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Introduction There is a balance between oxidative stress, antioxidant capacity and immune response. Their roles in physiological and behavioural mechanisms are important for the maintenance of the organism’s internal equilibrium. This study aimed to evaluate the antioxidant effects of the exogenous alga Spirulina platensis (Arthrospira platensis) in a stress-induced rat model, and to describe its possible mechanism of action. Material and Methods Thirty-six adult male Sprague Dawley rats were separated into four groups: control (C), stress (S), S. platensis (Sp), and S. platensis + stress (SpS). The rats in groups Sp and SpS were fed with 1,500 mg/kg b.w./day Spirulina platensis for 28 days. All rats were exposed to prolonged light phase conditions (18 h light : 6 h dark) for 14 days. The SpS and S groups were exposed to stress by being kept isolated and in a crowded environment. Blood samples were obtained by puncturing the heart on the 28th day. The effect of stress on serum corticosterone, oxidative stress markers (TOS, TAC, PON1, OSI) and immunological parameters (IL-2, IL-4, IFN-ɣ) were tested. Also, the brain, heart, intestines (duodenum, ileum, and colon), kidney, liver, spleen, and stomach of the rats were weighed. Results Serum corticosterone levels were higher in the S group than in the C group, and significantly lower in the SpS group than in the S group. Mean total antioxidant capacity were lower in the S group than in the C group, and Spirulina reversed this change. Although not significantly different, IL-2 was lower in the S group than in the C group. However, in the SpS group, IL-2 increased due to Spirulina platensis mitigating effects of stress. Conclusion Male rats fed a diet with Spirulina platensis could experience significantly milder physiological changes during stress, although stress patterns may be different. Exogenous antioxidant supplements merit further investigation in animals and humans where the endogenous defence mechanism against stress may not be sufficient.

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live mouse cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb (also known as the lupus autoantigen La) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female mouse embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain plays critical roles in XCI. In Ssb knockdown cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist. Ministry of Education (MOE) Ministry of Health (MOH) National Research Foundation (NRF) Published version L.C. was supported by the National Key R&D Program of China [2018YFC1313003, 2018YFA0107002]; National Natural Science Foundation of China [31871485]; Natural Science Foundation of Tianjin [18JCJQJC48400]; 111 Project Grant [B08011]; Fundamental Research Funds for the Central Universities; L.-F.Z. was supported by Singapore Ministry of Education Academic Research Fund [MOE2015-T2-1-093]; Singapore National Research Foundation under its Cooperative Basic Research Grant administered by the Singapore Ministry of Health’s National Medical Research Council [NMRC/CBRG/0092/2015]. Funding for open access charge: the National Key R&D Program of China [2018YFA0107002].

The blood hormone erythropoietin (EPO), upon binding to its receptor (EpoR), modulates high-fat diet-induced (HFD-induced) obesity in mice, improves glucose tolerance, and prevents white adipose tissue inflammation. Transgenic mice with constitutive overexpression of human EPO solely in the brain (Tg21) were used to assess the neuroendocrine EPO effect without increasing the hematocrit. Male Tg21 mice resisted HFD-induced weight gain; showed lower serum adrenocorticotropic hormone, corticosterone, and C-reactive protein levels; and prevented myeloid cell recruitment to the hypothalamus compared with WT male mice. HFD-induced hypothalamic inflammation (HI) and microglial activation were higher in male mice, and Tg21 male mice exhibited a lower increase in HI than WT male mice. Physiological EPO function in the brain also showed sexual dimorphism in regulating HFD response. Female estrogen production blocked reduced weight gain and HI. Targeted deletion of EpoR gene expression in neuronal cells worsened HFD-induced glucose intolerance in both male and female mice but increased weight gain and HI in the hypothalamus in male mice only. Both male and female Tg21 mice kept on normal chow and HFD showed significantly improved glycemic control. Our data indicate that cerebral EPO regulates weight gain and HI in a sex-dependent response, distinct from EPO regulation of glycemic control, and independent of erythropoietic EPO response.

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused-laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.

One of the major problems affecting the ecosystem health is the exposure of heavy metals due to anthropogenic activities. These exposures may induce toxiogenetic damage and carcinogenesis in aquatic organism. DNA damage biomarkers for fish species detect genotoxic parameters for ecological risk assessment. In the present study, the effect of heavy metals (Cr, Cd, Cu, Fe and Mn) on DNA damage and oxidative stress of Clarias gariepinus was examined by Comet assay at Reference site and two different sampling sites of the Orontes River. Moreover, the relationship between DNA damage and physiological response to oxidative stress caused in C. gariepinus was investigated by sampling seasonal water and fish samples for one year. In this study, Cr, Cd and Cu in water column of Orontes River and the concentrations of Fe and Mn in liver tissue of C. gariepinus were highly exceed the maximum permissible limit set by EPA (1999, 2016) WHO (1989) and TEG (2012) that can cause adverse effects on environment health. SOD activities in liver tissues were significantly higher than those in muscle tissues. SOD, CAT activity and MDA levels of both Site 1 and Site 2 at Orontes River were higher than those of the Reference site. In result of the COMET analysis, the highest levels of DNA damage were found at gill and liver tissues in Site 2 (17.746 ± 1.072% T-DNA; 16.014 ± 0.710% T-DNA respectively) at Orontes river. A higher level of DNA damage in gill cells compared to liver cells was observed in all regions of the Orontes River. In Principal component analysis (PCA), the heavy metals Cd, Cr and Cu (in the water column) and MDA in liver and muscle revealed strong contribution to the observed DNA damage that were scattered opposite to each other's along the principal components. Moreover, correlations between parameters revealed a positive significant relationship between Cd, Cr and Cu in water and DNA damage levels in C. gariepinus. Pearson correlation analysis detected a positive relationship between MDA, CAT and SOD and DNA damage levels in African catfish. Consequently, this study exposed genotoxic damage and oxidative stress of C. gariepinus due to metal pollution in Orontes River and lead to the better understanding of genotoxicty, oxidative stress and heavy metal relationships.

The mechanism of neurodegeneration in Parkinson’s disease (PD) remains unknown but it has been hypothesised that the intestinal tract could be an initiating and contributing factor to the neurodegenerative processes. In PD patients as well as in animal models for PD, alpha-synuclein-positive enteric neurons in the colon and evidence of colonic inflammation have been demonstrated. Moreover, several studies reported pro-inflammatory bacterial dysbiosis in PD patients. Here, we report for the first time significant changes in the composition of caecum mucosal associated and luminal microbiota and the associated metabolic pathways in a rotenone-induced mouse model for PD. The mouse model for PD, induced by the pesticide rotenone, is associated with an imbalance in the gut microbiota, characterised by a significant decrease in the relative abundance of the beneficial commensal bacteria genus Bifidobacterium. Overall, intestinal bacterial dysbiosis might play an important role in both the disruption of intestinal epithelial integrity and intestinal inflammation, which could lead or contribute to the observed alpha-synuclein aggregation and PD pathology in the intestine and central nervous system in the oral rotenone mouse model of PD.

Interleukin (IL)-6 is an important mediator of neurovascular dysfunction, neurodegeneration and/or neuroinflammation. We previously reported that brain pericytes released higher levels of IL-6 than did glial cells (astrocytes and microglia) in response to tumor necrosis factor (TNF)-?. Moreover, pericytes stimulated with TNF-? enhanced activation of BV-2 microglia. In this study, we investigated the mechanisms of TNF-? mediated induction of IL-6 release from brain pericytes and astrocytes and whether pericyte-derived IL-6 would facilitate activation of BV-2 microglia. Using rat brain pericyte and astrocyte primary cultures and pharmacological inhibitors, we found that, TNF-? induced the highest levels of IL-6 release from pericytes by activating the inhibitor kappa B (I?B)-nuclear factor kappa-light-chain-enhancer of activated B cells (NF?B) and Janus family of tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT)3 pathways. STAT3 contributed to TNF-? induced nuclear translocation of phospho-NF?B in pericytes. TNF-?-induced IL-6 release in astrocytes was mediated by NF?B but not by STAT3. The presence of pericytes amplified TNF-?-induced iNOS mRNA expression in BV-2 microglia. This effect was blocked by a neutralizing antibody for IL-6. These findings indicated that crosstalk between the I?B-NF?B and JAK-STAT3 pathways is a pericyte specific mechanism, not occurring in astrocytes, for TNF-?-induced IL-6 release. IL-6 derived from pericytes enhanced microglial activation. Our findings increase understanding of the role of pericyte-microglia crosstalk in the brain under neuroinflammatory conditions and suggest a potentially attractive therapeutic target for brain inflammation. © 2018 Elsevier B.V.

