Search results for: serine/threonine kinase

Authors: Boutheina Ben Abdelmoumen Mardassi, Salim Chibani, Safa Boujemaa, Amaury Vaysse, Julien Guglielmini, Elhem Yacoub

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Background and aim: Mycoplasma hominis (MH) is a pathogenic bacterium belonging to the Mollicutes class. It causes a wide range of gynecological infections and infertility among adults. Recently, we have explored for the first time the phylodistribution of Tunisian M. hominis clinical strains using an expanded MLST. We have demonstrated their distinction into two pure lineages, which each corresponding to a specific pathotype: genital infections and infertility. The aim of this project is to gain further insight into the evolutionary dynamics and the specific genetic factors that distinguish MH pathotypes Methods: Whole genome sequencing of Mycoplasma hominis clinical strains was performed using illumina Miseq. Denovo assembly was performed using a publicly available in-house pipeline. We used prokka to annotate the genomes, panaroo to generate the gene presence matrix and Jolytree to establish the phylogenetic tree. We used treeWAS to identify genetic loci associated with the pathothype of interest from the presence matrix and phylogenetic tree. Results: Our results revealed a clear categorization of the 62 MH clinical strains into two distinct genetic lineages, with each corresponding to a specific pathotype.; gynecological infections and infertility[AV1] . Genome annotation showed that GC content is ranging between 26 and 27%, which is a known characteristic of Mycoplasma genome. Housekeeping genes belonging to the core genome are highly conserved among our strains. TreeWas identified 4 virulence genes associated with the pathotype gynecological infection. encoding for asparagine--tRNA ligase, restriction endonuclease subunit S, Eco47II restriction endonuclease, and transcription regulator XRE (involved in tolerance to oxidative stress). Five genes have been identified that have a statistical association with infertility, tow lipoprotein, one hypothetical protein, a glycosyl transferase involved in capsule synthesis, and pyruvate kinase involved in biofilm formation. All strains harbored an efflux pomp that belongs to the family of multidrug resistance ABC transporter, which confers resistance to a wide range of antibiotics. Indeed many adhesion factors and lipoproteins (p120, p120', p60, p80, Vaa) have been checked and confirmed in our strains with a relatively 99 % to 96 % conserved domain and hypervariable domain that represent 1 to 4 % of the reference sequence extracted from gene bank. Conclusion: In summary, this study led to the identification of specific genetic loci associated with distinct pathotypes in M hominis.

Keywords: mycoplasma hominis, infertility, gynecological infections, virulence genes, antibiotic resistance

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Authors: Habtamu Abera Goshu, Ping Yan

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Copy number variation (CNV) is a significant marker of the genetic and phenotypic diversity among individuals that accounts for complex quantitative traits of phenotype and diseases via modulating gene dosage, position effects, alteration of downstream pathways, modification of chromosome structure, and position within the nucleus and disrupting coding regions in the genome. Associating copy number variations (CNVs) with growth and gene expression are a powerful approach for identifying genomic characteristics that contribute to phenotypic and genotypic variation. A previous study using next-generation sequencing illustrated that the choline kinase beta (CHKB), Krüpple-like factor 6 (KLF6), glypican 1(GPC1), and cholinergic receptor muscarinic 3 (CHRM3) genes reside within copy number variable regions (CNVRs) of yak populations that overlap with quantitative trait loci (QTLs) of meat quality and growth. As a result, this research aimed to determine the association of CNVs of the KLF6, CHKB, GPC1, and CHRM3 genes with growth traits in the Datong yak breed. The association between the CNV types of the KLF6, CHKB, GPC1, and CHRM3 genes and the growth traits in the Datong yak breed was determined by one-way analysis of variance (ANOVA) using SPSS software. The CNV types were classified as a loss (a copy number of 0 or 1), gain (a copy number >2), and normal (a copy number of 2) relative to the reference gene, BTF3 in the 387 individuals of Datong yak. These results indicated that the normal CNV types of the CHKB and GPC1 genes were significantly (P<0.05) associated with high body length, height and weight, and chest girth in six-month-old and five-year-old Datong yaks. On the other hand, the loss CNV types of the KLF6 gene is significantly (P<0.05) associated with body weight and length and chest girth at six-month-old and five-year-old Datong yaks. In the contrary, the gain CNV type of the CHRM3 gene is highly (P<0.05) associated with body weight, length, height, and chest girth in six-month-old and five-year-old. This work provides the first observation of the biological role of CNVs of the CHKB, KLF6, GPC1, and CHRM3 genes in the Datong yak breed and might, therefore, provide a novel opportunity to utilize data on CNVs in designing molecular markers for the selection of animal breeding programs for larger populations of various yak breeds. Therefore, we hypothesized that this study provided inclusive information on the application of CNVs of the CHKB, KLF6, GPC1, and CHRM3 genes in growth traits in Datong yaks and its possible function in bovine species.

