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Background DNA polymerase epsilon (POLE) is encoded by the POLE gene, and POLE-driven tumors are characterized by high mutational rates. POLE-driven tumors are relatively common in endometrial and colorectal cancer, and their presence is increasingly recognized in ovarian cancer (OC) of endometrioid type. POLE-driven cases possess an abundance of TCT > TAT and TCG > TTG somatic mutations characterized by mutational signature 10 from the Catalog of Somatic Mutations in Cancer (COSMIC). By quantifying the contribution of COSMIC mutational signature 10 in RNA sequencing (RNA-seq) we set out to identify POLE-driven tumors in a set of unselected Mayo Clinic OC. Methods Mutational profiles were calculated using expressed single-nucleotide variants (eSNV) in the Mayo Clinic OC tumors (n = 195), The Cancer Genome Atlas (TCGA) OC tumors (n = 419), and the Genotype-Tissue Expression (GTEx) normal ovarian tissues (n = 84). Non-negative Matrix Factorization (NMF) of the mutational profiles inferred the contribution per sample of four distinct mutational signatures, one of which corresponds to COSMIC mutational signature 10. Results In the Mayo Clinic OC cohort we identified six tumors with a predicted contribution from COSMIC mutational signature 10 of over five mutations per megabase. These six cases harbored known POLE hotspot mutations (P286R, S297F, V411L, and A456P) and were of endometrioid histotype (P = 5e−04). These six tumors had an early onset (average age of patients at onset, 48.33 years) when compared to non-POLE endometrioid OC cohort (average age at onset, 60.13 years; P = .008). Samples from TCGA and GTEx had a low COSMIC signature 10 contribution (median 0.16 mutations per megabase; maximum 1.78 mutations per megabase) and carried no POLE hotspot mutations. Conclusions From the largest cohort of RNA-seq from endometrioid OC to date (n = 53), we identified six hypermutated samples likely driven by POLE (frequency, 11%). Our result suggests the clinical need to screen for POLE driver mutations in endometrioid OC, which can guide enrollment in immunotherapy clinical trials.

The ATP-dependent chromatin remodeling proteins play an important role in DNA repair. The energy released by ATP hydrolysis is used for myriad functions ranging from nucleosome repositioning and nucleosome eviction to histone variant exchange. In addition, the distant member of the family, SMARCAL1, uses the energy to reanneal stalled replication forks in response to DNA damage. Biophysical studies have shown that this protein has the unique ability to recognize and bind specifically to DNA structures possessing double-strand to single-strand transition regions. Mutations in SMARCAL1 have been linked to Schimke immuno-osseous dysplasia, an autosomal recessive disorder that exhibits variable penetrance and expressivity. It has long been hypothesized that the variable expressivity and pleiotropic phenotypes observed in the patients might be due to the ability of SMARCAL1 to co-regulate the expression of a subset of genes within the genome. Recently, the role of SMARCAL1 in regulating transcription has been delineated. In this review, we discuss the biophysical and functional properties of the protein that help it to transcriptionally co-regulate DNA damage response as well as to bind to the stalled replication fork and stabilize it, thus ensuring genomic stability. We also discuss the role of SMARCAL1 in cancer and the possibility of using this protein as a chemotherapeutic target.

Pulmonary neuroendocrine neoplasms (NENs) are classified into low-grade neuroendocrine tumors and high-grade neuroendocrine carcinomas (NECs). There are significant differences in therapeutic strategies of the different NEN subtypes, and therefore, precise classification of pulmonary NENs is critical. However, challenges in pulmonary NEN classification include overlap of diagnostic histological features among the subtypes and reduced or negative expression of neuroendocrine markers in poorly differentiated pulmonary NECs. Recently, transcription factor insulinoma-associated protein 1 (INSM1) was identified as a sensitive marker of neuroendocrine and neuroepithelial differentiation. In this study, INSM1 expression was detected by immunohistochemistry in greater than 94% of pulmonary NENs, indicating that it is a highly sensitive marker of pulmonary NENs and is useful to detect poorly differentiated pulmonary NECs. Although there are well-established morphological and immunohistologic criteria to diagnose pulmonary NENs, there is no universal consensus regarding prognostic markers of pulmonary NENs. Studies have shown that non–small cell lung cancers express long non-coding RNAs (lncRNAs), which regulate gene expression, epithelial-to-mesenchymal transition, and carcinogenesis. We characterized expression and function of lncRNAs, including HOX transcript antisense RNA (HOTAIR), maternally expressed 3 (MEG3), and prostate cancer antigen 3 (PCA3) in pulmonary NENs, including typical carcinoid tumors, atypical carcinoid tumors, small cell lung carcinoma (SCLC/NEC), and large cell neuroendocrine carcinoma (LCNEC/NEC). In situ hybridization and real-time polymerase chain reaction studies showed higher expression (p

Enzymes are often immobilized on solid supports to enable their recovery from reaction solutions, facilitate their reuse and hence increase cost-effectiveness in their application. Immobilized enzymes may even be used for flow-through applications in continuous processes. However, the synthesis of traditional immobilization scaffolds and immobilization techniques lack sustainability as they are often based on fuel-based materials and tedious synthesis- and immobilization approaches. Here, we present the natural material wood as a green alternative for enzyme immobilization. Its natural structure provides a mechanically stable porous scaffold with a high inner surface area that allows for directional flow-through of liquids. Enzymes were immobilized by nanoparticle-mediated adsorption, a simple, versatile and completely water-based process. The resulting wood-enzyme hybrids were intensely investigated for the model enzyme laccase. Reaction kinetics, as well as catalytic activities at various pH-values, temperatures, and ionic strengths were determined. The wood-enzyme hybrids could quickly and completely be removed from the reaction solution. Hence, they allow for multifold reusability. We show a series of 25 consecutive reaction cycles with a remaining activity in the last cycle of 90% of the maximal activity. Moreover, the anisotropic porosity of wood enabled the application of the hybrid material as a biocatalytic flow-through reactor. Flow-rate dependent productivity of a single-enzyme reaction was determined. Moreover, we show a two-step reaction cascade in continuous flow by the immobilization of the enzymes glucose oxidase and horseradish peroxidase. Therefore, the natural material wood proved to be a promising material for application in continuous-flow biocatalysis.

Systemic lupus erythematosus (SLE) is a disease characterized by dysregulation and hyperreactivity of the immune response at various levels, including hyperactivation of effector cell subtypes, autoantibodies production, immune complex formation, and deposition in tissues. The consequences of hyperreactivity to the self are systemic and local inflammation and tissue damage in multiple organs. Lupus nephritis (LN) is one of the most worrying manifestations of SLE, and most patients have this involvement at some point in the course of the disease. Among the effector cells involved, the Th17, a subtype of T helper cells (CD4+), has shown significant hyperactivation and participates in kidney damage and many other organs. Th17 cells have IL-17A and IL-17F as main cytokines with receptors expressed in most renal cells, being involved in the activation of many proinflammatory and profibrotic pathways. The Th17/IL-17 axis promotes and maintains repetitive tissue damage and maladaptive repair; leading to fibrosis, loss of organ architecture and function. In the podocytes, the Th17/IL-17 axis effects include changes of the cytoskeleton with increased motility, decreased expression of health proteins, increased oxidative stress, and activation of the inflammasome and caspases resulting in podocytes apoptosis. In renal tubular epithelial cells, the Th17/IL-17 axis promotes the activation of profibrotic pathways such as increased TGF-β expression and epithelial-mesenchymal transition (EMT) with consequent increase of extracellular matrix proteins. In addition, the IL-17 promotes a proinflammatory environment by stimulating the synthesis of inflammatory cytokines by intrinsic renal cells and immune cells, and the synthesis of growth factors and chemokines, which together result in granulopoiesis/myelopoiesis, and further recruitment of immune cells to the kidney. The purpose of this work is to present the prognostic and immunopathologic role of the Th17/IL-17 axis in Kidney diseases, with a special focus on LN, including its exploration as a potential immunotherapeutic target in this complication.