Great strides have been made in triple negative breast cancer (TNBC) treatment, which represents 20% of total predicted annual US breast cancer (BC) cases. Despite the development of several therapeutics, TNBC patients have poor overall survival rate, compared to other BC patients, justifying the urgent need to discover new entities for use to control TNBC. Chalcones are important natural products with diverse bioactivities including anticancer effects. This study aimed to design, synthesize and validate novel chalcone leads as potential therapies for TNBC. Fourteen novel chalcone analogs were designed and synthesized comprising alicyclic amines (pyrrolidine, morpholine and piperidine) or nitrogen mustard (Bis-(2-chloroethyl) amine) substituents. Among them, compound 14 ((E)-3-(4-(Bis(2-chloroethyl) amino) phenyl)-1-(3-methoxyphenyl) prop-2-en-1-one) was identified as the most effective against TNBC and other BC phenotypes, with anti-proliferative IC50 values ranging between 3.94 and 9.22 μM against the TNBC cell lines MDA-MB-231 and MDA-MB-468, as well as against the estrogen positive MCF-7 cell line. Chalcone 14 effectively suppressed the colony formation capacity of MDA-MB-231, MDA-MB-468, and MCF-7 cell lines at 5 and 10 μM treatment concentrations. Furthermore, compound 14 has significantly inhibited cell invasion and migration of MDA-MB-231 and MCF-7 BC cell lines. Additionally, compound 14 had significantly promoted apoptosis by upregulating BAX and downregulating Bcl-2 proteins. Compound 14 induced significant cell cycle arrest of TNBC cells at the G2/M phase. It also induced a reversal of Epithelial Mesenchymal Transition (EMT) by upregulating the epithelial markers E-cadherin and Pan-cadherin and downregulating FAK. Furthermore, it had dramatically diminished new vessel formation (vasculogenesis) in chick chorioallantoic membrane (CAM) model by 60.20 ± 8.47%. Chalcone 14 inhibited 46.41 ± 0.71% of the TNBC MAD-MB-231 cells growth in a nude mouse orthotopic xenograft model in comparison with vehicle control treated animals. Collectively, this study results propose chalcone 14 as a promising lead molecule for the control of TNBC as well as other breast cancer phenotypes. This research work was supported by Grants# GCC-2017-2 and QUST-1-CPH-2018-18 from Qatar University ; and GSRA award# GSRA4-2-0418-17024 from Qatar National Research Fund (a member of Qatar Foundation). Scopus

Adult neurogenesis is emerging as an important player in brain functions and homeostasis, while impaired or altered adult neurogenesis has been associated with a number of neuropsychiatric diseases, such as depression and epilepsy. Here we investigated the possibility that synapsins (Syns) I and II, beyond their known functions in developing and mature neurons, also play a role in adult neurogenesis. We performed a systematic evaluation of the distinct stages of neurogenesis in the hippocampal dentate gyrus of Syn I and Syn II knockout (KO) mice, before (2-monthsold) and after (6-months-old) the appearance of the epileptic phenotype. We found that Syns I and II play an important role in the regulation of adult neurogenesis. In juvenile mice, Syn II deletion was associated with a specific decrease in the proliferation of neuronal progenitors, whereas Syn I deletion impaired the survival of newborn neurons. These defects were reverted after the appearance of the epileptic phenotype, with Syn I KO and Syn II KO mice exhibiting significant increases in survival and proliferation, respectively. Interestingly, long-term potentiation dependent on newborn neurons was present in both juvenile Syn mutants while, at later ages, it was only preserved in Syn II KO mice that also displayed an increased expression of brain-derived neurotrophic factor. This study suggests that Syns I and II play a role in adult neurogenesis and the defects in neurogenesis associated with Syn deletion may contribute to the alterations of cognitive functions observed in Syn-deficient mice. © Barbieri et al.

During my graduate years in Berkeley, I focused on the development of a new tool that can be used to attach DNA to live cells, and the subsequent use of the DNA modified live cells for different applications, including cell patterning, single cell analysis, and cell targeting. Chapter 1 focuses on a new and efficient bioconjugation method to attach single-stranded DNA to live cells. Chapter 2 describes the adaptation of this strategy into a highly-efficient system for multiplex cell patterning, capable of achieving single-cell resolution. Chapter 3 shows the application of this technique for the electrochemical analysis of individual live cells. Single cell metabolism is measured with the integrated device. Chapter 4 focuses on the attachment of DNA-modified cells to an AFM cantilever for direct live cell patterning and the measurement of cell adhesion force. Chapter 5 describes the measurement of the anchoring force of cell membrane protein receptor using AFM technique.

Background Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. Results Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b, miR-222a/b, miR-190a) or have now been found connected (miR-2188, miR-144, miR-731, miR-8157 and the novel n2) to the initiation of puberty. Conclusions This study has - for the first time - linked testis maturation to specific miRNAs and their inversely correlated expressed targets in Atlantic salmon. The study indicates a broad functional conservation of already known miRNAs and associated pathways involved in the transition into puberty in vertebrates. The analysis also reveals miRNAs not previously associated with testis tissue or its maturation, which calls for further functional studies in the testis. Electronic supplementary material The online version of this article (10.1186/s12864-017-4205-5) contains supplementary material, which is available to authorized users.

Metabolic syndrome (MS) increases cardiovascular risk and is associated with cardiac dysfunction and arrhythmias, although the precise mechanisms are still under study. Chronic inflammation in MS has emerged as a possible cause of adverse cardiac events. Male Wistar rats fed with 30% sucrose in drinking water and standard chow for 25–27 weeks were compared to a control group. The MS group showed increased weight, visceral fat, blood pressure, and serum triglycerides. The most important increases in serum cytokines included IL-1β(7-fold), TNF-α(84%), IL-6 (41%), and leptin (2-fold), the latter also showing increased gene expression in heart tissue (35-fold). Heart function ex vivo in MS group showed a decreased mechanical performance response to isoproterenol challenge (ISO). Importantly, MS hearts under ISO showed nearly twofold the incidence of ventricular fibrillation. Healthy rat cardiomyocytes exposed to MS group serum displayed impaired contractile function and Ca2+handling during ISO treatment, showing slightly decreased cell shortening and Ca2+transient amplitude (23%), slower cytosolic calcium removal (17%), and more frequent spontaneous Ca2+release events (7.5-fold). As spontaneous Ca2+releases provide a substrate for ventricular arrhythmias, our study highlights the possible role of serum proinflammatory mediators in the development of arrhythmic events during MS.

Extracellular bacterial ribonucleases such as binase from Bacillus pumilus possess cytotoxic activity against tumor cells with a potential for clinical application. Moreover, they may induce activation of tumor-derived macrophages either into the M1-phenotype with well-documented functions in the regulation of the antitumor immune response or into M2-macrophages that may stimulate tumor growth, metastasis, and angiogenesis. In this study, binase or endogenous RNase1 (but not RNA or short oligonucleotides) stimulated the expression of activated NF-κB p65 subunit in macrophages. Since no changes in MyD88 and TRIF adaptor protein expression were observed, toll-like receptors may not be involved in RNase-related NF-κB pathway activation. In addition, short exposure (0.5 hr) to binase induced the release of cytokines such as IL-6, МСР-1, or TNF-α (but not IL-4 and IL-10), indicative for the polarization into antitumor M1-macrophages. Thus, we revealed increased expression of activated NF-κB p65 subunit in macrophages upon stimulation by binase and RNase1, but not RNA or short oligonucleotides.

