Elucidation of genetic determinants of dyslipidaemia using a global screening array for the early detection of coronary artery disease.

Dyslipidemia is a major risk factor for the development of coronary artery disease (CAD). Understanding the genetic determinants of dyslipidemia can provide valuable information on the pathogenesis of CAD and aid in the development of early detection strategies. In this study, we used a Global Screening Array (GSA) to elucidate the genetic factors associated with dyslipidemia and their potential role in the prediction of CAD. We conducted a GSA-based association study in 265 subjects to identify the genetic loci associated with dyslipidemia traits using Multiple Linear Regression (MLR) and Logistic Regression (LR), Classification and Regression Tree (CART), and Manhattan plots. We identified an association between dyslipidemia and variants identified in genes such as JCAD, GLIS3, CD38, FN1, CELSR2, MTNR1B, GIPR, DYM, APOB, APOE, ADCY5. The MLR models explained 62%, 71%, and 81% of the variability in HDL, LDL, and triglycerides, respectively. The Area Under the Curve (AUC) values in the LR models of HDL, LDL, and triglycerides were 1.00, 0.94, and 0.95, respectively. CART models identified novel gene-gene interactions influencing the risk for dyslipidemia. To conclude, we have identified the association of 12 SNVs with dyslipidemia and demonstrated their clinical utility in four different models such as MLR, LR, CART, and Manhattan plots. The identified genetic variants and associated pathways shed light on the underlying biology of dyslipidemia and offer potential avenues for precision medicine strategies in the management of CAD.

STAT3 imparts BRCAness by impairing homologous recombination repair in Epstein-Barr virus-transformed B lymphocytes.

Epstein-Barr virus (EBV) causes lymphomas and epithelial cell cancers. Though generally silent in B lymphocytes, this widely prevalent virus can cause endemic Burkitt lymphoma and post-transplant lymphoproliferative disorders/lymphomas in immunocompromised hosts. By learning how EBV breaches barriers to cell proliferation, we hope to undermine those strategies to treat EBV lymphomas and potentially other cancers. We had previously found that EBV, through activation of cellular STAT3 prevents phosphorylation of Chk1, and thereby, suppresses activation of the intra-S phase cell-cycle checkpoint, a potent barrier to oncogene-driven proliferation. This observation prompted us to examine the consequences on DNA repair since homologous recombination repair, the most error-free form, requires phosphoChk1. We now report that the defect in Chk1 phosphorylation also curtails RAD51 nucleation, and thereby, homologous recombination repair of DNA double strand breaks. The resulting reliance on error-prone microhomology-mediated end-joining (MMEJ) repair makes EBV-transformed cells susceptible to PARP inhibition and simultaneous accrual of genome-wide deletions and insertions resulting from synthesis-dependent MMEJ. Analysis of transcriptomic and drug susceptibility data from hundreds of cancer lines reveals a STAT3-dependent gene-set predictive of susceptibility of cancers to synthetic lethal PARP inhibition. These findings i) demonstrate how the tumor virus EBV re-shapes cellular DNA repair, ii) provide the first genome-wide evidence for insertions resulting from MMEJ in human cells, and iii) expand the range of cancers (EBV-related and -unrelated) that are likely to respond to synthetic lethal inhibitors given the high prevalence of cancers with constitutively active STAT3.

Nascent Transcriptomics Reveal Cellular Prolytic Factors Upregulated Upstream of the Latent-to-Lytic Switch Protein of Epstein-Barr Virus.

Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle.IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular "prolytic" state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.

Understanding perspectives of signalling mechanisms regulating PEBP1 function.

UNLABELLED: Phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein, belongs to PEBP family of proteins. It is known to interact with many proteins that are mainly involved in pathways that monitor cell proliferation and differentiation. PEBP1 in many cells interacts with several pathways, namely MAPK, GRK2, NF-кB, etc. that keeps the cell proliferation and differentiation in check. This protein is expressed by many cells in humans, including neurons where it is predominantly involved in production of choline acetyltransferase. Deregulated PEBP1 is known to cause cancer, diabetic nephropathy and neurodegenerative diseases like Alzheimer's and dementia. Recent research led to the discovery of many drugs that mainly target the interaction of PEBP1 with its partners. These compounds are known to bind PEBP1 in its conserved domain which abrogate its association with interacting partners in several different pathways. We outline here the latest developments in understanding of PEBP1 function in maintaining cell integrity. Copyright © 2016 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDY: Phosphatidylethanolamine-binding protein is crucial in regulation of MAPK and PKC pathways. Its diverse roles, including regulating these pathways keep cell differentiation and proliferation in check. This review outlines some latest findings which greatly add to our current knowledge of phosphatidylethanolamine-binding protein.