Keywords: Copy number variation, growth traits, yak, genes

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Authors: Rashmi Shukla, Yamini Bhusan Tripathi

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Diabetic nephropathy (DN) is characterized as diabetic kidney disease which involves many pathways e.g. hyperactivated protein kinase c (PKC), polyol pathway, excess production of advanced glycation end product (AGEs) & free radical accumulation etc. All of them results to hypoxia followed by apoptosis of podocytes, glomerulosclerosis, extracellular matrix (ECM) accumulation and fibrosis resulting to irreversible changes in kidney. This is continuously rising worldwide and there are not enough specific drugs, to retard its progress. Due to increasing side effects of allopathic drugs, interest in herbal remedies is growing. Earlier, we have reported that PTY-2 (a phytomedicine, derived from Pueraria tuberosa Linn.) inhibits the accumulation of extracellular matrix (ECM) through activation of MMP-9. Present study exhibited the therapeutic potential of Pueraria tuberosa in the prevention of podocytes apoptosis and modulation of nephrin expression in streptozotocin (STZ) induced DN rats. DN rats were produced by maintaining persistent hyperglycemia for 8 weeks by intra-peritoneal injection of 55 mg/kg streptozotocin (STZ). These rats were randomly divided in 2 groups, i.e. DN control, and DN+ water extract of Pueraria tuberosa (PTW). One group of age-matched normal rats served as non-diabetic control (group-1), The STZ induced DN rats (group-2) and DN+PTW treated rats (group-3). The PTW was orally administered (0.3g/kg) daily to group-2 rats and drug vector (1 ml of 10% tween 20) in control rats. The treatments were continued for 20 days and blood and urine samples were collected. Rats were then sacrificed to investigate the expression Bcl2, Bax and nephroprotective protein i.e. nephrin in kidney glomerulus. The effect of PTW was evaluated, we have found that the PTW significantly(p < .001) reversed the raised serum urea, serum creatinine, urine protein and improved the creatinine clearance in STZ induce diabetic nephropathy in rats and also significantly(p < .001) prevented the rise in urine albumin excretion. The Western blot analysis of kidney tissue homogenate showed increased expression of Bcl2 in PTW treated rats. The RT-PCR showed the increased expression and accumulation of nephrin mRNA. The confocal photomicrographs also supported the reduction of Bax and a simultaneous increase in Bcl2 and nephrin in glomerular podocytes. Hence, our finding suggests that the nephroprotective role of PTW is mediated via restoration of nephrin thus prevents the podocytes apoptosis and ameliorates diabetic nephropathy. The clinical trial of PTW would prove to be a potential food supplement/ drug of alternative medicine for patients with diabetic nephropathy in early stage.

Keywords: Pueraria tuberosa, diabetic nephropathy, anti-apoptosis, nephrin

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Authors: Soma Banerjee, Aamen Talukdar, Argha Mandal, Dipankar Chaudhuri

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Japanese Encephalitis is a major infectious disease with nearly half the world’s population living in areas where it is prevalent. Currently, treatment for it involves only supportive care and symptom management through vaccination. Due to the lack of antiviral drugs against Japanese Encephalitis Virus (JEV), the quest for such agents remains a priority. For these reasons, simulation studies of drug targets against JEV are important. Towards this purpose, docking experiments of the kinase inhibitors were done against the chosen target NS3 helicase as it is a nucleoside binding protein. Previous efforts regarding computational drug design against JEV revealed some lead molecules by virtual screening using public domain software. To be more specific and accurate regarding finding leads, in this study a proprietary software Schrödinger-GLIDE has been used. Druggability of the pockets in the NS3 helicase crystal structure was first calculated by SITEMAP. Then the sites were screened according to compatibility with ATP. The site which is most compatible with ATP was selected as target. Virtual screening was performed by acquiring ligands from databases: KinaseSARfari, KinaseKnowledgebase and Published inhibitor Set using GLIDE. The 25 ligands with best docking scores from each database were re-docked in XP mode. Protein structure alignment of NS3 was performed using VAST against MMDB, and similar human proteins were docked to all the best scoring ligands. The low scoring ligands were chosen for further studies and the high scoring ligands were screened. Seventy-three ligands were listed as the best scoring ones after performing HTVS. Protein structure alignment of NS3 revealed 3 human proteins with RMSD values lesser than 2Å. Docking results with these three proteins revealed the inhibitors that can interfere and inhibit human proteins. Those inhibitors were screened. Among the ones left, those with docking scores worse than a threshold value were also removed to get the final hits. Analysis of the docked complexes through 2D interaction diagrams revealed the amino acid residues that are essential for ligand binding within the active site. Interaction analysis will help to find a strongly interacting scaffold among the hits. This experiment yielded 21 hits with the best docking scores which could be investigated further for their drug like properties. Aside from getting suitable leads, specific NS3 helicase-inhibitor interactions were identified. Selection of Target modification strategies complementing docking methodologies which can result in choosing better lead compounds are in progress. Those enhanced leads can lead to better in vitro testing.