International audience; Leptin stimulates fatty acid oxidation in muscle and heart; but, the mechanism by which these tissues provide additional intracellular fatty acids for their oxidation remains unknown. We examined, in isolated muscle and cardiac myocytes, whether leptin, via AMP-activated protein kinase (AMPK) activation, stimulated fatty acid translocase (FAT/CD36)-mediated fatty acid uptake to enhance fatty acid oxidation. In both mouse skeletal muscle and rat cardiomyocytes, leptin increased fatty acid oxidation, an effect that was blocked when AMPK phosphorylation was inhibited by adenine 9-β-D-arabinofuranoside or Compound C. In wild-type mice, leptin induced the translocation of FAT/CD36 to the plasma membrane and increased fatty acid uptake into giant sarcolemmal vesicles and into cardiomyocytes. In muscles of FAT/CD36-KO mice, and in cardiomyocytes in which cell surface FAT/CD36 action was blocked by sulfo-N-succinimidyl oleate, the leptin-stimulated influx of fatty acids was inhibited; concomitantly, the normal leptin-stimulated increase in fatty acid oxidation was also prevented, despite the normal leptin-induced increase in AMPK phosphorylation. Conversely, in muscle of AMPK kinase-dead mice, leptin failed to induce the translocation of FAT/CD36, along with a failure to stimulate fatty acid uptake and oxidation. Similarly, when siRNA was used to reduce AMPK in HL-1 cardiomyocytes, leptin failed to induce the translocation of FAT/CD36. Our studies have revealed a novel mechanism of leptin-induced fatty acid oxidation in muscle tissue; namely, this process is dependent on the activation of AMPK to induce the translocation of FAT/CD36 to the plasma membrane, thereby stimulating fatty acid uptake. Without increasing this leptin-stimulated, FAT/CD36-dependent fatty acid uptake process, leptin-stimulated AMPK phosphorylation does not enhance fatty acid oxidation. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

SETDB1 (SET Domain Bifurcated histone lysine methyltransferase 1) is a key lysine methyltransferase (KMT) required in embryonic stem cells (ESCs), where it silences transposable elements and DNA repeats via histone H3 lysine 9 tri-methylation (H3K9me3), independently of DNA methylation. The H3K9 methylation reader M-Phase Phosphoprotein 8 (MPP8) is highly expressed in ESCs and germline cells. Although evidence of a cooperation between H3K9 KMTs and MPP8 in committed cells has emerged, the interplay between H3K9 methylation writers and MPP8 in ESCs remains elusive. Here, we show that MPP8 interacts physically and functionally with SETDB1 in ESCs. Indeed, combining biochemical, transcriptomic and genomic analyses, we found that MPP8 and SETDB1 co-regulate a significant number of common genomic targets, especially the DNA satellite repeats. Together, our data point to a model in which the silencing of a class of repeated sequences in ESCs involves the cooperation between the H3K9 methylation writer SETDB1 and its reader MPP8. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thawing quality of drone sperm. Semen samples were collected from sexually mature drones. Pooled semen was diluted with extender without RJ (control) or supplemented with different concentrations of RJ (1, 2, 4 or 8%). Sperm motility, plasma membrane functional integrity, and acrosomal integrity were evaluated. At post thaw, the highest sperm motility and acrosomal integrity rates were obtained in the RJ1 group. Functional integrity of sperm membrane was better preserved in the RJ1 and RJ2 groups compare to the other groups. The study shows that RJ supplemented extenders have beneficial effects on drone semen parameters. The results of the present study demonstrated advantage of using 1% RJ supplemented extender.

Porcine cysticercosis is an endemic parasitic disease caused by infection with Taenia solium that is found predominantly in developing countries. In order to aid in the development of simple diagnostic approaches, identification and characterization of potential new antigens for immunodiagnostic purposes is desired. The cysteine protease family has previously been found to have important immunodiagnostic properties. These proteases are expressed as zymogens which contain a signal peptide, pro-peptide, and an active domain. Subsequent catalytic cleavage of the pro-peptide converts these zymogens into enzymes. With the use of bioinformatic tools we identified an active domain of a novel cathepsin L-like cysteine protease (TsolCL) in the T. solium genome. The TsolCL gene includes 705 nucleotides (nt) within a single intron and a 633 nt exonic sequence encoding an active protein of 211 amino acids. Sequence alignment and phylogenetic analysis suggest that the TsolCL gene is closely related to genes found in Echinoccocus granulosus and E. multiloculars. In addition, TsolCL was found to have a 61.9%–99.0% similarity to other cathepsin L proteins found in other helminths and mammals. We cloned, expressed, purified, and characterized the recombinant active TsolCL (27 kDa) using the baculovirus-insect cell expression system. TsolCL showed cysteine protease enzymatic activity with the capacity to hydrolyze the Z-Phe-Arg-AMC substrate as well as bovine serum albumin. However, TsolCL was not able to hydrolyze human immunoglobulin. In addition, TsolCL has cathepsin L conserved amino acid residues in the catalytic site (Gln8, Cys14, His159, Asn179 and Trp181) and the motif GCNGG. Using ELISA, TsolCL was able to distinguish circulating IgG antibodies between healthy animals and naturally infected pigs with cysticercosis, showing a moderate sensitivity of 83.33% (40/48; 95% CI: [69.8%–92.5 %]), and a specificity of 83.78% (31/37; 95% CI: [67.9%–93.8%]). In conclusion, a novel cathepsin L-like cysteine protease from a T. solium metacestode was expressed successfully in Baculovirus system and was evaluated as a candidate antigen to diagnose porcine cysticercosis using the ELISA immunoassay.

Spermatogonial stem cell (SSC) is known for its self-renewal capacity. We have studied the in vitro proliferation of isolated SSC from adult alpaca (Vicugna pacos) testes. A total of 107 samples were evaluated of which 31 were evaluated at baseline, 36 were cultivated in DMEM and 40 in STEMPRO media. Half of the cultivated samples was analysed after 14 days, and the rest after 21 days. Round cell subpopulations were identified with FITC-DBA by flow cytometry: strongly positive DBA (sDBA+) as SSC, weakly positive DBA (wDBA+) as in early differentiation and negative DBA as differentiated. At the beginning, 4.16% of the cells were SSC, 37.61% wDBA+ while 54.12% were DBA-. After 14 days, 42.28% of SSC, 44.68% wDBA+ and 11.07% DBA- were found in DMEM while 47.09% of SSC, 32.57% wDBA+ and 18.48% DBA- in STEMPRO. After 21 days 38.66% were SSC, 52.78% wDBA and 7.65% DBA- in DMEM and on STEMPRO media 47.92% SSC, 44.20% wDBA+, 4.93% DBA-. There is a significant difference between the number of initial and SSC cultivated, as well as between DBA- (p < 0.05), while there is no significant difference between the wDBA+ (p > 0.05). Our results suggest that both culture media are appropriate for the in vitro proliferation of alpacas SSC.

The aim of this study was to investigate the potential beneficial effects of β-glucan treatment against oxidative, histological and spermatological damage caused by cisplatin on the male reproductive system. Twenty-eight Sprague Dawley male rats were used in the study. The rats were randomly divided into four equal-sized groups: a control group, cisplatin group (7 mg/kg in a single-dose cisplatin administered intraperitoneally), β-glucan group (β-glucan given at a dose of 50 mg kg−1 d−1 for 14 day) and a cisplatin plus β-glucan group (cisplatin and β-glucan administered together at the same dose). Cisplatin administration induced an increase in the level of thiobarbituric acid-reactive substances, a lipid peroxidation indicator. It induced a decrease in enzymatic (superoxide dismutase, catalase and glutathione peroxidase) activities and nonenzymatic (reduced glutathione) antioxidant levels. In addition, cisplatin caused both histological and spermatological damage, as shown by a decrease in sperm motility and epididymal sperm concentrations and an increase in abnormal sperm rates. The β-glucan treatment improved cisplatin-induced oxidative, histological and spermatological damage. This study revealed that β-glucan treatment provided prevention against male reproductive system damage caused by cisplatin. These preventative effects were likely due to its antioxidant properties. © 2019 Blackwell Verlag GmbH