As patient derived xenograft (PDX) models are increasingly used for preclinical drug development, strategies to account for the nonhuman component of PDX RNA expression data are critical to its interpretation. A bioinformatics pipeline to separate donor tumor and mouse stroma transcriptome profiles was devised and tested. To examine the molecular fidelity of PDX versus donor tumors, we compared mRNA differences between paired PDX-donor tumors from nine ovarian cancer patients. 1,935 differentially expressed genes were identified between PDX and donor tumors. Over 90% (n = 1767) of these genes were down-regulated in PDX models and enriched in stroma-specific functions. Several protein kinases were also differentially expressed in PDX tumors, e.g. PDGFRA, PDGFRB and CSF1R. Upon in silico removal of these PDX-donor tumor differentially expressed genes, a stronger transcriptional resemblance between PDX-donor tumor pairs was seen (average correlation coefficient increases from 0.91 to 0.95). We devised and validated an effective bioinformatics strategy to separate mouse stroma expression from human tumor expression for PDX RNAseq. In addition, we showed most of the PDX-donor differentially expressed genes were implicated in stromal components. The molecular similarities and differences between PDX and donor tumors have implications in future therapeutic trial designs and treatment response evaluations using PDX models.

Simple Summary It is well known that actin forms a cytoplasmic network of microfilaments, the part of the cytoskeleton, in the cytoplasm of eukaryotic cells. The presence of nuclear actin was elusive for a very long time. Now, there is a very strong evidence that actin plays many important roles in the nucleus. Here, we discuss the recently discovered functions of the nuclear actin pool. Actin does not have nuclear localization signal (NLS), so its import to the nucleus is facilitated by the NLS-containing proteins. Nuclear actin plays a role in the maintenance of the nuclear structure and the nuclear envelope breakdown. It is also involved in chromatin remodeling, and chromatin and nucleosome movement necessary for DNA recombination, repair, and the initiation of transcription. It also binds RNA polymerases, promoting transcription. Because of the multifaceted role of nuclear actin, the future challenge will be to further define its functions in various cellular processes and diseases. Abstract Actin is one of the most abundant proteins in eukaryotic cells. There are different pools of nuclear actin often undetectable by conventional staining and commercial antibodies used to identify cytoplasmic actin. With the development of more sophisticated imaging and analytical techniques, it became clear that nuclear actin plays a crucial role in shaping the chromatin, genomic, and epigenetic landscape, transcriptional regulation, and DNA repair. This multifaceted role of nuclear actin is not only important for the function of the individual cell but also for the establishment of cell fate, and tissue and organ differentiation during development. Moreover, the changes in the nuclear, chromatin, and genomic architecture are preamble to various diseases. Here, we discuss some of the newly described functions of nuclear actin.

International audience; Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.

The omentum is the most common site of ovarian cancer metastasis. Immune cell clusters called milky spots are found throughout the omentum. It is however unknown if these immune cells contribute to ovarian cancer metastasis. Here we report that omental macrophages promote the migration and colonization of ovarian cancer cells to the omentum through the secretion of chemokine ligands that interact with chemokine receptor 1 (CCR1). We found that depletion of macrophages reduces ovarian cancer colonization of the omentum. RNA-sequencing of macrophages isolated from mouse omentum and mesenteric adipose tissue revealed a specific enrichment of chemokine ligand CCL6 in omental macrophages. CCL6 and the human homolog CCL23 were both necessary and sufficient to promote ovarian cancer migration by activating ERK1/2 and PI3K pathways. Importantly, inhibition of CCR1 reduced ovarian cancer colonization. These findings demonstrate a critical mechanism of omental macrophage induced colonization by ovarian cancer cells via CCR1 signaling., Krishnan et al. find that CCR1 ligands CCL6 and CCL23 secreted by murine and human macrophages, respectively, enhance metastatic colonization of ovarian cancer cells to the omentum in manner dependent on chemokine receptor 1 (CCR1). This study suggests that targeting CCR1 or CCL23 in ovarian cancer may be a therapeutic strategy.

Pseudenhygromyxa WMMC2535, a representative of the myxobacteria (family Nannocystaceae ), was isolated from a ragged sea hare in the Florida Keys, and its genome was sequenced using PacBio technology. The WMMC2535 genome sequence is the first of this genus and validates the notion that myxobacteria represent outstanding sources of structurally diverse natural products.

Epidemiological data in COVID-19 mortality indicate that men are more prone to die of SARS-CoV-2 infection than women, but biological causes for this sexual dimorphism are unknown. We discuss the prospective behavioral and biological differences between the sexes that could be attributed to this sex-based differentiation. The female sex hormones and the immune stimulatory genes, including Toll-like receptors, interleukins, and micro-RNAs present on X-chromosome, may impart lesser infectivity and mortality of the SARS-CoV-2 in females over males. The sex hormone estrogen interacts with the renin-angiotensin-aldosterone system, one of the most critical pathways in COVID-19 infectivity, and modulates the vasomotor homeostasis. Testosterone on the contrary enhances the levels of the two most critical molecules, angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease serine-type 2 (TMPRSS2), transcriptionally and posttranslationally, thereby increasing viral load and delaying viral clearance in men as compared with women. We propose that modulating sex hormones, either by increasing estrogen or antiandrogen, may be a therapeutic option to reduce mortality from SARS-CoV-2.

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment1. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes1. Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes—interstitial cells—by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR–Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals—and, more generally, micronutrients—to energy homeostasis.