Keywords: antivirals, docking, glide, high-throughput virtual screening, Japanese encephalitis, ns3 helicase

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Authors: Markus Bredel, Sindhu Nair, Hoa Q. Trummell, Rajani Rajbhandari, Christopher D. Willey, Lewis Z. Shi, Zhuo Zhang, William J. Placzek, James A. Bonner

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Purpose: EGFR-targeted monoclonal antibodies (mAbs) provide clinical benefit in some patients with H&N squamous cell carcinoma (HNSCC), but others progress with minimal response. Missense mutations in the EGFR ectodomain (ECD) can be acquired under mAb therapy by mimicking the effect of large deletions on receptor untethering and activation. Little is known about the contribution of EGFR ECD mutations to EGFR activation and anti-EGFR response in HNSCC. Methods: We selected patient-derived HNSCC cells (UM-SCC-1) for resistance to mAb Cetuximab (CTX) by repeated, stepwise exposure to mimic what may occur clinically and identified two concurrent EGFR ECD mutations (UM-SCC-1R). We examined the competence of the mutants to bind EGF ligand or CTX. We assessed the potential impact of the mutations through visual analysis of space-filling models of the native sidechains in the original structures vs. their respective side-chain mutations. We performed CRISPR in combination with site-directed mutagenesis to test for the effect of the mutants on ligand-independent EGFR activation and sorting. We determined the effects on receptor internalization, endocytosis, downstream signaling, and radiation sensitivity. Results: UM-SCC-1R cells carried two non-synonymous missense mutations (G33S and N56K) mapping to domain I in or near the EGF binding pocket of the EGFR ECD. Structural modeling predicted that these mutants restrict the adoption of a tethered, inactive EGFR conformation while not permitting association of EGFR with the EGF ligand or CTX. Binding studies confirmed that the mutant, untethered receptor displayed a reduced affinity for both EGF and CTX but demonstrated sustained activation and presence at the cell surface with diminished internalization and sorting for endosomal degradation. Single and double-mutant models demonstrated that the G33S mutant is dominant over the N56K mutant in its effect on EGFR activation and EGF binding. CTX-resistant UM-SCC-1R cells demonstrated cross-resistance to mAb Panitumuab but, paradoxically, remained sensitive to the reversible receptor tyrosine kinase inhibitor Erlotinib. Conclusions: HNSCC cells can select for EGFR ECD mutations under EGFR mAb exposure that converge to trap the receptor in an open, constitutively activated state. These mutants impede the receptor’s competence to bind mAbs and EGF ligand and alter its endosomal trafficking, possibly explaining certain cases of clinical mAb and radiation resistance.

Keywords: head and neck cancer, EGFR mutation, resistance, cetuximab

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Authors: Asma'u Mahe, Abdullahi A. Imam, Adamu J. Alhassan, Nasiru Abdullahi, Sadiya A. Bichi, Nura Lawal, Kamaluddeen Babagana