Paclitaxel, which isolated from Taxus brevifolia, is recently started to be used against prostate cancer treatment and it is a very effective compound against cancer. In this study, we aimed to test the synergistic effect of two plant active compounds (sulphoraphane (SFN) and silymarin (SILY)) and several endemic plant species from Turkey (such as Phlomis leucophracta, Rubia davisiana, Alkanna tinctoria), which are known to have anticarcinogenic effect on androgen-independent PC3 and DU145, and androgen-dependent VCaP prostate cancer cell lines, with paclitaxel on the expression of cell cycle signaling and apoptosis regulator genes. Herbal substances and endemic herbal extracts were combined with Paclitaxel drug. IC50 doses were identified as real-time online. The most effective synergistic doses were determined according to isobologram analysis. The apoptotic effects of effective combined doses were evaluated by TUNEL, Annexin V, and JC-1 methods. Apoptotic and/or cell cycle arrest effects of confirmed combined doses on the expression of genes in these pathways were assessed by real-time online. Endemic plant extracts (Alkanna tinctoria, Phlomis leucophracta and Rubia davisiana, IC50 < 220 μg/ml) and herbal substances (SILY, and SFN IC50 < 130 μM) indicated antiproliferative and apoptotic effects in prostate cancer cell lines. They testified to the synergistic effect of paclitaxel with endemic plant extracts (Combination Index CI, ED50 < 0.41). The combinations, which indicate the synergistic effect was increased to the Bax/Bcl‑2 ratio by suppressing Bcl‑2 gene expression into the prostate cancer cell lines. Besides, they increased the expression of TNFRSF10A, TNFRSF1A, CHEK1, CDKN1A, CDKN2B, CDK8, CDKN3 and CASP14 and decreased BAD, CDK5RAP1, CDC20, cyclin H, CDK5RAP1, CDC20. The effective doses of paclitaxel were reduced and G2/M arrest was induced by the endemic plant extracts and herbal substances that indicate a synergistic effect with paclitaxel. By using different combination of herbal extracts or active substances with paclitaxel, more economical and efficient treatment strategies can be developed. © 2018 Elsevier B.V.

Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare severe cutaneous adverse reactions to drugs. Granulysin (GNLY) plays a key role in keratinocyte apoptosis during SJS/TEN pathophysiology. To determine if GNLY-encoding mutations might be related to the protein’s functional disturbances, contributing to SJS/TEN pathogenesis, we performed direct sequencing of GNLY’s coding region in a group of 19 Colombian SJS/TEN patients. A GNLY genetic screening was implemented in a group of 249 healthy individuals. We identified the c.11G > A heterozygous sequence variant in a TEN case, which creates a premature termination codon (PTC) (p.Trp4Ter). We show that a mutant protein is synthesised, possibly due to a PTC-readthrough mechanism. Functional assays demonstrated that the mutant protein was abnormally located in the nuclear compartment, potentially leading to a toxic effect. Our results argue in favour of GNLY non-synonymous sequence variants contributing to SJS/TEN pathophysiology, thereby constituting a promising, clinically useful molecular biomarker. © 2019, Springer-Verlag GmbH Germany, part of Springer Nature.

Malaria continues being a high-impact disease regarding public health worldwide; the WHO report for malaria in 2018 estimated that ~219 million cases occurred in 2017, mostly caused by the parasite Plasmodium falciparum. The disease cost the lives of more than 400,000 people, mainly in Africa. In spite of great efforts aimed at developing better prevention (i.e., a highly effective vaccine), diagnosis, and treatment methods for malaria, no efficient solution to this disease has been advanced to date. The Fundación Instituto de Inmunología de Colombia (FIDIC) has been developing studies aimed at furthering the search for vaccine candidates for controlling P. falciparum malaria. However, vaccine development involves safety and immunogenicity studies regarding their formulation in animal models before proceeding to clinical studies. The present work has thus been aimed at evaluating the safety and immunogenicity of a mixture of 23 chemically synthesised, modified peptides (immune protection-inducing protein structure (IMPIPS)) derived from different P. falciparum proteins. Single and repeat dose assays were thus used with male and female BALB/c mice which were immunised with the IMPIPS mixture. It was found that single and repeat dose immunisation with the IMPIPS mixture was safe, both locally and systemically. It was observed that the antibodies so stimulated recognised the parasite’s native proteins and inhibited merozoite invasion of red blood cells in vitro when evaluating the humoral immune response induced by the IMPIPS mixture. Such results suggested that the IMPIPS peptide mixture could be a safe candidate to be tested during the next stage involved in developing an antimalarial vaccine, evaluating local safety, immunogenicity, and protection in a nonhuman primate model.

Tuberculosis is an infectious disease caused byMycobacterium tuberculosis(Mtb, i.e., the aetiological agent); the WHO has established this disease as high priority due to its ensuing mortality.Mtbuses a range of mechanisms for preventing its elimination by an infected host; new, viable alternatives for blocking the host-pathogen interaction are thus sought constantly. This article updates our laboratory’s systematic search for antigens using bioinformatics tools to clarify theMtbH37Rv Rv3632 protein’s topology and location. This article reports a C-terminal region consisting of peptides 39255 and 39256 (81Thr-Arg114) having high specific binding regarding two infection-related cell lines (A549 and U937); they inhibited mycobacterial entry to U937 cells in a concentration-dependent manner. Rv3632 forms part of the mycobacterial cell envelope, formed by six linear synthetic peptides. Circular dichroism enabled determining the protein’s secondary structure. It was also found that peptide 39254 (61Gly-Thr83) was a HABP for alveolar epithelial cells and inhibited mycobacteria entry to these cells regardless of concentration. Sera from active or latent tuberculosis patients did not recognise HABPs 39254 and 39256. These sequences represent a promising approach aiming at their ongoing modification and for including them when designing a multi-epitope, anti-tuberculosis vaccine.

Flavobacterium psychrophilum is the etiological agent of bacterial cold water disease (BCWD), also referred to as rainbow trout fry syndrome (RTFS), a disease with great economic impact in salmonid aquaculture. Despite this, to date, not many studies have analyzed in depth how the immune system is regulated during the course of the disease. In the current study, we have studied the transcription of several immune genes related to T and B cell activity in the skin of rainbow trout (Oncorhynchus mykiss) naturally infected with F. psychrophilum in a farm located in Lake Titicaca (Peru). The levels of expression of these genes were tested and compared to those obtained in asymptomatic and apparently healthy rainbow trout. In the case of symptomatic fish, skin samples containing characteristic ulcerative lesions were taken, as well as skin samples with no lesions. Our results pointed to a significant local up-regulation of IgD, CD4, CD8, perforin and IFNγ within the ulcerative lesions. On the other hand, no differences between the levels of expression of these genes were visible in the spleen. To confirm these results, the distribution of IgD+ and CD3+ cells was studied through immunohistochemical techniques in the ulcerative lesions. Our results demonstrate a strong local response to F. psychrophilum in rainbow trout in which IgD and T cells seem to play a major role.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that—in vertebrates—over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations., This research was funded primarily by the European Research Council (ERC) under the European Union’s Horizon 2020 and Seventh Framework Program FP7 research and innovation programs (ERC-AdG-LS8-740041 to J.L.G.-S., ERC-StG-LS2-637591 to M.I., a Marie Sklodowska-Curie Grant (658521) to I.M. and a FP7/2007-2013-ERC-268513 to P.W.H.H.), the Spanish Ministerio de Economía y Competitividad (BFU2016-74961-P to J.L.G.-S., RYC-2016-20089 to I.M., BFU2014-55076-P and BFU2017-89201-P to M.I. and BFU2014-55738-REDT to J.L.G.-S, M.I. and J.R.M.-M), the ‘Centro de Excelencia Severo Ochoa 2013-2017’(SEV-2012-0208), the ‘Unidad de Excelencia María de Maetzu 2017-2021’(MDM-2016-0687), the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7 under REA grant agreement number 607142 (DevCom) to J.L.G.-S., and the CNRS and the ANR (ANR16-CE12-0008-01) to H.E. O.B. was supported by an Australian Research Council Discovery Early Career Researcher Award (DECRA; DE140101962).