BACKGROUND: Transplantation of autologous mesenchymal stem cells (MSCs) may be a viable option for treatment of several diseases. MSCs efficacy depends on adequate function of their mitochondria, which might be impaired in a noxious milieu. OBJECTIVES: We hypothesized that obesity compromises MSCs mitochondrial structure and function, possibly via micro-RNA (miRNA)-based mechanisms. METHODS: MSCs were collected from swine abdominal adipose tissue after 16 weeks of Lean or Obese diet (n=7 each). Mitochondrial structure was assessed by electron microscopy and function by membrane potential and cytochrome-c oxidase (COX)-IV activity. Oxidative stress was assessed by Mito-SOX and dihydroethidium staining. Next-generation sequencing (RNA-seq) was performed to identify miRNAs expression in MSCs, and predicted mitochondrial target genes were then identified (MitoCarta). RESULTS: Compared to Lean-MSCs, mitochondria from Obese-MSCs were smaller and showed cristae remodeling and loss. Mitochondrial membrane potential and COX-IV activity decreased in Obese-MSCs, associated with increased mitochondrial oxidative stress. RNA-seq generated reads for 413 miRNAs, of which 5 miRNAs were upregulated in Obese-MSCs (fold change >2, p

Autologous transplantation of mesenchymal stem cells (MSCs) is a viable option for the treatment of several diseases. Evidence indicates that MSCs release extracellular vesicles (EVs) and that EVs shuttle miRNAs to damaged parenchymal cells to activate an endogenous repair program. We hypothesize that comorbidities may interfere with the packaging of cargo in MSC-derived EVs. Therefore, we examined whether metabolic syndrome (MetS) modulates the miRNA content packed within MSC-derived EVs. MSCs were collected from swine abdominal adipose tissue after 16 weeks of lean or obese diet (n = 7 each). Next-generation RNA sequencing of miRNAs (miRNA-seq) was performed to identify miRNAs enriched in MSC-derived EVs and their predicted target genes. Functional pathway analysis of the top 50 target genes of the top 4 miRNAs enriched in each group was performed using gene ontology analysis. Lean- and MetS-EVs were enriched in, respectively, 14 and 8 distinct miRNAs. Target genes of miRNAs enriched in MetS-EVs were implicated in the development of MetS and its complications, including diabetes-related pathways, validated transcriptional targets of AP1 family members Fra1 and Fra2, Class A/1 (Rhodopsin-like receptors), and Peptide ligand-binding receptors. In contrast, miRNAs enriched in Lean EVs target primarily EphrinA-EPHA and the Rho family of GTPases. MetS alters the miRNA content of EVs derived from porcine adipose tissue MSCs. These alterations could impair the efficacy and limit the therapeutic use of autologous MSCs in subjects with MetS. Our findings may assist in developing adequate regenerative strategies to preserve the reparative potency of MSCs in individuals with MetS. © 2017 International Society for Advancement of Cytometry.

In this work, we show that different enzymes, such as horseradish peroxidase (HRP), glucose oxidase, laccase, and catalase, can be directly immobilized onto plasmonic gold nanoparticles (NPs) and superparamagnetic iron oxide NPs simply via unspecific physical adsorption, yielding catalytically active and colloidally stable NP systems. The enzyme coating on the NP surface is highly robust and enzymatically active. The colloidal stability and the enzymatic performance (Vmax and Km) of the enzyme-coated NPs (Au@enzyme) are strongly dependent on the pH of the dispersion and physicochemical properties of the respective enzyme. In particular, we observe that the colloidal stability of the enzyme-coated NPs does not necessarily lie in the pH range of the optimal catalytic performance of the enzymes (Au@HRP is unstable at pH 3, but kcat is the highest, 7.4 × 103 min–1). Understanding the relationship between the colloidal stability of the different enzyme-coated NPs at different pH values and the optimal catalyti...

Transcriptional control has been earnestly pursued for the regulation of cellular proliferation associated with cancer progression. The foundational paradigm of targeting transcription factors has yielded exquisite specificity, but many factors cannot yet be targeted. In contrast, targeting epigenetic factors to control chromatin structure and consequential gene expression generally yields more global effects on transcription. Our working paradigm targets neither specific transcription factors nor global epigenetic factors but ATP-dependent chromatin remodeling factors that regulate expression of a limited set of genes. Active DNA-dependent ATPase A Domain inhibitor (ADAADi) synthesized by aminoglycoside phosphotransferases is the first-in-class inhibitor of ATP-dependent chromatin remodeling proteins that targets the ATPase domain of these proteins. Mammalian cells are sensitive to ADAADi but cell lines are variable in their individual responses to the inhibitor. The ADAADi product can be generated from a variety of aminoglycoside substrates with cells showing differential responses to ADAADi depending on the starting aminoglycoside. RNA seq analysis demonstrated that targeting the chromatin remodeling by treatment with a sub-lethal concentration of ADAADi yields alterations to the transcriptional network of the cell. Predominantly, the tumor-promoting genes were repressed while pro-apoptotic and tumor suppressors genes were upregulated on treatment with ADAADi, leading to apoptotic-type cell death. Treatment with ADAADi reversed the EMT process as well as inhibited migration of cells and their colony forming ability. In conjunction with the previous report that treatment with ADAADi regresses tumors in mouse model, this chromatin remodeling inhibitor shows promising anti-tumor properties by targeting the main hallmarks of cancer.

The properties and behaviour of stem cells rely heavily on signaling from the local microenvironment. At the apical end of Drosophila testis, self-renewal and differentiation of germline stem cells (GSCs) are tightly controlled by distinct somatic cells that comprise a specialised stem cell niche known as the hub. The hub maintains GSC homeostasis through adhesion and cell signaling. The Salvador/Warts/Hippo (SWH) pathway, which suppresses the transcriptional co-activator YAP/Yki via a kinase cascade, is a known regulator of stem cell proliferation and differentiation. Here, we show that increasing YAP/Yki expression in the germline, as well as reducing Warts levels, blocks the decrease of GSC numbers observed in aging flies, with only a small increase on their proliferation. An increased expression of YAP/Yki in the germline or a reduction in Warts levels also stymies an age-related reduction in hub cell number, suggesting a bilateral relationship between GSCs and the hub. Conversely, RNAi-based knockdown of YAP/Yki in the germline leads to a significant drop in hub cell number, further suggesting the existence of such a SC-to-niche relationship. All together, our data implicate the SWH pathway in Drosophila GSC maintenance and raise questions about its role in stem cell homeostasis in aging organisms.

CD4 and CD8 T cells are vital components of the immune system. We found that histone deacetylase 3 (HDAC3) is critical for the development of CD4 T cells, as HDAC3-deficient DP thymocytes generate only CD8SP thymocytes in mice. In the absence of HDAC3, MHC Class II-restricted OT-II thymocytes are redirected to the CD8 cytotoxic lineage, which occurs with accelerated kinetics. Analysis of histone acetylation and RNA-seq reveals that HDAC3-deficient DP thymocytes are biased towards the CD8 lineage prior to positive selection. Commitment to the CD4 or CD8 lineage is determined by whether persistent TCR signaling or cytokine signaling predominates, respectively. Despite elevated IL-21R/γc/STAT5 signaling in HDAC3-deficient DP thymocytes, blocking IL-21R does not restore CD4 lineage commitment. Instead, HDAC3 binds directly to CD8-lineage promoting genes. Thus, HDAC3 is required to restrain CD8-lineage genes in DP thymocytes for the generation of CD4 T cells.