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Malaria is a disease with global health significance. It is caused by parasites and transmitted by Anopheles mosquitoes. Increase in insecticide resistance threatens the disease vector control. The strength of selection pressure acting on a mosquito population in relation to insecticide resistance can be assess by determining the genetic diversity of a fragment spanning exon- 20 of IIS6 of the voltage gated sodium channel (VGSC). Larval samples reared to adulthood were identified and kdr (knock down resistance) profile was determined. The DNA sequences were used to assess the patterns of genetic differentiation by determining the levels of genetic variability between the Anopheles mosquitoes. Genetic differentiation of the Anopheles mosquitoes based on a portion of the voltage gated sodium channel gene was obtained. Polymorphisms were detected; sequence variation and analysis were presented as a phylogenetic tree. Phylogenetic tree of VGSC haplotypes was constructed for samples of the Anopheles mosquitoes using the maximum likelihood method in MEGA 6.0 software. DNA sequences were edited using BioEdit sequence editor. The edited sequences were aligned with reference sequence (Kisumu strain). Analyses were performed as contained in dnaSP 5.10. Results of genetic parameters of polymorphism and haplotype reconstruction were presented in count. Twenty sequences were used for the analysis. Regions selected were 1- 576, invariable (monomorphic) sites were 460 while variable (polymorphic) sites were 5 giving the number of total mutations observed in this study. Mutations obtained from the study were at codon 105: TTC- Phenylalanine replaces TCC- Serine, codon 513: TAG- Termination replaces TTG- Leucine, codon 153, 300 and 553 mutations were non-synonymous. From the constructed phylogenetic tree, some groups were shown to be closer with Exon20Gambiae Kisumu (Reference strain) having some genetic distance, while 5-Exon20Gambiae-F I13.ab1, 18-Exon20Gambiae-F C17.ab1, and 2-Exon20Gambiae-F C13.ab1 clustered together genetically differentiated away from others. Mutations observed in this study can be attributed to the high insecticide resistance profile recorded in the study areas. Haplotype networks of pattern of genetic variability and polymorphism for the fragment of the VGSC sequences of sampled Anopheles mosquitoes revealed low haplotypes for the present study. Haplotypes are set of closely linked DNA variation on X-chromosome. Haplotypes were scaled accordingly to reflect their respective frequencies. Low haplotype number, four VGSC-1014F haplotypes were observed in this study. A positive association was previously established between low haplotype number of VGSC diversity and pyrethroid resistance through kdr mechanism. Significant values at (P < 0.05) of Tajima D and Fu and Li D’ were observed for some of the results indicating possible signature of positive selection on the fragment of VGSC in the study. This is the first report of VGSC-1014F in the study site. Based on the results, the mutation was present in low frequencies. However, the roles played by the observed mutations need further investigation. Mutations, environmental factors among others can affect genetic diversity. The study area has recorded increase in insecticide resistance that can affect vector control in the area. This finding might affect the efforts made against malaria. Sequences were deposited in GenBank for Accession Number.

Keywords: anopheles mosquitoes, insecticide resistance, kdr, malaria, voltage gated sodium channel

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Authors: N. Garrós, P. Bustos, N. Beirampour, R. Mohammadi, M. Mallandrich, A.C. Calpena, H. Colom

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Atopic dermatitis (AD) is a persistent skin condition characterized by chronic inflammation caused by an autoimmune response. It is a prevalent clinical issue that requires continual treatment to enhance the patient's quality of life. Systemic therapy often involves the use of glucocorticoids or immunosuppressants to manage symptoms. Our objective was to create and assess topical liposomal formulations containing Baricitinib (BNB), a reversible inhibitor of Janus-associated kinase (JAK), which is involved in various immune responses. These formulations were intended to address flare-ups and improve treatment outcomes for AD. We created three distinct liposomal formulations by combining different amounts of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), cholesterol (CHOL), and ceramide (CER): (i) pure POPC, (ii) POPC mixed with CHOL (at a ratio of 8:2, mol/mol), and (iii) POPC mixed with CHOL and CER (at a ratio of 3.6:2.4:4.0 mol/mol/mol). We conducted various tests to determine the formulations' skin tolerance, irritancy capacity, and their ability to cause erythema and edema on altered skin. We also assessed the transepidermal water loss (TEWL) and skin hydration of rabbits to evaluate the efficacy of the formulations. Histological analysis, the HET-CAM test, and the modified Draize test were all used in the evaluation process. The histological analysis revealed that liposome POPC and POPC:CHOL avoided any damage to the tissues structures. The HET-CAM test showed no irritation effect caused by any of the three liposomes, and the modified Draize test showed a good Draize score for erythema and edema. Liposome POPC effectively counteracted the impact of xylol on the skin, and no erythema or edema was observed during the study. TEWL values were constant for all the liposomes with similar values to the negative control (within the range 8 - 15 g/h·m2, which means a healthy value for rabbits), whereas the positive control showed a significant increase. The skin hydration values were constant and followed the trend of the negative control, while the positive control showed a steady increase during the tolerance study. In conclusion, the developed formulations containing BNB exhibited no harmful or irritating effects, they did not demonstrate any irritant potential in the HET-CAM test and liposomes POPC and POPC:CHOL did not cause any structural alteration according to the histological analysis. These positive findings suggest that additional research is necessary to evaluate the efficacy of these liposomal formulations in animal models of the disease, including mutant animals. Furthermore, before proceeding to clinical trials, biochemical investigations should be conducted to better understand the mechanisms of action involved in these formulations.