Quantitative polymerase chain reaction (qPCR) for Toxoplasma gondii multicopy genes has emerged as a promising strategy for sensitive detection of parasite DNA. qPCR can be performed from blood samples, which are minimally invasive to collect. However, there is no consensus about what type of blood specimen yields the best sensitivity. The development of a novel protocol for qPCR detection of T. gondii using blood clot, involving an appropriate DNA extraction method and the use of an internal amplification control to monitor the reaction is presented in the current study. Assays directed to the B1 and REP529 genes were performed in spiked specimens of whole blood, guanidine–ethylenediaminetetraacetic acid blood, and clot. The clot-based qPCR was shown to be more sensitive when compared with other types of specimens, detecting five and 0.05 T. gondii genomes, using B1 and REP529 targets, respectively. Finally, a comparative analysis with samples from HIV patients with clinical suspicion of toxoplasmosis was performed, demonstrating the detection of four positive suspected cases with clots compared with only one using guanidine–ethylenediaminetetraacetic acid blood. The high analytical sensitivity and the cost-effective advantages offered by clot supports this methodology as a good laboratory tool to monitor parasite burden.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

Brain aging is marked by a decline in cognitive abilities and associated with neurodegenerative disorders. Recent studies have shown, neurogenesis continues into adulthood but is known to be decreasing during advancing age and these changes may contribute to cognitive alterations. Advances, which aim to promote better aging are of paramount importance. Dietary restriction (DR) is the only non-genetic intervention that reliably extends life and health-span. Mechanism's of how and why DR and age affect neurogenesis are not well-understood, and have not been utilized much in the zebrafish, which has become a popular model to study brain aging and neurodegenerative disease due to widely available genetic tools. In this study we used young (8-8.5 months) and old (26-32.5 months) zebrafish as the model to investigate the effects of a short-term DR on actively proliferating cells. We successfully applied a 10-week DR to young and old fish, which resulted in a significant loss of body weight in both groups with no effect on normal age-related changes in body growth. We found that age decreased cell proliferation and increased senescence associated beta-galactosidase, as well as shortened telomere lengths. In contrast, DR shortened telomere lengths only in young animals. Neither age nor DR changed the differentiation patterns of glial cells. Our results suggest that the potential effects of DR could be mediated by telomere regulation and whether these are beneficial or negative remains to be determined. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikkϵ in Wnt-driven tumor development. We found that Ikkϵ was activated in intestinal tumors forming upon loss of the tumor suppressor Apc. Genetic ablation of Ikkϵ in β-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikkϵ to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikkϵ was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding pro-inflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikkϵ-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikkϵ phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikkϵ to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. Cancer Res; 76(9); 2587–99. ©2016 AACR.

Control of spindle orientation is a fundamental process for embryonic development, morphogenesis and tissue homeostasis, while defects are associated with tumorigenesis and other diseases. Force sensing is one of the mechanisms through which division orientation is determined. Here we show that integrin β1 plays a critical role in this process, becoming activated at the lateral regions of the cell cortex in a ligand-independent manner. This activation is force dependent and polar, correlating with the spindle capture sites. Inhibition of integrin β1 activation on the cortex and disruption of its asymmetric distribution leads to spindle misorientation, even when cell adhesion is β1 independent. Examining downstream targets reveals that a cortical mechanosensory complex forms on active β1, and regulates spindle orientation irrespective of cell context. We propose that ligand-independent integrin β1 activation is a conserved mechanism that allows cell responses to external stimuli., During cell division, the orientation of the mitotic spindle is sensitive to forces exerted on the cell cortex. Here Petridou and Skourides show that such cues are sensed by a mechanosensory complex established on force activated integrin β1 independent of ligand binding.

Transcriptomics in combination with in vitro cell systems is a powerful approach to unravel modes of action of toxicants. An important question is to which extent the modes of action as revealed by transcriptomics depend on cell type, species and study type (in vitro or in vivo). To acquire more insight into this, we assessed the transcriptomic effects of the immunosuppressive drug cyclosporine A (CsA) upon 6 h of exposure of the mouse cytotoxic T cell line CTLL-2, the thymoma EL-4 and primary splenocytes and compared these to the effects in spleens of mice orally treated with CsA for 7 days. EL-4 and CTLL-2 cells showed the highest similarities in response. CsA affected many genes in primary splenocytes that were not affected in EL-4 or CTLL-2. Pathway analysis demonstrated that CsA upregulated the unfolded protein response, endoplasmic reticulum stress and NRF2 activation in EL-4 cells, CTLL-2 cells and primary mouse splenocytes but not in mouse spleen in vivo. As expected, CsA downregulated cell cycle and immune response in splenocytes in vitro, spleens in vivo as well as CTLL-2 in vitro. Genes up- and downregulated in human Jurkat, HepG2 and renal proximal tubular cells were similarly affected in CTLL-2, EL-4 and primary splenocytes in vitro. In conclusion, of the models tested in this study, the known mechanism of immunotoxicity of CsA is best represented in the mouse cytotoxic T cell line CTLL-2. This is likely due to the fact that this cell line is cultured in the presence of a T cell activation stimulant (IL-2) making it more suitable to detect inhibitory effects on T cell activation. © 2014, Springer-Verlag Berlin Heidelberg. Chemicals/CAS: cyclosporin A, 59865-13-3, 63798-73-2; interleukin 2, 85898-30-2 Manufacturers: Sigma, Netherlands

Pradhan, Ajay/0000-0003-2305-8574, WOS: 000509791400034, PubMed ID: 31676350, In the present study, we investigated the detrimental effects of ethoxyquin (EQ) on zebrafish embryonic development using different endpoints including lethality, malformations, locomotion and gene expression. EQ is primarily used as a preservative in animal feed and it has been shown to have negative impacts on different laboratory animals. However, studies on the adverse effects of EQ in aquatic animals are still limited. In this study, zebrafish eggs were exposed to different concentrations of EQ ranging from 1 to 100 mu M for six days. In the 100 mu M treated groups 95 and 100% mortality was observed at 24 and 48 h, respectively. Delayed development, decreased pigmentation and pericardial edema were observed in larvae. Behavioral analysis of larvae demonstrated a distinct locomotive pattern in response to EQ both in light and dark indicating a possible developmental neurotoxicity and deficits in locomotion. The expression levels of genes involved in several physiological pathways including stress response, cell cycle and DNA damage were altered by EQ. Our results demonstrate that EQ could cause developmental and physiological toxicity to aquatic organisms. Hence, its toxic effect should be further analyzed and its use and levels in the environment must be monitored carefully., Knowledge Foundation, Sweden; Orebro University, This study was financed by Knowledge Foundation, Sweden, and Orebro University.

Accumulation of oxidative insults on molecular and supramolecular levels could compromise renewal potency and architecture in the aging skin. To examine and compare morphological and ultrastructural changes with redox alterations during chronological skin aging, activities of antioxidant defense (AD) enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), thioredoxin reductase (TR), and methionine sulfoxide reductase A (MsrA), and the markers of oxidative damage of biomolecules—4-hydroxynonenal (HNE) and 8-oxoguanine (8-oxoG)—were examined in the rat skin during life (from 3 days to 21 months). As compared to adult 3-month-old skin, higher activities of CAT, GSH-Px, and GR and a decline in expression of MsrA are found in 21-month-old skin. These changes correspond to degenerative changes at structural and ultrastructural levels in epidermal and dermal compartments, low proliferation capacity, and higher levels of HNE-modified protein aldehydes (particularly in basal lamina) and 8-oxoG positivity in nuclei and mitochondria in the sebaceous glands and root sheath. In 3-day-old skin, higher activities of AD enzymes (SOD, CAT, GR, and TR) and MsrA expression correspond to intensive postnatal development and proliferation. In contrast to 21-month-old skin, a high level of HNE in young skin is not accompanied by 8-oxoG positivity or any morphological disturbances. Observed results indicate that increased activity of AD enzymes in elderly rat skin represents the compensatory response to accumulated oxidative damage of DNA and proteins, accompanied by attenuated repair and proliferative capacity, but in young rats the redox changes are necessary and inherent with processes which occur during postnatal skin development. Мorphological and ultrastructurаl changes are in line with the redox profile in the skin of young and old rats.

Safety is prerequisite for preventive medicine, but non-toxic agents are generally ineffective as clinical chemoprevention. Here we propose a strategy overcoming this challenge by delivering molecular-targeted agent specifically to the effector cell type to achieve sufficient potency, while circumventing toxicity in the context of cancer chemoprevention. Hepatic myofibroblasts drive progressive fibrosis that results in cirrhosis and liver cancer. In a rat model of cirrhosis-driven liver cancer, a small molecule epidermal growth factor receptor inhibitor, erlotinib, was delivered specifically to myofibroblasts by a versatile nanoparticle-based system, targeting platelet-derived growth factor receptor-beta uniquely expressed on their surface in the liver. With systemic administration of erlotinib, tumor burden was reduced to 31%, which was further improved to 21% by myofibroblast-targeted delivery even with reduced erlotinib dose (7.3-fold reduction with equivalent erlotinib dose) and less hepatocyte damage. These findings demonstrate a strategy, cell type-specific kinase inhibition, for more effective and safer precision cancer chemoprevention. (c) 2017 Elsevier Inc. All rights reserved.