Active DNA-dependent ATPase A Domain inhibitor (ADAADi) is the only known inhibitor of ATP-dependent chromatin remodeling proteins that targets the ATPase domain of these proteins. The molecule is synthesized by aminoglycoside phosphotransferase enzyme in the presence of aminoglycosides. ADAADi interacts with ATP-dependent chromatin remodeling proteins through motif Ia present in the conserved helicase domain, and thus, can potentially inhibit all members of this family of proteins. We show that mammalian cells are sensitive to ADAADi but with variable responses in different cell lines. ADAADi can be generated from a wide variety of aminoglycosides; however, cells showed differential response to ADAADi generated from various aminoglycosides. Using HeLa and DU145 cells as model system we have explored the effect of ADAADi on cellular functions. We show that the transcriptional network of a cell type is altered when treated with sub-lethal concentration of ADAADi. Although ADAADi has no known effects on DNA chemical and structural integrity, expression of DNA-damage response genes was altered. The transcripts encoding for the pro-apoptotic proteins were found to be upregulated while the anti-apoptotic genes were found to be downregulated. This was accompanied by increased apoptosis leading us to hypothesize that the ADAADi treatment promotes apoptotic-type of cell death by upregulating the transcription of pro-apoptotic genes. ADAADi also inhibited migration of cells as well as their colony forming ability leading us to conclude that the compound has effective anti-tumor properties.

Introduction We hypothesized that common Alzheimer's disease (AD)-associated variants within the triggering receptor expressed on myeloid ( TREM ) gene cluster influence disease through gene expression. Methods Expression microarrays on temporal cortex and cerebellum from ∼400 neuropathologically diagnosed subjects and two independent RNAseq replication cohorts were used for expression quantitative trait locus analysis. Results A variant within a DNase hypersensitive site 5′ of TREM2 , rs9357347-C, associates with reduced AD risk and increased TREML1 and TREM2 levels (uncorrected P = 6.3 × 10 −3 and 4.6 × 10 −2 , respectively). Meta-analysis on expression quantitative trait locus results from three independent data sets ( n = 1006) confirmed these associations (uncorrected P = 3.4 × 10 −2 and 3.5 × 10 −3 , Bonferroni-corrected P = 6.7 × 10 −2 and 7.1 × 10 −3 , respectively). Discussion Our findings point to rs9357347 as a functional regulatory variant that contributes to a protective effect observed at the TREM locus in the International Genomics of Alzheimer's Project genome-wide association study meta-analysis and suggest concomitant increase in TREML1 and TREM2 brain levels as a potential mechanism for protection from AD.

The pharmacological management of insomnia has lately become a challenge for researchers worldwide. As per the third International Classification of Sleep disorders (ICSD-3) insomnia can be defined as a state with repeated difficulty in sleep initiation, duration, consolidation, or quality that occurs despite adequate opportunity and circumstances for sleep, and results in some form of daytime impairment. The conventional treatments approved for management of insomnia were benzodiazepines (BZDs) (estazolam, quazepam, triazolam, flurazepam and temazepam) and non-BZDs, also known as z-drugs (zaleplon, zolpidem, and eszopiclone), tricyclic antidepressant (TCA) doxepin as well as melatonin agonists, e.g. ramelteon. But the potential of these agents to address sleep problems has been limited due to substantial side effects associated with them like hangover, dependence and tolerance, rebound insomnia, muscular atonia, inhibition of respiratory system, cognitive dysfunctions, and increased anxiety. Recently, orexin neuropeptides have been identified as regulators of transition between wakefulness and sleep and documented to aid an initial transitory effect towards wakefulness by activating cholinergic/monoaminergic neural pathways of the ascending arousal system. This has led to the development of orexin peptides and receptors, as possible therapeutic targets for the treatment of sleep disorders with the advantage of having lesser side effects as compared to conventional treatments. The present review focuses on the orexin peptides and receptors signifying their physiological profile as well as the development of orexin receptor antagonists as novel strategies in sleep medicine.

Sleep deprivation (SD) is an experience of inadequate or poor quality of sleep that may produce significant alterations in multiple neural systems. Centella asiatica (CA) is a psychoactive medicinal herb with immense therapeutic potential. The present study was designed to explore the possible nitric oxide (NO) modulatory mechanism in the neuroprotective effect of CA against SD induced anxiety like behaviour, oxidative damage and neuroinflammation. Male laca mice were sleep deprived for 72 h, and CA (150 and 300 mg/kg) was administered alone and in combination with NO modulators for 8 days, starting five days before 72-h SD exposure. Various behavioural (locomotor activity, elevated plus maze) and biochemical (lipid peroxidation, reduced glutathione, catalase, nitrite levels and superoxide dismutase activity), neuroinflammation marker (TNF-alpha) were assessed subsequently. CA (150 and 300 mg/kg) treatment for 8 days significantly improved locomotor activity, anti-anxiety like effect and attenuated oxidative damage and TNF α level as compared to sleep-deprived 72-h group. Also while the neuroprotective effect of CA was increased by NO antagonists, it was diminished by NO agonists. The present study suggests that NO modulatory mechanism could be involved in the protective effect of CA against SD-induced anxiety-like behaviour, oxidative damage and neuroinflammation in mice. Copyright © 2016 John Wiley & Sons, Ltd.

Introduction: Acquired resistance to platinum remains a major clinical problem in ovarian cancer since systemic therapies have limited efficacy in this setting. There is growing evidence that the tumor microenvironment (TME) contributes to such resistance, as well as the pathogenesis of the disease. The study of the TME is limited by technical challenges of separating the epithelial from the stromal component. To overcome this limitation, we performed RNA sequencing (RNA-Seq) for three pairs of sensitive and induced resistant patient-derived xenograft models since the epithelial transcripts originate from human tissue and the stroma component is murine. Methods: To determine which TME genes might be contributing to chemotherapy resistance, three platinum-sensitive ovarian cancer PDX lines were treated repeatedly with carboplatin/paclitaxel until resistance occurred. Primary platinum-sensitive and induced platinum resistant tumors were characterized histologically by H&E and Masson Trichrome staining. The three pairs of sensitive and resistant PDX models were then subjected to RNA-Seq. Raw sequencing data were processed through Xenome to classify the reads into epithelial carcinoma (human) or TME (mouse) through Mayo Clinic’s internal RNA-Seq pipeline for transcriptome quantification. Differential expression analysis between induced resistant and sensitive models was carried out for both carcinoma and TME components using edgeR paired analysis. Gene set enrichment analysis was used to identify overrepresented biologic functions for differentially expressed genes between the pairs. Top significantly varied, differentially expressed genes between the pairs were validated using species-specific qPCR. Results: Histologic analysis of the matched sensitive/resistant pairs revealed a marked increase of the stromal component in the resistant tumors evident by H&E and Masson trichrome staining. A total number of 95 and 312 genes were differentially expressed in the epithelial and TME component, respectively (FDR1.5). The stemness marker SOX2 was significantly upregulated in the human component of the resistant tumors as confirmed by qPCR. Genes involved in stroma remodeling were upregulated in the “murine” component of the resistant tumors, and upregulation was confirmed by qPCR (normalized to a murine housekeeper gene). Gene set enrichment analysis showed that the upregulated genes in TME of resistant models were enriched for cytoskeleton related functions, while the downregulated genes were enriched for extracellular matrix-related functions. Conclusion: A bioinformatics strategy able to map human and murine reads separately may identify resistance mechanisms attributed to the two tumor subcompartments and could lead to strategies to potentially target each separately or concurrently. Citation Format: Valentina Zanfagnin, Yuanhang Liu, Pritha Chanana, Xiaonan Pritha, Beverly Long, Joe Pathoulas, Brad Evans, Jaime Davila, Chen Wang, Saravut John Weroha. Transcriptional profiling of tumor stroma using ovarian cancer PDX models with induced platinum-resistance [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B18.