Characterization of high-yield Bacillus subtilis cysteine protease for diverse industrial applications.

Bacterial proteases have extensive applications in various fields of industrial microbiology. In this study, protease-producing organisms were screened on skimmed milk agar media using serial dilution. Through microbial biomass production, biochemical tests, protease-specific activity, and 16 s RNA gene sequencing, the isolates were identified as Bacillus subtilis and submitted to NCBI. The strain accession numbers were designated as A1 (MT903972), A2 (MT903996), A4 (MT904091), and A5 (MT904796). The strain A4 Bacillus subtilis showed highest protease-specific activity as 76,153.84 U/mg. A4 Bacillus subtilis was unaffected by Ca2+, Cu2+, Fe2+, Hg2+, Mg2+, Na, Fe2+, and Zn2+ but was inhibited by 80% by Mn2+ (5 mM). The protease activity was inhibited by up to 30% by iodoacetamide (5 mM). These findings confirm the enzyme to be a cysteine protease which was further confirmed by MALDI-TOF. The identified protease showed 71% sequence similarity with Bacillus subtilis cysteine protease. The crude cysteine protease significantly aided in fabric stain removal when added to a generic detergent. It also aided in the recovery of silver from used X-ray films and de-hairing of goat skin hides and showed decent application in meat tenderization. Thus, the isolated cysteine protease has high potential for industrial applications.

Methanol extraction revealed anticancer compounds Quinic Acid, 2(5H)­Furanone and Phytol in Andrographis paniculata.

Andrographis paniculata (Ap) has been a part of traditional medicine for its anti-inflammatory effects, treatment of snake bites and liver abnormalities. Several investigations have revealed its bioactive components to be andrographolides. The methanolic extracts of leaves from Ap were characterized, the non-andrographolides were identified and screened for anti-proliferative activity. The extracts showed significant toxicity against numerous cancer cells including HeLa, MCF7, BT549, 293 and A549 cells. Anti-proliferative activity and effect on cancer cell invasion (metastatic potential) and cell migration were examined. The extracts revealed significant inhibition of the ability of HeLa cells in repairing the gap created by a vertical wound made on a confluent cell monolayer. Similarly, a 40% reduction in cell migration was observed in presence of the extracts. Significant anti-angiogenic activity in terms of reduced blood capillary formation was observed on the Chorioallantoic membranes of embryonated hen eggs co-inoculated with HeLa cells and the extracts. Analysis of HeLa cells treated with the extracts using flow cytometry indicated the arrest of cell cycle progression at the G2/M phase. Variation in the Bax/Bcl-2 ratio and elevated caspase-3 levels by immunoblotting confirmed cell death induction via the apoptotic pathway. Investigation of the extracts by gas chromatography-mass spectrometry displayed the predominant components to be 2(5H)-Furanone (14.73%), Quinic acid (17.32%), and Phytol (11.43%). These components have been previously known to have anticancer activity, while being studied individually in other plants. This is the first study, to the best of our knowledge, on the anti-proliferative and anti-angiogenic activity of the non-andrographolide components from Ap.

Melanin pigment of Streptomyces puniceus RHPR9 exhibits antibacterial, antioxidant and anticancer activities.

The present study reveals the production of dark, extracellular melanin pigment (386 mg/L) on peptone yeast extract iron agar medium by Streptomyces puniceus RHPR9 using the gravimetric method. UV-Visible, Fourier Transform Infrared (FTIR), and Nuclear Magnetic Resonance (1H) (NMR) spectroscopy confirmed the presence of melanin. Extracted melanin showed antibacterial activity against human pathogens such as Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli except for Klebsiella pneumoniae. A potent free radical scavenging activity was observed at 100 µg/mL of melanin by the DPPH method with a concentration of 89.01±0.05% compared with ascorbic acid 96.16±0.01%. Antitumor activity of melanin was evaluated by MTT assay against HEK 293, HeLa, and SK-MEL-28 cell lines with IC50 values of 64.11±0.00, 14.43±0.02, and 13.31±0.01 µg/mL respectively. Melanin showed maximum anti-inflammatory activity with human red blood cells (hRBC) (78.63 ± 0.01%) and minimum hemolysis of 21.37±0.2%. The wound healing potential of the pigment was confirmed on HeLa cells, cell migration was calculated, and it was observed that cell migration efficiency decreased with an increase in the concentration of melanin. To our knowledge, this is the first evidence of melanin produced from S. puniceus RHPR9 that exhibited profound scavenging, anti-inflammatory and cytotoxic activities.