Ethnopharmacological importance Decoction and infusion prepared from aerial parts of Lavandula stoechas L. (L. stoechas) have been traditionally used as remedy against several components of metabolic syndrome (MetS) and associated disorders including type II diabetes and cardiovascular diseases by Anatolian people. Aim of the study The aim is to elucidate the potential ameliorative effects of L. stoechas aqueous extracts on insulin resistance and inflammation models through multitarget in vitro approaches and also to elucidate mechanism of action by analyzing transcriptional and metabolic responses. Materials and methods An aqueous extract was prepared and fractionated to give rise to ethyl acetate (EE) and butanol (BE) extracts. The anti-insulin resistance effects of BE and EE were evaluated on palmitate induced insulin resistance model of H4IIE, C2C12 and 3T3L1 cells by using several metabolic parameters. Specifically, whole genome transcriptome analysis was performed by using microarray over 55.000 genes in control, insulin resistant and EE (25 µg/mL) treated insulin resistant H4IIE cells. Anti-inflammatory effects of both extracts were analyzed in LPS-stimulated RAW264.7 macrophages. Results Both EE and BE at low doses (25–50 µg/mL) significantly decreased hepatic gluconeogenesis in H4IIE cell line by suppressing the expression of PEPCK and G6Pase. In C2C12 myotubes, both extracts increased the insulin stimulated glucose uptake more effectively than metformin. Both extracts decreased the isoproterenol induced lipolysis in 3T3L1 cell line. Moreover, they also effectively increased the expression of lipoprotein lipase protein level in insulin resistant myotubes at low doses. EE increased the protein level of PPARγ and stimulated the activation AKT in insulin resistant H4IIE and C2C12 cell lines. The results obtained from biochemical assays, mRNA/protein studies and whole genome transcriptome analyses were found to be complementary and provided support for the hypothesis that EE might be biologically active against insulin resistance and act through the inhibition of liver gluconeogenesis and AKT activation. Besides, LPS induced inflammation in RAW264.7 macrophages was mainly inhibited by EE through suppression of iNOS/NO signaling, IL1β and COX-2 genes. HPLC-TOF/MS analysis of EE of L. stoechas mainly resulted in caffeic acid, apigenin, luteolin, rosmarinic acid and its methyl ester, 4-hydroxybenzoic acid, vanillic acid, ferrulic acid and salicylic acid. Conclusion Data suggest that EE of L. stoechas contains phytochemicals that can be effective in the treatment/prevention of insulin resistance and inflammation. These results validate the traditional use of L. stoechas in Anatolia against several metabolic disorders including metabolic syndrome.

Neurocysticercosis is a parasitic brain disease caused by the larval form (Cysticercus cellulosae) of the Taenia solium and is the leading cause of preventable epilepsy world-wide. However, the pathophysiology and relation to the wide range of clinical features remains poorly understood. Axonal swelling is emerging as an important early pathological finding in multiple neurodegenerative diseases and causes of brain injury, but has not been well-described in neurocysticercosis. Histological analysis was performed on human, rat and porcine NCC brain specimens to identify axonal pathology. Rat infection was successfully carried out via two routes of inoculation: direct intracranial injection and oral feeding. Extensive axonal swellings, in the form of spheroids, were observed in both humans and rats and to a lesser extent in pigs with NCC. Spheroids demonstrated increased immunoreactivity to amyloid precursor protein and neurofilament indicating probable impairment of axonal transport. These novel findings demonstrate that spheroids are present in NCC which is conserved across species. Not only is this an important contribution towards understanding that pathogenesis of NCC, but it also provides a model to analyze the association of spheroids with specific clinical features and to investigate the reversibility of spheroid formation with antihelmintic treatment.

Lipid metabolism involves complex pathways, which are regulated in a similar way across vertebrates. Hormonal and hypolipidemic deregulations cause lipid imbalance from fish to humans, but the underlying mechanisms are far from understood. This study explores the potential of using juvenile brown trout to evaluate the in vivo interferences caused by estrogenic (17α-ethinylestradiol – EE2), androgenic (testosterone – T), and hypolipidemic (clofibrate – CLF) compounds in lipidic and/or peroxisomal pathways. Studied endpoints were from blood/plasma biochemistry, plasma fatty acid profile, ultrastructure of hepatocytes and abundance of their peroxisomes to mRNA expression in the liver. Both T and CLF caused minimal effects when compared to EE2. Estrogenized fish had significantly higher hepatosomatic indexes, increased triglycerides and very-low density lipoproteins (VLDL) in plasma, compared with solvent control. Morphologically, EE2 fish showed increased lipid droplets in hepatocytes, and EE2 and T reduced volume density of peroxisomes in relation to the hepatic parenchyma. Polyunsaturated fatty acids (PUFA) in plasma, namely n−3 PUFA, increased with EE2. EE2 animals had increased mRNA levels of vitellogenin A (VtgA), estrogen receptor alpha (ERα), peroxisome proliferator-activated receptor alpha (PPARα), PPARαBa and acyl-CoA long chain synthetase 1 (Acsl1), while ERβ-1, acyl-CoA oxidase 1–3I (Acox1–3I), Acox3, PPARγ, catalase (Cat), urate oxidase (Uox), fatty acid binding protein 1 (Fabp1) and apolipoprotein AI (ApoAI) were down-regulated. In summary, in vivo EE2 exposure altered lipid metabolism and peroxisome dynamics in brown trout, namely by changing the mRNA levels of several genes. Our model can be used to study possible organism-level impacts, viz. in gonadogenesis. © 2018 Elsevier This study was primarily funded by FCT - Foundation for Science and Technology , through the project PTDC/CVT/115618/2009 and grants given to Tânia V. Madureira ( SFRH/BPD/97139/2013 ) and Tiago Simões ( SFRH/BD/98266/2013 ). It was also partially supported by the Strategic Funding UID/Multi/04423/2013 and UID/MAR/04292/2013 granted to CIMAR and MARE, respectively, through national funds provided by FCT and European Regional Development Fund (ERDF), in the framework of the program PT2020 . The work was implemented in the Framework of the Structured Program of R&D&I INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources (NORTE-01-0145-FEDER-000035), namely within the Research Line ECOSERVICES, supported by the Northern Regional Operational Programme ( NORTE2020 ), through ERFD. This study is also included in the Integrated Program of SR&TD "SmartBioR" (reference Center-01-0145-FEDER-000018), cofunded by Centro 2020 program, by PT2020, through the ERDF.

Renalase, a recently identified oxidoreductase, is emerging as a novel regulator of cardiovascular and metabolic disease states. The mechanism of regulation of renalase gene, especially at the post-transcriptional level, is completely unknown. We set out to investigate the possible role of microRNAs in regulation of renalase gene in this study. Computational predictions using multiple algorithms coupled with systematic functional analysis revealed specific interactions of miR-29a/b/c and miR-146a/b with mouse and human renalase 3?-UTR (untranslated region) in cultured cells. Next, we estimated miR-29b and miR-146a, as well as renalase expression, in genetically hypertensive blood pressure high and genetically hypotensive blood pressure low mice. Kidney tissues from blood pressure high mice showed diminished (? 1.6- to 1.8-fold) renalase mRNA/protein levels and elevated (? 2.2-fold) miR-29b levels as compared to blood pressure low mice. A common single nucleotide polymorphism in human renalase 3?-UTR (C/T, rs10749571) creates a binding site for miR-146a, consistently, miR-146a down-regulated human renalase 3?-UTR/luciferase activity in case of the T allele suggesting its potential role in regulation of renalase in humans. Indeed, genome-wide association studies revealed directionally concordant association of rs10749571 with diastolic blood pressure, glucose and triglyceride levels in large human populations (n ? 58,000-96,000 subjects). This study provides evidence for post-transcriptional regulation of renalase gene by miR-29 and miR-146 and has implications for inter-individual variations on cardiometabolic traits. � 2015 Elsevier Ltd. All rights reserved.

With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs.

