200). Images were captured and processed using Adobe Photoshop (version 8.0). Images were analyzed by two investigators. Histological changes were evaluated semiquantitatively by a pathologist unaware of the type of treatment. For quantification of tubular injury score, sections were assessed by counting the percentage of tubular necrosis in the outer stripe of outer medulla (OSOM) using the following scoring criteria: 0 = normal; 1 = <10%; 2 = 10–25%; 3 = 26–75%; 4 = >75%. Tubular necrosis was defined as the loss of the proximal tubular brush border, blebbing of apical membranes, tubular epithelial cell detachment from the basement membrane or intraluminal aggregation of cells and proteins Small pieces were separated from the left kidney, rinsed in ice-cold saline and cut into fragments (diameter = 1 mm). Fragments were then processed and ultra-thin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope  and photographed. Electron micrographs were taken systematically at ×2,000 and ×10,000 magnifications. Images were analyzed by two investigators. Percentage of mitochondrial cross-sectional area/cytoplasmic area was estimated where non-cytoplasmic area was first manually excluded from analysis. The optical density threshold was manually set to include all mitochondria and then the percentage area of mitochondria was calculated per cytoplasmic volume 6 Ehrlich ascites carcinoma cells were transplanted subcutaneously in the right thigh of the lower limb of each mouse. Mice with a palpable solid tumor mass (100 mm3) that developed within 7 days after implantation were divided into four groups, 6 animals each. Two groups were injected with either cisplatin as previously mentioned in the experimental design or an equivalent volume of normal saline. The other two groups received tempol orally in a dose of 100 mg/kg/day or a combination of tempol and cisplatin. The change in tumor volume was measured after 3 days using a Vernier caliper and was calculated by the following formula according to Osman et al. In this experiment, 2×10All data obtained were presented as mean ± S.E.M. Results were analyzed using one way analysis of variance test (One-way ANOVA) followed by Student-Newman-Keuls multiple comparison test. Statistical analysis was performed using GraphPad Prism software (version 5). For all the statistical tests, the level of significance was fixed at p<0.05.Cisplatin-treated group showed significant decrease in survival% (71%) and body weight (20.42±0.98 vs. 23.33±0.70 g) as well as significant increase in the relative kidney weight (1.64±0.03 vs. 1.10±0.02); which were pronouncedly alleviated with tempol pretreatment .Cisplatin raised both serum creatinine and urea levels by five and nine folds, respectively compared to the normal group. Cisplatin-treated animals also exhibited a significant renal tubular dysfunction as evidenced by marked glucosuria and proteinuria . PretreaCisplatin-treated group showed significant elevation of TBARS content and significant reduction of GSH content  as well The enhanced state of oxidative stress in cisplatin-treated group was associated with significant reduction of mitochondrial oxidative phosphorylation capacity and complexes I and III activities. These changes were correlated with a pronounced reduction of ATP content . On the Cisplatin-induced mitochondrial disruption was accompanied by significant elevation of caspase-3 activity and mNOS protein expression which was attenuated by tempol pretreatment .Cisplatin-treated group showed a marked tubular damage compared to the normal group. Pretreatment with tempol significantly decreased tubular damage score . the mitTreatment of mice with cisplatin (25 mg/kg) revealed a cessation of solid Ehrlich carcinoma tumor growth where the tumor volume was significantly decreased compared to the control group. Co-administration of tempol with cisplatin suppressed tumor growth in a similar pattern to cisplatin-treated animals. Therefore, tempol did not interfere with the cytotoxic effect of cisplatin against the growth of solid Ehrlich carcinoma .Despite being widely used, the use of cisplatin as chemotherapy is limited by its nephrotoxicity with about 25–35% of patients experiencing a significant decline of renal function after a single dose of cisplatin treatment The present data show that administration of cisplatin to mice caused marked elevation of serum creatinine and urea levels as well as significant glucosuria and proteinuria. Cisplatin causes acute renal failure due to its preferential accumulation within the proximal tubular cells in the outer medulla of the kidney The present results clearly indicate a significant degree of oxidative stress in both mitochondrial and postmitochondrial fractions in renal tissues of cisplatin-treated mice. This was demonstrated by a significant elevation of TBARS content and a significant reduction of GSH content along with inhibition of SOD and catalase activities in both mitochondrial and postmitochondrial fractions. The enhanced state of oxidative stress in cisplatin-treated group was associated with significant reduction of mitochondrial oxidative phosphorylation capacity and complexes I and III activities together with significant elevation of mNOS protein expression and caspase-3 activity. This was correlated with a pronounced reduction of ATP content indicating marked deterioration of mitochondrial respiration and energy production. GSH is one of the essential components for maintaining cell integrity because of its reducing properties and participation in the cell metabolism. The depletion of the renal GSH level has been observed in response to oxidative stress caused by cisplatin treatment 2O2 and lipid peroxides. The inhibition of mitochondrial and postmitochondrial antioxidant enzyme activities may occur due to direct binding of cisplatin to essential sulfhydryl groups at the active sites of these enzymes and depletion of copper and manganese which are essential for SOD activity Cisplatin affects many enzymes that protect the cells from oxidative damage, among which Cu, Zn-SOD, Mn-SOD and catalase. SOD plays an important role in the dismutation of superoxide anions. Decreased SOD activity as observed in this study could lead to incomplete scavenging of superoxide anions that are produced during the normal metabolic process with further initiation and propagation of lipid peroxidation Cisplatin exposure induces a mitochondria-dependent ROS generation that significantly contributes to cisplatin-induced nephrotoxicity Partial ATP depletion as observed in the present study may constitute a common biochemical pathway that initiates a cascade of events leading to further cellular dysfunctions and activation of programmed cell death. This in turn may accelerate ROS formation by the damaged cells, which may contribute to an amplification of ROS-mediated cell death of the same cell or even the neighboring cells 2O2 and accept an electron to form the antioxidant hydroxylamine 2O2 by oxidizing transition metal ions, such as iron. Moreover, a direct superoxide anion scavenging activity of nitroxides has been reported Tempol pretreatment succeeded to alleviate the changes induced by the state of oxidative stress in both mitochondrial and postmitochondrial fractions in addition to amelioration of disruption of mitochondrial function and caspase-3 elevation. Several studies have previously demonstrated the role of antioxidants in prevention of cisplatin-induced nephrotoxicity 2O2 production and preserve the mitochondrial antioxidant enzyme activities including MnSOD and catalase. This was correlated with preservation of mitochondrial respiratory function  and prevention of mitochondria-induced apoptosis as indicated by normalization of caspase-3 activity and histological examinations of renal tubules and mitochondria. Tempol treatment has previously restored mitochondrial membrane potential and reduced tissue oxidative damage in Atm-deficient mice, both in vitro and in vivo 2O2-induced cellular injury and death in a previous in vitro study Tempol, a membrane-permeable antioxidant, through its superoxide anion scavenging activity could prevent the increase in mitochondrial superoxide and HIn conclusion, the present study highlights the potential role of tempol in alleviating cisplatin-induced mitochondrial dysfunction without affecting its antitumor activity via reducing ROS formation, protecting electron transport complexes, favoring energy production and inhibiting apoptosis in addition to improving histological changes. The beneficial effect of tempol in cisplatin-induced nephrotoxicity is an important outcome which needs to be evaluated in clinical studies.