Keywords: baricitinib, HET-CAM test, histological study, JAK inhibitor, liposomes, modified draize test

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Authors: Abdulaziz Alakeel, Roaa Alamri, Abdulrahman Alomair, Mohammed Fouda

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Introduction: Ibrutinib is a selective, potent, and irreversible small-molecule inhibitor of Bruton's tyrosine kinase (BTK). It forms a covalent bond with a cysteine residue (CYS-481) at the active site of Btk, leading to inhibition of Btk enzymatic activity. The drug is indicated to treat certain type of cancers such as mantle cell lymphoma (MCL), chronic lymphocytic leukaemia and Waldenström's macroglobulinaemia (WM). Cardiac failure is a condition referred to inability of heart muscle to pump adequate blood to human body organs. There are multiple types of cardiac failure including left and right-sided heart failure, systolic and diastolic heart failures. The aim of this review is to evaluate the risk of cardiac failure associated with the use of ibrutinib and to suggest regulatory recommendations if required. Methodology: Signal Detection team at the National Pharmacovigilance Center (NPC) of Saudi Food and Drug Authority (SFDA) performed a comprehensive signal review using its national database as well as the World Health Organization (WHO) database (VigiBase), to retrieve related information for assessing the causality between cardiac failure and ibrutinib. We used the WHO- Uppsala Monitoring Centre (UMC) criteria as standard for assessing the causality of the reported cases. Results: Case Review: The number of resulted cases for the combined drug/adverse drug reaction are 212 global ICSRs as of July 2020. The reviewers have selected and assessed the causality for the well-documented ICSRs with completeness scores of 0.9 and above (35 ICSRs); the value 1.0 presents the highest score for best-written ICSRs. Among the reviewed cases, more than half of them provides supportive association (four probable and 15 possible cases). Data Mining: The disproportionality of the observed and the expected reporting rate for drug/adverse drug reaction pair is estimated using information component (IC), a tool developed by WHO-UMC to measure the reporting ratio. Positive IC reflects higher statistical association while negative values indicates less statistical association, considering the null value equal to zero. The results of (IC=1.5) revealed a positive statistical association for the drug/ADR combination, which means “Ibrutinib” with “Cardiac Failure” have been observed more than expected when compared to other medications available in WHO database. Conclusion: Health regulators and health care professionals must be aware for the potential risk of cardiac failure associated with ibrutinib and the monitoring of any signs or symptoms in treated patients is essential. The weighted cumulative evidences identified from causality assessment of the reported cases and data mining are sufficient to support a causal association between ibrutinib and cardiac failure.

Keywords: cardiac failure, drug safety, ibrutinib, pharmacovigilance, signal detection

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Authors: Jong H. Kim, Kathleen L. Chan, Luisa W. Cheng

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Control of fungal pathogens, such as foodborne mycotoxin producers, is problematic as effective antimycotic agents are often very limited. Mycotoxin contamination significantly interferes with the safe production of foods or crops worldwide. Moreover, expansion of fungal resistance to commercial drugs or fungicides is a global human health concern. Therefore, there is a persistent need to enhance the efficacy of commercial antimycotic agents or to develop new intervention strategies. Disruption of the cellular antioxidant system should be an effective method for pathogen control. Such disruption can be achieved with safe, redox-active compounds. Natural phenolic derivatives are potent redox cyclers that inhibit fungal growth through destabilization of the cellular antioxidant system. The goal of this study is to identify novel, redox-active compounds that disrupt the fungal antioxidant system. The identified compounds could also function as sensitizing agents to conventional antimycotics (i.e., chemosensitization) to improve antifungal efficacy. Various benzo derivatives were tested against fungal pathogens. Gene deletion mutants of the yeast Saccharomyces cerevisiae were used as model systems for identifying molecular targets of benzo analogs. The efficacy of identified compounds as potent antifungal agents or as chemosensitizing agents to commercial drugs or fungicides was examined with methods outlined by the Clinical Laboratory Standards Institute or the European Committee on Antimicrobial Susceptibility Testing. Selected benzo derivatives possessed potent antifungal or antimycotoxigenic activity. Molecular analyses by using S. cerevisiae mutants indicated antifungal activity of benzo derivatives was through disruption of cellular antioxidant or cell wall integrity system. Certain benzo analogs screened overcame tolerance of Aspergillus signaling mutants, namely mitogen-activated protein kinase mutants, to fludioxonil fungicide. Synergistic antifungal chemosensitization greatly lowered minimum inhibitory or fungicidal concentrations of test compounds, including inhibitors of mitochondrial respiration. Of note, salicylaldehyde is a potent antimycotic volatile that has some practical application as a fumigant. Altogether, benzo derivatives targeting cellular antioxidant system of fungi (along with cell wall integrity system) effectively suppress fungal growth. Candidate compounds possess the antifungal, antimycotoxigenic or chemosensitizing capacity to augment the efficacy of commercial antifungals. Therefore, chemogenetic approaches can lead to the development of novel antifungal intervention strategies, which enhance the efficacy of established microbe intervention practices and overcome drug/fungicide resistance. Chemosensitization further reduces costs and alleviates negative side effects associated with current antifungal treatments.