Tri-ortho-cresyl phosphate (ToCP) is a multipurpose organophosphorus compound that is neurotoxic and suspected to be involved in aerotoxic syndrome in humans. It has been reported that not ToCP itself but a metabolite of ToCP, namely, 2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one (CBDP), may be responsible for this effect as it can irreversibly bind to human butyrylcholinesterase (BuChE) and human acetylcholinesterase (AChE). The bioactivation of ToCP into CBDP involves Cytochrome P450s (P450s). However, the individual human P450s responsible for this bioactivation have not been identified yet. In the present study, we aimed to investigate the metabolism of ToCP by different P450s and to determine the inhibitory effect of the in vitro generated ToCP-metabolites on human BuChE and AChE. Human liver microsomes, rat liver microsomes, and recombinant human P450s were used for that purpose. The recombinant P450s 2B6, 2C18, 2D6, 3A4 and 3A5 showed highest activity of ToCP-bioactivation to BuChE-inhibitory metabolites. Inhibition experiments using pooled human liver microsomes indicated that P450 3A4 and 3A5 were mainly involved in human hepatic bioactivation of ToCP. In addition, these experiments indicated a minor role for P450 1A2. Formation of CBDP by in-house expressed recombinant human P450s 1A2 and 3A4 was proven by both LC-MS and GC-MS analysis. When ToCP was incubated with P450 1A2 and 3A4 in the presence of human BuChE, CBDP-BuChE-adducts were detected by LC-MS/MS which were not present in the corresponding control incubations. These results confirmed the role of human P450s 1A2 and 3A4 in ToCP metabolism and demonstrated that CBDP is the metabolite responsible for the BuChE inactivation. Interindividual differences at the level of P450 1A2 and 3A4 might play an important role in the susceptibility of humans in developing neurotoxic effects, such as aerotoxic syndrome, after exposure to ToCP. © 2015 American Chemical Society

Tumor vaccine design requires prediction and validation of immunogenic MHC class I epitopes expressed by target cells as well as MHC class II epitopes expressed by antigen-presenting cells essential for the induction of optimal immune responses. Epitope prediction methods are based on different algorithms and are instrumental for a first screening of possible epitopes. However, their results do not reflect a one-to-one correlation with experimental data. We combined several in silico prediction methods to unravel the most promising C57BL/6 mouse-restricted Hepatitis C virus (HCV) MHC class I epitopes and validated these epitopes in vitro and in vivo. Cytotoxic T lymphocyte (CTL) epitopes within the HCV non-structural proteins were identified, and proteasomal cleavage sites and helper T cell (Th) epitopes at close proximity to these CTL epitopes were analyzed using multiple prediction algorithms. This combined in silico analysis enhances the precision of identification of functional HCV-specific CTL epitopes. This approach will be applicable to the design of human vaccines not only for HCV, but also for other antigens in which T-cell responses play a crucial role.

The skin is an attractive organ for immunization because of the presence of antigen-presenting cells. Intradermal delivery via tattooing has demonstrated superior vaccine immunogenicity of DNA vaccines in comparison to conventional delivery methods. In this study, we explored the efficacy of tattoo injection of a tumor vaccine based on recombinant Semliki Forest virus replicon particles (rSFV) targeting human papillomavirus (HPV). Tattoo injection of rSFV particles resulted in antigen expression in both the skin and draining lymph nodes. In comparison with intramuscular injection, the overall antigen expression determined at the site of administration and draining lymph nodes was 10-fold lower upon tattoo injection. Delivery of SFV particles encoding the E6 and E7 antigens of human papillomavirus type 16 (SFVeE6,7) via tattooing resulted in HPV-specific cytotoxic T cells and in vivo therapeutic antitumor response. Strikingly, despite the observed lower overall transgene expression, SFVeE6,7 delivered via tattoo injection resulted in higher or equal levels of immune responses as compared to intramuscular injection. The intrinsic immunogenic potential of tattooing provides a benefit for immunotherapy based on an alphavirus.

A chemical genomic screen of an in-house library of small molecule heterocycles was carried out using Xenopus laevis embryos. This led to the identification of N-(4-chloro-5H-1,2,3-dithiazol-5-ylidene)-4-methoxyaniline (1c), which elicits loss of pigmentation in melanophores and the retinal pigment epithelium (RPE) of developing embryos, independent of the developmental stage of initial exposure. The phenotype was reversible, since pigmentation returned upon compound removal while analysis of neural crest cell markers (Pax7) and melanophore markers (Dct/Xtrp2) revealed that both neural crest precursors and fully differentiated melanophores were present in the dithiazole 1c treated embryos. A subsequent focused structure-activity relationship (SAR) study identified the more active dithiazole 4-benzyloxy-N-(4-chloro-5H-1,2,3-dithiazol-5-ylidene)-aniline (1l) and the need for a chlorine substituent at the dithiazole C-4 position. Both the initial chemical genomic screen and the focused SAR study highlighted the toxicity of (dithiazolylidene)aminoazines, and also of methoxyaniline (anisidine) analogues that hosted strong electron-withdrawing or electronegative substituents or acidic hydroxyl groups on the anisidine moiety. This study suggests that 1,2,3-dithiazoles can act as reversible melanin synthesis inhibitors, revealing a new biological activity for this class of compounds. The inhibition of melanin synthesis is medically relevant as a potential treatment for pigmentation disorders such as melasma. © The Royal Society of Chemistry 2015. 6 935 946 Cited By :2

Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods. © Kasper WJ Derks, Branislav Misovic, Mirjam CGN van den Hout, Christel EM Kockx, Cesar Payan Gomez, Rutger WW Brouwer, Harry Vrieling, Jan HJ Hoeijmakers, Wilfred FJ van IJcken, and Joris Pothof.

716749 Made available in DSpace on 2019-09-11T20:51:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2015 This study aimed to evaluate the capacity of Lactobacillus rhamnosus and/or its products to induce the synthesis of cytokines (TNF-α, IL-1β, IL-4, IL-6, IL-10, and IL-12) by mouse macrophages (RAW 264.7). Three microorganism preparations were used: live L. rhamnosus (LLR) suspension, heat-killed L. rhamnosus (HKLR) suspension, and the supernatant of a heat-killed L. rhamnosus (SHKLR) suspension, which were cultured with macrophages (37°C, 5% CO2) for 2 h and 30 min. After that, cells were cultured for 16 h. The supernatants were used for the quantitation of cytokines, by ELISA. The results were compared with the synthesis induced by lipopolysaccharide (LPS) and analysed, using ANOVA and Tukey test, 5%. LLR and HKLR groups were able to significantly increase the production of TNF-α, IL-6, and IL-10 (P < 0. 05). SHKLR also significantly increased the production of TNF-α and IL-10 (P < 0. 05) but not IL-6 (P > 0. 05). All the L. rhamnosus suspensions were not able to produce detectable levels of IL-1β or significant levels of IL-4 and IL-12 (P > 0. 05). In conclusion, live and heat-killed L. rhamnosus suspensions were able to induce the synthesis of different cytokines with proinflammatory (TNF-α and IL-6) or regulatory (IL-10) functions, suggesting the role of strain L. rhamnosus ATCC 7469 in the modulation or in the stimulation of immune responses. © 2015 Adeline Lacerda Jorjão et al. Jorjão, A.L., Department of Biosciences and Oral Diagnosis, Laboratory of Microbiology and Immunology, Institute of Science and Technology, Universidade Estadual Paulista (UNESP), Avenida Engenheiro Francisco José Longo, São José dos Campos, SP, 12245-000, Brazil De Oliveira, F.E., Department of Biosciences and Oral Diagnosis, Laboratory of Microbiology and Immunology, Institute of Science and Technology, Universidade Estadual Paulista (UNESP), Avenida Engenheiro Francisco José Longo, São José dos Campos, SP, 12245-000, Brazil Leão, M.V.P., Bioscience Basic Institute, University of Taubaté, Street Expedicionário Ernesto Pereira 3, Taubaté, SP, 12020-130, Brazil Carvalho, C.A.T., Department of Restorative Dentistry, Institute of Science and Technology, Universidade Estadual Paulista (UNESP), Avenida Engenheiro Francisco José Longo, São José dos Campos, SP, 12245-000, Brazil Jorge, A.O.C., Department of Biosciences and Oral Diagnosis, Laboratory of Microbiology and Immunology, Institute of Science and Technology, Universidade Estadual Paulista (UNESP), Avenida Engenheiro Francisco José Longo, São José dos Campos, SP, 12245-000, Brazil De Oliveira, L.D., Department of Biosciences and Oral Diagnosis, Laboratory of Microbiology and Immunology, Institute of Science and Technology, Universidade Estadual Paulista (UNESP), Avenida Engenheiro Francisco José Longo, São José dos Campos, SP, 12245-000, Brazil