The danger molecule HMGB1 cooperates with the NLRP3 inflammasome to sustain expression of the EBV lytic switch protein in Burkitt lymphoma cells.

High mobility group box 1 (HMGB1) is an important chromatin protein and a pro-inflammatory molecule. Though shown to enhance target DNA binding by the Epstein-Barr virus (EBV) lytic switch protein ZEBRA, whether HMGB1 actually contributes to gammaherpesvirus biology is not known. In investigating the contribution of HMGB1 to the lytic phase of EBV, important for development of EBV-mediated diseases, we find that compared to latently-infected cells, lytic phase Burkitt lymphoma-derived cells and peripheral blood lytic cells during primary EBV infection express high levels of HMGB1. Our experiments place HMGB1 upstream of ZEBRA and reveal that HMGB1, through the NLRP3 inflammasome, sustains the expression of ZEBRA. These findings indicate that in addition to the NLRP3 inflammasome's recently discovered role in turning the EBV lytic switch on, NLRP3 cooperates with the danger molecule HMGB1 to also maintain ZEBRA expression, thereby sustaining the lytic signal.

ß-N-oxalyl-L-α,ß-diaminopropionic acid regulates mitogen-activated protein kinase signaling by down-regulation of phosphatidylethanolamine-binding protein 1.

ß-N-Oxalyl-L-α,ß-diaminopropionic acid (l-ODAP) an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor agonist activates protein kinase C in white leghorn chick brain. The current study focuses on the protein kinase C downstream signaling targets associated with L-ODAP excitotoxicity in SK-N-MC human neuroblastoma cells and white leghorn male chick (Gallus domesticus) brain extracts. L-ODAP treatment in SK-N-MC cells (1.5 mM) and chicks (0.5 mg/g body weight) results in a decreased expression and increased phosphorylation of phosphatidylehthanolamine-binding protein 1 (PEBP1) up to 4 h which however, returns to normal by 8 h. D-ODAP, the non-toxic enantiomer however, did not affect PEBP1 levels in either chick brain or SK-N-MC cells. Decreased PEBP1 expression correlated with subsequent activation of Raf-1, MEK and ERK signaling components of the mitogen-activated protein kinase cascade and nuclear translocation of hypoxia inducible factor-1α (HIF-1α) in chick brain nuclear extracts and SK-N-MC cells. SK-N-MC cells over-expressing PEBP1 inhibited nuclear translocation of HIF-1α when treated with l-ODAP, indicating that down-regulation of PEBP1 is responsible for HIF-1α stabilization and nuclear localization. Excitotoxicity of L-ODAP may thus be the result of phosphorylation and down-regulation of PEBP1, a crucial signaling protein regulating diverse signaling cascades. L-ODAP induced convulsions and seizures in chicks could be the result of a hypoxic insult to brain.

Selenium Nanoparticles Synthesized Using Pseudomonas stutzeri (MH191156) Show Antiproliferative and Anti-angiogenic Activity Against Cervical Cancer Cells.