Background Neuroinflammation plays an important role in ischemic brain injury and recovery, however the interplay between brain development and the neuroinflammatory response is poorly understood. We previously described age-dependent differences in the microglial response and the effect of microglial inhibition. Here we investigate whether age-dependent microglial responses may be related to pre-injury developmental differences in microglial phenotype. Methods Measures of microglia morphology were quantified using semi-automated software analysis of immunostained sections from postnatal day 2 (P2), P9, P30 and P60 mice using IMARIS. Microglia were isolated from P2, P9, P30 and P60 mice, and expression of markers of classical and alternative microglial activation was assessed, as well as transforming growth factor beta (TGF-β) receptor, Serpine1, Mer Tyrosine Kinase (MerTK), and the suppressor of cytokine signaling (SOCS3). Hypoxia-ischemia (HI) was induced in P9 and P30 mice using unilateral carotid artery ligation and exposure to 10% oxygen for 50 min. Microglia morphology and microglial expression of genes in the TGF-β and MerTK pathways were determined in ipsilateral and contralateral hippocampus. Results A progressive and significant increase in microglia branching morphology was seen in all brain regions from P2 to P30. No consistent classical or alternative activation profile was seen in isolated microglia. A clear transition to increased expression of TGF-β and its downstream effector serpine1 was seen between P9 and P30. A similar increase in expression was seen in MerTK and its downstream effector SOCS3. HI resulted in a significant decrease in branching morphology only in the P9 mice, and expression of TGF-β receptor, Serpine1, MerTK, and SOCS3 were elevated in P30 mice compared to P9 post-HI. Conclusion: Microglia maturation is associated with changes in morphology and gene expression, and microglial responses to ischemia in the developing brain differ based on the age at which injury occurs.

MicroRNAs (miRNAs) are involved in growth, development, and occurrence and progression of many diseases. MiRNA-mediated post-transcriptional regulation is poorly understood in vascular biology and pathology. The purpose of this is to determine circulatory miRNAs as early detectable peripheral biomarkers in patients with ischemic stroke (IS). MiRNAs expression levels were measured in IS serum samples and healthy controls using Illumina deep sequencing analysis and identified differentially expressed miRNAs. Differentially expressed miRNAs were further validated using SYBR-green-based quantitative real-time PCR (qRT-PCR) assay in postmortem IS brains, lymphoblastoid IS cell lines, oxygen and glucose deprivation/reoxygenation -treated human and mouse neuroblastoma cells, and mouse models of hypoxia and ischemia (HI)-induced stroke. A total of 4656 miRNAs were differentially expressed in IS serum samples relative to healthy controls. Out of 4656 miRNAs, 272 were found to be significantly deregulated in IS patients. Interestingly, we found several novel and previously unreported miRNAs in IS patients relative to healthy controls. Further analyses revealed that some candidate miRNAs and its target genes were involved in the regulation of the stroke. To the best of our knowledge, this is the first study identified potential novel candidate miRNAs in IS serum samples from the residents of rural West Texas. MiRNAs identified in this study could potentially be used as a biomarker and the development of novel therapeutic approaches for stroke. Further studies are necessary to better understand miRNAs-regulated stroke cellular changes.

Age is the common risk factor for both neurodegenerative and neuromuscular diseases. Alzheimer disease (AD), a neurodegenerative disorder, causes dementia with age progression while GNE myopathy (GNEM), a neuromuscular disorder, causes muscle degeneration and loss of muscle motor movement with age. Individuals with mutations in presenilin or amyloid precursor protein (APP) gene develop AD while mutations in GNE (UDP N-acetylglucosamine 2 epimerase/N-acetyl Mannosamine kinase), key sialic acid biosynthesis enzyme, cause GNEM. Although GNEM is characterized with degeneration of muscle cells, it is shown to have similar disease hallmarks like aggregation of Aβ and accumulation of phosphorylated tau and other misfolded proteins in muscle cell similar to AD. Similar impairment in cellular functions have been reported in both disorders such as disruption of cytoskeletal network, changes in glycosylation pattern, mitochondrial dysfunction, oxidative stress, upregulation of chaperones, unfolded protein response in ER, autophagic vacuoles, cell death, and apoptosis. Interestingly, AD and GNEM are the two diseases with similar phenotypic condition affecting neuron and muscle, respectively, resulting in entirely different pathology. This review represents a comparative outlook of AD and GNEM that could lead to target common mechanism to find a plausible therapeutic for both the diseases.

Objective: The present work is an attempt to assess the in vitro antimicrobial activity of the leaves of Caesalpinia decapetala (Roth) fabaceae family collected from forest area of Tamilnadu, India. Methods: The crude drug was successively extracted by Soxhlet assembly using Petroleum ether, dichloromethane, ethyl acetate and methanol as solvents. Preliminary phytochemical screening of different extracts was carried out using several colour and precipitative chemical reagents as per described methods. Antimicrobial activity of the extracts was evaluated against fungal strains (Aspergillus fumigatus and Candida albicans), Gram +ve bacteria (Staphylococcus aureus and Streptococcus pyogenes) and Gram –ve bacteria (Escherichia coli and Pseudomonas aeruginosa) using agar wells dilution method. Nutrient agar medium at37 oC and sabouraud dextrose e agar medium at 28 oC were used in antimicrobial activity evaluation and antifungal activity evaluation respectively. Results: Preliminary phytochemical screening of C. decepetala leaves showed the presence of alkaloids, glycosides, phenols, phytosterols, saponins and flavonoids crude drug. C. decapetala leaf extracts exhibited marked dose dependent antibacterial activity in vitro against tested bacteria. Methanolic extract was found to be more potent particularly against Staphylococcus aureus (Gram +ve bacteria) and staphylococcus aeruginosa (Gram -ve bacteria). Conclusion: Various phytochemicals were found to be present in C. decepetala leaves. Methanolic extract of C. decepetala leaves exhibited better antimicrobial activity in vitro and can be used as a good therapeutic approach for infectious disease management and therapy. Further studies on isolation of phyto-constituents and both in vitro and in vivo evaluation of pharmacological activities of isolated bioactive constituents of the crude drug are recommended as future works.