High concentrations of adenosine in tumor microenvironments inhibit antitumor cytotoxic lymphocyte responses. Although T cells express inhibitory adenosine A2A receptors (A2AR) that suppress their activation and inhibit immune killing of tumors, a role for myeloid cell A2ARs in suppressing the immune response to tumors has yet to be investigated. In this study, we show that the growth of transplanted syngeneic B16F10 melanoma or Lewis lung carcinoma cells is slowed in Adora2af/f–LysMCre+/− mice, which selectively lack myeloid A2ARs. Reduced melanoma growth is associated with significant increases in MHCII and IL12 expression in tumor-associated macrophages and with >90% reductions in IL10 expression in tumor-associated macrophages, dendritic cells (DC), and Ly6C+ or Ly6G+ myeloid-derived suppressor cells (MDSC). Myeloid deletion of A2ARs significantly increases CD44 expression on tumor-associated T cells and natural killer (NK) cells. Depletion of CD8+ T cells or NK cells in tumor-bearing mice indicates that both cell types initially contribute to slowing melanoma growth in mice lacking myeloid A2A receptors, but tumor suppression mediated by CD8+ T cells is more persistent. Myeloid-selective A2AR deletion significantly reduces lung metastasis of melanomas that express luciferase (for in vivo tracking) and ovalbumin (as a model antigen). Reduced metastasis is associated with increased numbers and activation of NK cells and antigen-specific CD8+ T cells in lung infiltrates. Overall, the findings indicate that myeloid cell A2ARs have direct myelosuppressive effects that indirectly contribute to the suppression of T cells and NK cells in primary and metastatic tumor microenvironments. The results indicate that tumor-associated myeloid cells, including macrophages, DCs, and MDSCs all express immunosuppressive A2ARs that are potential targets of adenosine receptor blockers to enhance immune killing of tumors. Cancer Res; 74(24); 7250–9. ©2014 AACR.

Adenosine A2A receptor (A2AR) blockade enhances innate and adaptive immune responses. However, mouse genetic studies have shown that A2AR deletion does not inhibit the growth of all tumor types. In the current study, we showed that growth rates for ectopic melanoma and bladder tumors are increased in Adora2a−/− mice within 2 weeks of tumor inoculation. A2AR deletion in the host reduced numbers of CD8+ T cells and effector–memory differentiation of all T cells. To examine intrinsic functions in T cells, we generated mice harboring a T-cell–specific deletion of A2AR. In this host strain, tumor-bearing mice displayed increased growth of ectopic melanomas, decreased numbers of tumor-associated T cells, reduced effector–memory differentiation, and reduced antiapoptotic IL7Rα (CD127) expression on antigen-experienced cells. Intratumoral pharmacologic blockade similarly reduced CD8+ T-cell density within tumors in wild-type hosts. We found that A2AR-proficient CD8+ T cells specific for melanoma cells displayed a relative survival advantage in tumors. Thus, abrogating A2AR signaling appeared to reduce IL7R expression, survival, and differentiation of T cells in the tumor microenvironment. One implication of these results is that the antitumor effects of A2AR blockade that can be mediated by activation of cytotoxic T cells may be overcome in some tumor microenvironments as a result of impaired T-cell maintenance and effector–memory differentiation. Thus, our findings imply that the efficacious application of A2AR inhibitors for cancer immunotherapy may require careful dose optimization to prevent activation-induced T-cell death in tumors. Cancer Res; 74(24); 7239–49. ©2014 AACR.

Tachykinins are comprised of the family of related peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). NKB has emerged as regulator of kisspeptin release in the arcuate nucleus (ARC), whereas the roles of SP and NKA in reproduction remain unknown. This work explores the roles of SP and NKA in the central regulation of GnRH release. First, central infusion of specific agonists for the receptors of SP (neurokinin receptor 1, NK1R), NKA (NK2R) and NKB (NK3R) each induced gonadotropin release in adult male and ovariectomized, estradiol-replaced female mice, which was absent in Kiss1r-/- mice, indicating a kisspeptin-dependent action. The NK2R agonist, however, decreasedLHrelease in ovariectomized-sham replaced females, as documented forNK3R agonists but in contrast to the NK1R agonist, which further increased LH release. Second, Tac1 (encoding SP and NKA) expression in the ARC and ventromedial nucleus was inhibited by circulating estradiol but did not colocalize with Kiss1 mRNA. Third, about half of isolated ARC Kiss1 neurons expressed Tacr1 (NK1R) and 100% Tacr3 (NK3R); for anteroventral-periventricular Kiss1 neurons andGnRHneurons, approximately one-fourth expressed Tacr1 and one-tenth Tacr3; Tacr2 (NK2R) expression was absent in all cases. Overall, these results identify a potent regulation of gonadotropin release by the SP/NK1R and NKA/NK2R systems in the presence of kisspeptin-Kiss1r signaling, indicating that they may, along with NKB/NK3R, control GnRH release, at least in part through actions on Kiss1 neurons. Copyright © 2015 by the Endocrine Society.

Somitogenesis is one of the major hallmarks of bilateral symmetry in vertebrates. This symmetry is lost when retinoic acid (RA) signalling is inhibited, allowing the left-right determination pathway to influence somitogenesis. In all three studied vertebrate model species, zebrafish, chicken and mouse, the frequency of somite formation becomes asymmetric, with slower gene expression oscillations driving somitogenesis on the right side. Still, intriguingly, the resulting left-right asymmetric phenotypes differ significantly between these model species. While somitogenesis is generally considered as functionally equivalent among different vertebrates, substantial differences exist in the subset of oscillating genes between different vertebrate species. Variation also appears to exist in the way oscillations cease and somite boundaries become patterned. In addition, in absence of RA, the FGF8 gradient thought to constitute the determination wavefront becomes asymmetric in zebrafish and mouse, extending more anteriorly to the right, while remaining symmetric in chicken. Here we use a computational modelling approach to decipher the causes underlying species differences in asymmetric somitogenesis. Specifically, we investigate to what extent differences can be explained from observed differences in FGF asymmetry and whether differences in somite determination dynamics may also be involved. We demonstrate that a simple clock-and-wavefront model incorporating the observed left-right differences in somitogenesis frequency readily reproduces asymmetric somitogenesis in chicken. However, incorporating asymmetry in FGF signalling was insufficient to robustly reproduce mouse or zebrafish asymmetry phenotypes. In order to explain these phenoptypes we needed to extend the basic model, incorporating species-specific details of the somitogenesis determination mechanism. Our results thus demonstrate that a combination of differences in FGF dynamics and somite determination cause species differences in asymmetric somitogenesis. In addition,they highlight the power of using computational models as well as studying left-right asymmetry to obtain more insight in somitogenesis.

Primary ovarian insufficiency (POI) affects ~1% of women in the general population. Despite numerous attempts at identifying POI genetic aetiology, coding mutations in only a few genes have been functionally related to POI pathogenesis. It has been suggested that mutant BMPR2 might contribute towards the phenotype. Several BMP15 (a BMPR2 ligand) coding mutations in human species have been related to POI pathogenesis. The BMPR2 p.Ser987Phe mutation, previously identified in a woman with POI, might therefore lead to cellular dysfunction contributing to the phenotype. To explore such an assumption, the present study assessed potential pathogenic subcellular localization/aggregation patterns associated with the p.Ser987Phe mutant form of BMPR2 in a relevant model for studying ovarian function. A significant increase in protein-like aggregation patterns was identified at the endoplasmic reticulum (ER) which permitted us to establish, for the first time, a potential functional association between mutant BMPR2 and POI aetiology. Since BMPR2 mutant forms were previously related to idiopathic pulmonary arterial hypertension, BMPR2 mutations may be related to an as-yet-to-be described syndromic form of POI involving pulmonary dysfunction. Additional assays are necessary to confirm that BMPR2 abnormal subcellular patterns are composed by aggregates. Abbreviations: POI: primary ovarian insufficiency; ER: endoplasmic reticulum; NGS: next generation sequencing. © 2017 Taylor and Francis.