PURPOSE: Selenium nanoparticles (SeNP) have several applications in the field of biotechnology, including their use as anti-cancer drugs. The purpose of the present study is to analyze the efficacy of green synthesis on the preparation of SeNP and its effect on their anti-cancer properties. METHODS: A bacterial strain isolated from a freshwater source was shown to efficiently synthesize SeNP with potential therapeutic properties. The quality and stability of the NP were studied by scanning electron microscopy, X-ray diffraction, zeta-potential and FTIR analysis. A cost-effective medium formulation from biowaste having 6% banana peel extract enriched with 0.25 mM tryptophan was used to synthesize the NP. The NP after optimization was used to analyze their anti-tumor and anti-angiogenic activity. For this purpose, first, the cytotoxicity of the NP against cancer cells was analyzed by MTT assay and then chorioallantoic membrane assay was performed to assess anti-angiogenic activity. Further, cell migration assay and clonogenic inhibition assay were performed to test the anti-tumor properties of SeNP. To assess the cytotoxicity of SeNP on healthy RBC, hemolysis assay was performed. RESULTS: The strain identified as Pseudomonas stutzeri (MH191156) produced phenazine carboxylic acid, which aids the conversion of Se oxyanions to reduced NP state, resulting in particles in the size range of 75 nm to 200 nm with improved stability and quality of SeNP, as observed by zeta (ξ) potential of the particles which was found to be -46.2 mV. Cytotoxicity of the SeNP was observed even at low concentrations such as 5 µg/mL against cervical cancer cell line, ie, HeLa cells. Further, neovascularization was inhibited by upto 30 % in CAMs of eggs coinoculated with SeNp when compared with untreated controls, indicating significant anti-angiogenic activity of SeNP. The NP also inhibited the invasiveness of HeLa cells as observed by decreased cell migration and clonogenic proliferation. These observations indicate significant anti-tumor and anti-angiogenic activity of the SeNP in cervical cancer cells. CONCLUSION: P. stutzeri (MH191156) is an efficient source of Se NP production with potential anti-angiogenic and anti-tumor properties, particularly against cervical cancer cells.

STAT3 activates the anti-apoptotic form of caspase 9 in oncovirus-infected B lymphocytes.

The cancer-causing Epstein-Barr virus (EBV) activates the transcription factor STAT3 upon infecting B-lymphocytes. STAT3 then activates caspase 7 to degrade cellular claspin, resulting in impaired Chk1 phosphorylation. This blockade of ATR-Chk1 signaling allows EBV-transformed cells to proliferate despite DNA lesions from virus-induced replication stress. In addressing the mechanism of caspase 7 activation, we now report that in newly-infected B-cells, STAT3 transcriptionally activates the initiator caspase, caspase 9. Caspase 9 then activates caspase 7 to impair phosphorylation of Chk1 at S345. Importantly, although cleaved products of caspase 9 are detectable in infected cells, there is simultaneous increase in the alternatively-spliced dominant-negative form of caspase 9 - and - expression of dominant-negative caspase 9 is abrogated when STAT3 activation is impaired. Thus EBV, via STAT3, activates caspase 9 but also shifts the balance of transcripts towards its dominant-negative form to allow activation of caspase 7 while avoiding death of EBV-infected cells.

Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity-independent pyroptosis, apoptosis, necroptosis, and inflammatory disease.

RIPK1 kinase activity has been shown to be essential to driving pyroptosis, apoptosis, and necroptosis. However, here we show a kinase activity-independent role for RIPK1 in these processes using a model of TLR priming in a TAK1-deficient setting to mimic pathogen-induced priming and inhibition. TLR priming of TAK1-deficient macrophages triggered inflammasome activation, including the activation of caspase-8 and gasdermin D, and the recruitment of NLRP3 and ASC into a novel RIPK1 kinase activity-independent cell death complex to drive pyroptosis and apoptosis. Furthermore, we found fully functional RIPK1 kinase activity-independent necroptosis driven by the RIPK3-MLKL pathway in TAK1-deficient macrophages. In vivo, TAK1 inactivation resulted in RIPK3-caspase-8 signaling axis-driven myeloid proliferation and a severe sepsis-like syndrome. Overall, our study highlights a previously unknown mechanism for RIPK1 kinase activity-independent inflammasome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis) that could be targeted for treatment of TAK1-associated myeloid proliferation and sepsis.

Haptoglobin improves acute phase response and endotoxin tolerance in response to bacterial LPS.

Sepsis is characterized by delayed acute phase response and lowered immune tolerance in patients. Acute phase serum proteins, like Haptoglobin (Hp), have been associated with increased mortality in bacteria mediated acute lung inflammation and sepsis in neonates. However, it's direct role in modulating the immune response by regulating pro-inflammatory mediators leading to immune tolerant state and if gender affects its expression levels during bacterial infection, especially in blood has not been fully explored. To understand its specific role in endotoxin-mediated immune response, we investigated the correlation between the rise in Hp levels on bacterial infection and its influence on the expression of pro-inflammatory mediators in male and female Whole blood (WHB) and PBMCs. Here, we observed pathogen-specific and gender-specific expression of Hp. Gonadal steroid hormones differentially influenced the Hp expression in LPS-induced WHB, where the addition of Estrogen increased Hp expression, with suppression of TNFα, in both genders. Further on evaluating, the influence of Hp on TNFα expression in endotoxin tolerance (ET), we show that increased Hp levels directly reduced TNFα expression in ET models. Interestingly, blockade of secreted Hp significantly reversed the (ET) state, confirmed by a significant rise in TNFα expression in both ex vivo and in vitro ET models, indicating a possible feedback inhibition by Hp on inflammatory mediators like TNFα. We also investigated the role of PKCδ in the regulation of LPS induced secretion of acute phase proteins (Hp) in serum, where inhibition of PKCδ, reduced secretion of anti-microbial proteins in response to LPS shown by restored bacterial growth. These findings clearly highlight the crucial role of Hp in maintaining immune tolerance via suppressing the pro-inflammatory mediators and also in preventing bacterial proliferation in blood during infection.