Keywords: antifungals, antioxidant system, benzo derivatives, chemosensitization

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Authors: Rahaba Marima, Clement Penny

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The Health-related quality of life (HRQoL) for HIV positive patients has improved since the introduction of the highly active antiretroviral treatment (HAART). However, in the present HAART era, HIV co-morbidities such as lung cancer, a non-AIDS (NAIDS) defining cancer have been documented to be on the rise. Under normal physiological conditions, cells grow, repair and proliferate through the cell-cycle as cellular homeostasis is important in the maintenance and proper regulation of tissues and organs. Contrarily, the deregulation of the cell-cycle is a hallmark of cancer, including lung cancer. The association between lung cancer and the use of HAART components such as Efavirenz (EFV) is poorly understood. This study aimed at elucidating the effects of EFV on the cell-cycle genes’ expression in lung cancer. For this purpose, the human cell-cycle gene array composed of 84 genes was evaluated on both normal lung fibroblasts (MRC-5) cells and adenocarcinoma (A549) lung cells, in response to 13µM EFV or 0.01% vehicle. The ±2 up or down fold change was used as a basis of target selection, with p < 0.05. Additionally, RT-qPCR was done to validate the gene array results. Next, In-silico bio-informatics tools, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), Reactome, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Ingenuity Pathway Analysis (IPA) were used for gene/gene interaction studies as well as to map the molecular and biological pathways influenced by the identified targets. Interestingly, the DNA damage response (DDR) pathway genes such as p53, Ataxia telangiectasia mutated and Rad3 related (ATR), Growth arrest and DNA damage inducible alpha (GADD45A), HUS1 checkpoint homolog (HUS1) and Role of radiation (RAD) genes were shown to be upregulated following EFV treatment, as revealed by STRING analysis. Additionally, functional enrichment analysis by the KEGG pathway revealed that most of the differentially expressed gene targets function at the cell-cycle checkpoint such as p21, Aurora kinase B (AURKB) and Mitotic Arrest Deficient-Like 2 (MAD2L2). Core analysis by IPA revealed that p53 downstream targets such as survivin, Bcl2, and cyclin/cyclin dependent kinases (CDKs) complexes are down-regulated, following exposure to EFV. Furthermore, Reactome analysis showed a significant increase in cellular response to stress genes, DNA repair genes, and apoptosis genes, as observed in both normal and cancerous cells. These findings implicate the genotoxic effects of EFV on lung cells, provoking the DDR pathway. Notably, the constitutive expression of this pathway (DDR) often leads to uncontrolled cell proliferation and eventually tumourigenesis, which could be the attribute of HAART components’ (such as EFV) effect on human cancers. Targeting the cell-cycle and its regulation holds a promising therapeutic intervention to the potential HAART associated carcinogenesis, particularly lung cancer.