In this work, we investigate the ligand exchange of cetyltrimethylammonium bromide (CTAB) with bovine serum albumin for gold nanorods. We demonstrate by surface-enhanced Raman scattering measurements that CTAB, which is used as a shape-directing agent in the particle synthesis, is completely removed from solution and particle surface. Thus, the protein-coated nanorods are suitable for bioapplications, where cationic surfactants must be avoided. At the same time, the colloidal stability of the system is significantly increased, as evidenced by spectroscopic investigation of the particle longitudinal surface plasmon resonance, which is sensitive to aggregation. Particles are stable at very high concentrations (cAu 20 mg/mL) in biological media such as phosphate buffer saline or Dulbecco’s Modified Eagle’s Medium and over a large pH range (2–12). Particles can even be freeze-dried (lyophilized) and redispersed. The protocol was applied to gold nanoparticles with a large range of aspect ratios and sizes with main absorption frequencies covering the visible and the near-IR spectral range from 600 to 1100 nm. Thus, these colloidally stable and surfactant-free protein-coated nanoparticles are of great interest for various plasmonic and biomedical applications., ACS Applied Materials & Interfaces, 7 (10), ISSN:1944-8244, ISSN:1944-8252

BACE1, a key protein involved in Alzheimer's progression, initiates Aβ42 generation that induce senile plaques in brain. However, the role of chaperone synergy or antagonism on BACE1-mediated amyloid processing is unknown. We have discovered that BACE1 as well as Aβ42 are antagonistically controlled by ER chaperone ORP150 and cellular chaperone CHIP. We have shown ORP150 as a chaperone interacts with and stabilizes BACE1 at post-translational level. Furthermore, ORP150 enhances BACE1-mediated amyloid processing thus masking CHIP-mediated BACE1 degradation. Conversely, siORP150 reversed the chaperone function of ORP150 resulting in BACE1 degradation. ORP150 and CHIP demonstrate antagonism under normal and stress conditions wherein they inversely regulate each other thus affecting BACE1 level. In conclusion, we have uncovered for the first time a phenomenon of chaperone antagonism on BACE1-mediated Aβ42 generation. Future strategy would require both suppression of ORP150 as well as activation of E3-ligase activity of CHIP that might prevent Aβ42 in Alzheimer's disease.

GNE myopathy is a rare neuromuscular genetic disorder characterized by early adult onset and muscle weakness due to mutation in sialic acid biosynthetic enzyme, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). More than 180 different GNE mutations are known all over the world with unclear pathomechanism. Although hyposialylation of glycoproteins is speculated to be the major cause, but cellular mechanism leading to loss of muscle mass has not yet been deciphered. Besides sialic acid biosynthesis, GNE affects other cellular functions such as cell adhesion and apoptosis. In order to understand the effect of mutant GNE protein on cellular functions, differential proteome profile of HEK293 cells overexpressing pathologically relevant recombinant mutant GNE protein (D207V and V603L) was analyzed. These cells, along with vector control and wild-type GNE-overexpressing cells, were subjected to two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF MS/MS). In the study, 10 differentially expressed proteins were identified. Progenesis same spots software revealed downregulation of peroxiredoxin IV (PrdxIV), an ER-resident H2O2 sensor that regulates neurogenesis. Significant reduction in mRNA and protein levels of PrdxIV was observed in GNE mutant cell lines compared with vector control. However, neither total reactive oxygen species was altered nor H2O2 accumulation was observed in GNE mutant cell lines. Interestingly, ER redox state was significantly affected due to reduced normal GNE enzyme activity. Our study indicates that downregulation of PrdxIV affects ER redox state that may contribute to misfolding and aggregation of proteins in GNE myopathy.

Rediscovery of known natural products hinders the discovery of new, unique scaffolds. Efforts have mostly focused on streamlining the determination of what compounds are known vs. unknown (dereplication), but an alternative strategy is to focus on what is different. Utilizing statistics and assuming that common actinobacterial metabolites are likely known, focus can be shifted away from dereplication and towards discovery. LC-MS-based principal component analysis (PCA) provides a perfect tool to distinguish unique vs. common metabolites, but the variability inherent within natural products leads to datasets that do not fit ideal standards. To simplify the analysis of PCA models, we developed a script that identifies only those masses or molecules that are unique to each strain within a group, thereby greatly reducing the number of data points to be inspected manually. Since the script is written in R, it facilitates integration with other metabolomics workflows and supports automated mass matching to databases such as Antibase.

Bacterial communities associated with marine invertebrates such as sponges and ascidians have demonstrated potential as sources of bio-medically relevant small molecules. Metagenomic analysis has shown that many of these invertebrates harbor populations of Actinobacteria, many of which are cultivable. While some populations within invertebrates are transmitted vertically, others are obtained from the environment. We hypothesized that cultivable diversity from sponges living in brackish mangrove habitats have associations with Actinobacterial populations that differ from those found in clear tropical waters. In this study, we analyzed the cultivable Actinobacterial populations from sponges found in these two distinct habitats with the aim of understanding the secondary metabolite potential. Importantly, we wanted to broadly evaluate the potential differences among these groups to guide future Actinobacterial collection strategies for the purposes of drug discovery.

Stress is often marked by a state of hyperarousal to aid the initiation of necessary stress response for the successful management of stressful stimuli. It can be manifested as a challenge (stimulus) that requires behavioral, psychological, and physiological adaptations for the maintenance of a state of homeostasis in response to stressful stimuli. In an organism, miscellaneous stressors trigger a wide spectrum of alterations in hormonal and neuronal physiologies, resulting in behavioral (anxiety and depression disorders, diminished food intake and gastrointestinal dysfunctions, decline in sexual behavior, diabetes, and loss of cognitive function) and other physiological responses. Stress serves as a potent etiological link to development of several neuropsychiatric diseases such as depression, anxiety, and cognitive impairments. Exposure to stressful stimuli has been found to be associated with activation of nitric oxide synthase and generation of NO which reacts with spontaneous oxygen species to aid formation of active nitrogen radicals. High concentrations of reactive nitrogen radicals may cause damage to intracellular proteins, in addition to causing impairment to components of the mitochondrial transport chain, leading to cellular energy deficiency. This may further serve as an etiological link to the development of secondary neurological diseases associated with chronic stress. Also, during stress exposure, pharmacological inhibition of nitric oxide production displays reduction in indicators of anxiety- and depressive-like behavior in animal models. Therefore, the purpose of this chapter is to present an overview on the role of NO in stress-evoked emergence of secondary neurological disorders like anxiety as well as citing examples where NO has been used as a therapeutic target for the management of stress-induced anxiety-like behavior.

Introduction: Sleep deprivation [SD] often raises several neurological problems by causing significant alterations at multiple neural systems. Centella asiatica is a psychoactive medicinal herb with immense therapeutic potential. Objective: The present study has been designed with aim to target mitoprotective and anti-stress pathways involved in the protective effect of Centella asiatica in addition to nitric oxide mechanism. Methods: Male laca mice were sleep deprived for 72 hours using grid suspended over water method and Centella asiatica (150 and 300mg/kg) was administered alone and in combination with NO modulators for 8 days, starting five days before 72 hours sleep deprivation. Various behavioural (pentobarbitone induced sleep time i.e. sleep latency as well as total sleep time and assessment of anxiety like behaviour), mitochondrial enzyme complex activities, serum corticosterone levels as well as pyknotic cell density in thalamo-cortical region were assessed subsequently. Results: Centella asiatica (150 and 300mg/kg) treatment for 8 days significantly improved locomotor activity, anti-anxiety like effect, reversed mitochondrial enzyme complex activities, attenuated corticosterone level as well as improved neuroinflammatory and apoptotic responses (pyknotic cell density) as compared to 72 hours sleep deprived animals. Upon treatment with Centella asiatica, the parameters of pentobarbitone induced hypnosis though modified but were not found to be significant in comparison to the sleep deprived animals. Further, L-arginine (100 mg/kg) pre-treatment significantly reversed protective effect of Centella asiatica (150 mg/ kg) in 72 hour sleep deprived animals. However, L-NAME (10 mg/ kg) pretreatment with Centella asiatica (150 mg/kg) significantly potentiated their protective effect which was significant as compared to their effect per se. Conclusion: The present study suggests mitoprotective; antistress pathways are also involved in the protective effect of Centella asiatica via nitric oxide modulatory mechanisms against sleep deprivation induced anxiety-like behaviour in mice.