Continuous evaluation of changes in the serum proteome from early to late stages of sepsis caused by Klebsiella pneumoniae.

Serum protein profiles of patients with bacterial sepsis from the day of diagnosis until recovery/mortality were compared from early to late stages in response to severe sepsis using two dimensional electrophoresis. The proteins exhibiting changes during the course of sepsis (20­28 day mortality) were selected and identified by matrix­assisted laser desorption ionization­time of flight­tandem mass spectrometry. Among the proteins identified, haptoglobin (Hp), transthyretin (TTR), orosomucoid 1/α1 acid glycoprotein (ORM1), α1 antitrypsin (A1AT), serum amyloid A (SAA) and S100A9 exhibited differential expression patterns between survivors (S; n=6) and non­survivors (NS; n=6), particularly during the early stages of sepsis. Expression factors (EFs), taken as the ratio between the NS and S during early stages, showed ratios of Hp, 0.39 (P≤0.012); TTR, 3.96 (P≤0.03); ORM1, 0.69 (P≤0.79); A1AT, 0.92 (P≤0.87) and SAA, 0.69 (P≤0.01). S100A9, an acute phase protein, exhibited an EF ratio of 1.68 (P≤0.004) during the end stages of sepsis. A delayed rise in levels was observed in Hp, A1AT, ORM1, S100A9 and SAA, whereas TTR levels increased during the early stages of sepsis in NS. Analysis of inflammatory responses in the early stages of sepsis revealed increased mRNA expression in leukocytes of interleukin (IL)­6 (EF, 2.50), IL­10 (EF, 1.70) and prepronociceptin (EF, 1.6), which is a precursor for nociceptin in NS compared with S, and higher Toll­like receptor­4 (EF, 0.30) levels in S compared with NS. Therefore, a weaker acute phase response in the early stages of sepsis in NS, combined with an inefficient inflammatory response, may contribute to sepsis mortality.

Downregulation of PEBP1 in rat brain cortex in hypoxia.

In order to understand dementia and other ailments associated with high altitude hypoxia, adult Sprague Dawley male rats were exposed to simulated conditions of high altitude (7,500 m above sea level, 59 mmHg) for a period of 5 days and analyzed for changes in neuronal proteome by 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein extracts obtained from the brain cortex and hippocampus of the hypoxic rats were separated by 2-D gel electrophoresis. Differentially expressed proteins (analysis by 2-D gel analysis software, Bio-2D, Vilber-Lourmat, France and Delta2d, Decodon, Germany) were subjected to matrix-assisted laser desorption/ionization time-of-flight analysis. Among the proteins identified, the spot corresponding to pI 5.4 and molecular weight 21 kDa, identified as phosphatidylethanolamine binding protein (PEBP1), was consistently lowered (54%) in hypoxic cortex samples. PEBP1, also known as Raf kinase inhibitor protein, is a precursor of hippocampus cholinergic neurostimulatory peptide (HCNP). Western blot analysis revealed elevated phospho-extracellular signal-regulated kinase in hypoxic rat cortex samples, indicating activation of Raf/mitogen-activated protein kinase pathway under hypoxia. Lowered HCNP levels leading to 23% decrease in choline acetyltransferase and 63% increase in acetylcholinesterase activity were detected in hypoxic rat brain cortex, while no significant change was noted in hippocampus. Since PEBP1 is lowered in a number of neurological disorders associated with dementia, we speculate that lowered expression of PEBP1 might be responsible for dementia associated with high-altitude hypoxia. Further studies targeting PEBP1 might give clues about signaling pathways associated with hypoxia and dementia.