Keywords: cell-cycle, DNA damage response, Efavirenz, lung cancer

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Authors: Mariia A. Parfenenko, Ilya S. Dantsev, Sergei V. Bochenkov, Natalia V. Vinogradova, Olga S. Groznova, Victoria Yu. Voinova

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Introduction: Autism is the most common neurodevelopmental disorder. Autism is characterized by difficulties in social interaction and adherence to stereotypic behavioral patterns and frequently co-occurs with epilepsy, intellectual disabilities, connective tissue disorders, and other conditions. CHD8 codes for chromodomain-helicase-DNA-binding protein 8 - a chromatin remodeler that regulates cellular proliferation and neurodevelopment in embryogenesis. CHD8 is one of the genes most frequently involved in autism. Patients and methods: 2 unrelated male patients, P3 and P12, aged 3 and 12 years old, underwent whole genome sequencing, which determined that they both had different likely pathogenic variants, both previously undescribed in literature. Sanger sequencing later determined that P12 inherited the variant from his affected mother. Results: P3 and P12 presented with autism, a developmental delay, ataxia, sleep disorders, overgrowth, and macrocephaly, as well as other clinical features typically present in patients with causative variants in CHD8. The mother of P12 also has autistic traits, as well as ataxia, hypotonia, sleep disorders, and other symptoms. However, P3 and P12 also have different cardiac abnormalities. P3 had signs of a repolarization disorder: a flattened T wave in the III and aVF derivations and a negative T wave in the V1-V2 derivations. He also had structural valve anomalies with associated regurgitation, local contractility impairment of the left ventricular, and diastolic dysfunction of the right ventricle. Meanwhile, P12 had Wolff-Parkinson-White syndrome and underwent radiofrequency ablation at the age of 2 years. At the time of observation, P12 had mild sinus arrhythmia and an incomplete right bundle branch block, as well as arterial hypertension. Discussion: Cardiac abnormalities were not previously reported in patients with causative variants in CHD8. The underlying mechanism for the formation of those abnormalities is currently unknown. However, the two hypotheses are either a disordered interaction with CHD7 – another chromodomain remodeler known to be directly involved in the cardiophenotype of CHARGE syndrome – a rare condition characterized by coloboma, heart defects and growth abnormalities, or the disrupted functioning of CHD8 as an A-Kinase Anchoring Protein, which are known to modulate cardiac function. Conclusion: We observed 2 unrelated autistic males with likely pathogenic variants in CHD8 that presented with typical symptoms of CHD8-related neurodevelopmental disorder, as well as cardiac abnormalities. Cardiac abnormalities have, until now, been considered uncharacteristic for patients with causative variants in CHD8. Further accumulation of data, including experimental evidence of the involvement of CHD8 in heart formation, will elucidate the mechanism underlying the cardiophenotype of those patients. Acknowledgements: Molecular genetic testing of the patients was made possible by the Charity Fund for medical and social genetic aid projects «Life Genome.»

Keywords: autism spectrum disorders, chromodomain-helicase-DNA-binding protein 8, neurodevelopmental disorder, cardio phenotype

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Authors: Natalia V. Naryzhnaya, Alexandr V. Mukhomedzyanov, Ivan A. Derkachev, Boris K. Kurbatov, Leonid N. Maslov

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Background: Techniques of adaptation to hypoxia and remote postconditioning (RPost) have great prospects for use in the clinic. However, recent studies have shown low efficacy of remote postconditioning in patients with AMI. We hypothesize that the reasons for this inefficiency may be metabolic disorders, which are very common, especially in patients with cardiovascular disease, and age of patients. The purpose of the study was to reveal the effectiveness of adaptation to chronic hypoxia and RPost. To determine the possible relationship between the decrease in the effectiveness of projective impacts and disorders of carbohydrate and lipid metabolism. Design: The study was carried out on Wistar rats 60 day old. MetS was induced by high-carbohydrate, high-fat diet (HСHFD). Modeling MS led to the formation of obesity, hypertension, impaired lipid and carbohydrate metabolism, hyperleptinemia, and moderate stress. Groups with adaptation to chronic hypoxia were subjected to hypoxia for 21 days at 12% O2 and 0.3% CO2 after complete of HСHFD. All animals were subjected to 45 min coronary occlusion and 120 min reperfusion. Groups with RPost, immediately after the end of ischemia, tourniquets were applied to the hind limbs in the area of the hip joint (3 times in the mode of 5 min ischemia, 5 min reperfusion). Results: RPost led to a twofold reduction of infarct size in rats with intact metabolism (р < 0.0001), while in rats with MetS, a decrease in infarct size during RPost was 25 % (p = 0.00003). A direct correlation was found between of infarct size during RPost and the serum leptin level of rats with MetC (r = 0.85). The presented data suggested that a decrease in the efficiency of remote postconditioning in rats with diet-induced metabolic syndrome depends on serum leptin. Chronic hypoxia resulted in a 38% reduced in infarct size in metabolically intact rats. The decrease of cardioprotection was observed in rats with chronic hypoxia and MetS. Infarct size showed a direct correlation with impaired glucose tolerance (AUC, glucose tolerance test, r = 0.034) and serum triglyceride levels (r = 0.39). Our study showed the dependence of cardioprotection in rats with metabolic syndrome during chronic hypoxia and DPost on opioids in the blood serum and myocardium, protein kinase C and NO synthase activity. Conclusion: The results obtained showed that the infarct-limiting efficiency of adaptation to hypoxia and remote postconditioning is reduced or completely absent in animals with metabolic syndrome. The increase in the infarction, in this case, directly depends on the disturbances in carbohydrate. lipid metabolism and opioids signaling. Funding: Investigation of effectiveness of chronic hypoxia at the metabolic syndrome was carried out within the support of Russian Science Foundation Grant 22-15-00048. Studies of the mechanisms of arterial hypertension in induced metabolic syndrome were carried out within the framework of the state assignment (122020300042-4). The work was performed using the Center for Collective Use "Medical Genomics".