A 25-year-old woman was diagnosed with anaemic retinopathy following Plasmodium falciparum malaria. The serial clinical and spectral domain optical coherence tomography (SD-OCT) changes are described, including sub-internal limiting membrane (sub-ILM) haemorrhage at the macula with the formation of a corresponding sub-ILM cavity evident on SD-OCT. The patient was managed conservatively and showed complete recovery of fundus and SD-OCT changes after one year. To the best of our knowledge, this is the first documentation of formation of a temporary sub-ILM cavity following anaemic retinopathy.

BACKGROUND: Metastatic ovarian cancer remains an urgent clinical problem. The homing and invasion of cancer cells into the omental adipose tissue, which is the preferred site of ovarian cancer metastasis, is a critical step in disease progression. Omental adipose tissue contains adipocytes, blood vessels, and clusters of leukocytes known as milky spots. We have previously demonstrated in several mouse models that intraperitoneally injected ovarian cancer cells localize to the omentum and specifically to the milky spots. However, we found that B, T, or NK cells were not required for omental colonization. We hypothesize that macrophages within the milky spots play a crucial role in promoting ovarian cancer progression. METHODS AND RESULTS: Depleting macrophages disrupts cancer cells' localization to the omentum. This result supports our hypothesis that macrophages play an essential role in attracting ovarian cancer cells to the omental milky spots. Correspondingly, factors secreted by omental macrophages promote ovarian cancer migration in vitro, in contrast to factors secreted by macrophages isolated from other peritoneal adipose tissues. In order to understand the specific mechanism(s) that regulate metastatic colonization of the omentum, we performed RNA-Seq of CD45+CD11b+F4/80+ flow-sorted macrophages from the omentum and the mesenteric fat of naïve and cancer-bearing mice. We found that omental macrophages have a distinct and dynamic gene expression pattern even when compared to a very similar cell type. The chemokine CCL6 is one of the most highly upregulated genes during metastatic colonization. The human homolog of CCL6 is CCL23 and is also expressed by human omental macrophages. Genomic inactivation of CCR1, the gene encoding receptor for CCL6 in mice and CCL23 in humans, in mouse ovarian cancer cells abolishes their ability to home to omental milky spots. Our findings establish a role for chemokine signaling in the early colonization of the omentum by ovarian cancer. CONCLUSION: We demonstrate that omental milky spots are the preferential sites for cancer colonization of peritoneal adipose. Further, we have identified omental macrophages as the critical mediators of colonization via the CCL6/CCR1 chemokine axis. Future studies are focused on understanding the cancer cell-macrophage interactions that can be targeted therapeutically to disrupt metastatic growth and extend disease-free survival. Citation Format: Venkatesh Krishnan, Supreeti Tallapragada, Bruce Schaar, Anita Chanana, Carrie Rinker-Schaeffer, Oliver Dorigo. OMENTAL MACROPHAGES REGULATE OVARIAN CANCER METASTATIC COLONIZATION THROUGH THE CCL6-CCR1 SIGNALING AXIS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-076.

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

BackgroundRNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5-35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data.MethodsWe describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization.ResultsWe demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance.ConclusionsThe approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis.

In this study, we investigated the effects of a bioactive high-affinity TrkB receptor agonist 7,8- dihydroxyflavone (7,8 DHF) on neonatal brain injury in female and male mice after hypoxia ischemia (HI). HI was induced by exposure of postnatal day 9 (P9) mice to 10% O2 for 50 minutes at 37°C after unilateral ligation of the left common carotid artery. Animals were randomly assigned to HI-vehicle control group [phosphate buffered saline (PBS), intraperitoneally (i.p.)] or HI + 7,8 DHF-treated groups (5 mg/kg in PBS, i.p at 10 min, 24 h, or with subsequent daily injections up to 7 days after HI). The HI-vehicle control mice exhibited neuronal degeneration in the ipsilateral hippocampus and cortex with increased Fluoro-Jade C positive staining and loss of microtubule associated protein 2 expression. In contrast, the 7,8 DHF-treated mice showed less hippocampal neurodegeneration and astrogliosis, with more profound effects in female than in male mice. Moreover, 7,8 DHF-treated mice improved motor learning and spatial learning at P30-60 compared to the HI-vehicle control mice. Diffusion tensor imaging of ex vivo brain tissues at P90 after HI revealed less reduction of fractional anisotropy values in the ipsilateral corpus callosum of 7,8 DHF-treated brains, which was accompanied with better preserved myelin basic protein expression and CA1 hippocampal structure. Taken together, these findings strongly suggest that TrkB agonist 7,8 DHF is protective against HI-mediated hippocampal neuronal death, white matter injury, and improves neurological function, with a more profound response in female than in male mice.

Nonalcoholic fatty liver disease (NAFLD) is an increasing problem worldwide and is associated with negative outcomes such as cirrhosis, hepatocellular carcinoma, insulin resistance, diabetes, and cardiovascular events. Current evidence shows that the immune response has an important participation driving the initiation, maintenance, and progression of the disease. So, various immune imbalances, from cellular to cytokines levels, have been studied, either for better compression of the disease pathophysiology or as biomarkers for severity assessment and outcome prediction. In this article, we performed a thorough review of studies that evaluated the role of inflammatory/immune imbalances in the NAFLD. At the cellular level, we gave special focus on the imbalance between neutrophils and lymphocytes counts (the neutrophil to lymphocyte ratio [NLR]), and that which occurs between T helper 17 (Th17) and regulatory T cells (Treg) as emerging biomarkers. By extension, we reviewed the reflection of these imbalances at the molecular level through proinflammatory cytokines including those involved in Th17 differentiation (IL-6, IL-21, IL-23, and transforming growth factor-beta [TGF-β]), and those released by Th17 cells (IL-17A, IL-17F, IL-21, and IL-22). We gave particular attention to the role of IL-17, either produced by Th17 cells or neutrophils, in fibrogenesis and steatohepatitis. Finally, we reviewed the potential of this pathways as new therapeutics targets in NAFLD.

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

In vitro shoot regeneration was carried out for Kinnow mandarin ( Citrus reticulata Blanco) through shoot tip explants obtained from in vitro germinated seedling. The medium supplemented with 2.5 mg/L benzylaminopurine (BAP) supported maximum shoot proliferation (2.45 shoots/explant). This rate was further enhanced (7.23 shoots/explant) by adding 25 mg/L glutamine, 50 mg/L adenine sulphate and 100 mg/L casein hydrolysate. From the in vitro proliferated shoots, the shoot apices and nodes were excised and sub-cultured on four different medium namely: SCM-1, SCM-2, SCM-3 and SCM-4 for six consecutive months. Problems like defoliation, chlorosis and loss of vigour in shoots were observed at different stages of subculture. These problems were successfully controlled by SCM-4 having 1.5 mg/L of BAP, 0.5% charcoal, 25 mg/L glutamine, 50 mg/L adenine sulphate and 100 mg/L casein hydrolysate. The SCM-4 also maintained the high proliferation rate throughout the subculturing process. Rooting was best induced on 2 mg/L indole-butyric acid (IBA) in combination with 0.1 mg/L indole-3-acetic acid (IAA). Rooting was significantly enhanced by 100 mg/L casein hydrolysate. The in vitro raised plantlets were acclimatized under culture room conditions in different potting mixture, of which the combination of garden soil, sand and vermiculite mixture in 1:1:2 ratio was found most supportive. After 30 days of acclimatization, plantlets were transferred to soil, where established plants showed more than 90% survival. Key words: In vitro shoot regeneration, micro-shoots, enhanced shoot multiplication, subculture, acclimatization.

Our recent study reveals that Na⁺/H⁺ exchanger isoform 1 (NHE-1) mediates H⁺ extrusion during "respiratory bursting", which is important for microglial activation. In the present study, we further investigated whether NHE-1 plays a role in proinflammatory activation of microglia in vivo using a mouse model of transient focal cerebral ischemia and reperfusion (I/R). Activated microglial cells were identified by their expression of two microglial marker proteins (CD11b and Iba1) as well as by their transformation from a "ramified" to an "amoeboid" morphology. An immediate increase in activated microglial numbers was detected in the ipsilateral ischemic core area of NHE-1⁺/⁺ brains at 1 hour (h) I/1 h R, which gradually decreased during 6-24 h I/R. This was followed by a sharp rise in microglial activation in the peri-infarct area and an increase in proinflammatory cytokine formation at 3 day after I/R. Interestingly, HOE 642 (a potent NHE-1 inhibitor) -treated or NHE-1 heterozygous (NHE-1⁺/⁻) mice exhibited less microglia activation, less NADPH oxidase activation, or a reduced proinflammatory response at 3-7 day after I/R. Blocking NHE-1 activity also significantly decreased microglial phagocytosis in vitro. In contrast, astrogliosis formation in the peri-infarct area was not affected by NHE-1 inhibition. Taken together, our results demonstrate that NHE-1 protein was abundantly expressed in activated microglia and astrocytes. NHE-1 inhibition reduced microglial proinflammatory activation following ischemia.

Neuronal dendrites are vulnerable to injury under diverse pathological conditions. However, the underlying mechanisms for dendritic Na(+) overload and the selective dendritic injury remain poorly understood. Our current study demonstrates that activation of NHE-1 (Na(+)/H(+) exchanger isoform 1) in dendrites presents a major pathway for Na(+) overload. Neuronal dendrites exhibited higher pH(i) regulation rates than soma as a result of a larger surface area/volume ratio. Following a 2-h oxygen glucose deprivation and a 1-h reoxygenation, NHE-1 activity was increased by ∼70-200% in dendrites. This elevation depended on activation of p90 ribosomal S6 kinase. Moreover, stimulation of NHE-1 caused dendritic Na(+)(i) accumulation, swelling, and a concurrent loss of Ca(2+)(i) homeostasis. The Ca(2+)(i) overload in dendrites preceded the changes in soma. Inhibition of NHE-1 or the reverse mode of Na(+)/Ca(2+) exchange prevented these changes. Mitochondrial membrane potential in dendrites depolarized 40 min earlier than soma following oxygen glucose deprivation/reoxygenation. Blocking NHE-1 activity not only attenuated loss of dendritic mitochondrial membrane potential and mitochondrial Ca(2+) homeostasis but also preserved dendritic membrane integrity. Taken together, our study demonstrates that NHE-1-mediated Na(+) entry and subsequent Na(+)/Ca(2+) exchange activation contribute to the selective dendritic vulnerability to in vitro ischemia.

Invasive Salmonella has been reported to induce apoptosis of macrophages as part of its infection process, which may allow it to avoid detection by the innate immune system. However, the induction of apoptosis under the different host environments remains to be examined, including the oxidative stress experienced by pathogens in the macrophage milieu. To simulate in vivo oxidative conditions, Salmonella enterica serovar Typhi was grown in the presence of hydrogen peroxide and its ability to induce apoptosis of murine macrophages was assessed. Analysis of data revealed that oxidative stressed S. Typhi caused apoptotic cell death in 51% of macrophages, whereas S. Typhi grown under normal conditions accounted for apoptotic cell death in only 32% of macrophages. A significant increase in the levels of oxidants and decrease in the antioxidant was also observed which correlated with the increased generation of tumour necrosis factor alpha, interleukin-1alpha and interleukin-6. These results suggest that tumour necrosis factor alpha in conjunction with other cytokines may induce apoptotic cell death through the up-regulation of lipid peroxidation and down-regulation of superoxide dismutase. This finding may help us to understand better the host-pathogen interactions and may be of clinical importance in the development of preventive intervention against infection.

Summary The present study is aimed at investigating the effect of curcumin (CMN) in salvaging endotoxin-induced hepatic dysfunction and oxidative stress in the liver of rodents. Hepatotoxicity was induced by administering lipopolysaccharide (LPS) in a single dose of 1 mg/kg intraperitoneally to the animals, which were being treated with CMN daily for 7 days. Liver enzymes serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST) and alkaline phosphatase (ALP), total bilirubin and total protein were estimated in serum. Oxidative stress in liver tissue homogenates was estimated by measuring thiobarbituric acid reactive substances (TBARS), glutathione (GSH) content and superoxide dismutase (SOD) activity. Serum and tissue nitrite was estimated using Greiss reagent and served as an indicator of NO production. A separate set of experiments was performed to estimate the effect of CMN on cytokine levels in mouse serum after LPS challenge. LPS induced a marked hepatic dysfunction evident by rise in serum levels of ALT, AST, ALP and total bilirubin (P

Pathogenic microorganisms are known to sense and process signals within their hosts, including those resulting from starvation. Therefore, an attempt was made to evaluate the extent and the possible underlying mechanism of Salmonella typhimurium-induced hepatic damage using pre-starved laboratory mice. The following parameters were analysed, comparing control, fed infected, starved, and starved infected mice: the bacterial load in the liver, fluctuations in liver-derived enzymes alanine-aminotransferase and aspartate-aminotransferase, histopathological changes, lipid peroxidation as well as estimation of reduced glutathione, superoxide dismutase and catalase, along with the TNF content in livers. The number of bacterial cells recovered from starved infected livers at 3 days post-S. typhimurium inoculation was comparable to the number recovered from fed infected livers at 5 days post-Salmonella inoculation, indicating an early increase in the development of the bacteria in starved mice. A marked elevation in liver-derived enzymes in mouse serum and significant histopathological changes are markers of liver damage of higher amplitude in starved infected mice. Analysis of the liver indicated a significant increase in lipid peroxidation in starved infected mice compared to their control counterparts, a process coupled with increased TNF level. Although the reduced glutathione levels showed a marked increase in the starved infected mice, there was a significant decrease in superoxide dismutase and catalase activities in this group.