Keywords: chronic hypoxia, opioids, remote postconditioning, metabolic syndrome

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Authors: Parul Kulshreshtha, Subrata Sinha, Rakesh Bhatnagar

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This study was conducted by taking B. anthracis as a model pathogen. On infecting a host, B. anthracis secretes three proteins, namely, protective antigen (PA, 83kDa), edema factor (EF, 89 kDa) and lethal factor (LF, 90 kDa). These three proteins are the components of two anthrax toxins. PA binds to the cell surface receptors, namely, tumor endothelial marker (TEM) 8 and capillary morphogenesis protein (CMG) 2. TEM8 and CMG2 interact with LDL-receptor related protein (LRP) 6 for endocytosis of EF and LF. On entering the cell, EF acts as a calmodulin-dependent adenylate cyclase that causes a prolonged increase of cytosolic cyclic adenosine monophosphate (cAMP). LF is a metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MAPKK/MEK) close to their N-terminus. By secreting these two toxins, B.anthracis ascertains death of the host. Once the systemic levels of the toxins rise, antibiotics alone cannot save the host. Therefore, toxin-specific inhibitors have to be developed. In this wake, monoclonal antibodies have been developed for the neutralization of toxic effects of anthrax toxins. We created hybridomas by using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor of B. anthracis) to obtain anti-toxin antibodies. Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immunized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies from all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H8 and H10) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). The protective efficacy of H7, H8, H10 and H11 was investigated. H7, H8 and H10 were found to be protective. H11 was found to have disease enhancing characteristics in-vitro and in mouse model of challenge with B. anthracis. In this study the disease enhancing character of H11 monoclonal antibody and anti-rLFn polyclonal sera was investigated. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature both in-vitro and in-vivo. But combination of H11 with LETscFv (an scFv with VH and VL identical to H10 but lacking Fc region) could not abrogate the disease-enhancing character of H11 mAb. Therefore it was concluded that for suppression of disease enhancement, Fc portion was absolutely essential for interaction of H10 with H11. Our study indicates that the protective potential of an antibody depends equally on its idiotype/ antigen specificity and its isotype. A number of monoclonal and engineered antibodies are being explored as immunotherapeutics but it is absolutely essential to characterize each one for their individual and combined protective potential. Although new in the sphere of toxin-based diseases, it is extremely important to characterize the disease-enhancing nature of polyclonal as well as monoclonal antibodies. This is because several anti-viral therapeutics and vaccines have failed in the face of this phenomenon. The passive –immunotherapy thus needs to be well formulated to avoid any contraindications.

Keywords: immunotherapy, polyclonal, monoclonal, antibody-dependent disease enhancement

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Authors: B. Atika, S. Lehmann, E. Wimmer

Abstract:

The defense strategy is very common in the insect world. Defensive substances play a wide variety of functions for beetles, such as repellents, toxicants, insecticides, and antimicrobics. Beetles react to predators, invaders, and parasitic microbes with the release of toxic and repellent substances. Defensive substances are directed against a large array of potential target organisms or may function for boiling bombardment or as surfactants. Usually, Coleoptera biosynthesize and store their defensive compounds in a complex secretory organ, known as odoriferous defensive stink glands. The red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae), uses these glands to produce antimicrobial p-benzoquinones and 1-alkenes. In the past, the morphology of stink gland has been studied in detail in tenebrionid beetles; however, very little is known about the genes that are involved in the production of gland secretion. In this study, we studied a subset of genes that are essential for the benzoquinone production in red flour beetle. In the first phase, we selected 74 potential candidate genes from a genome-wide RNA interference (RNAi) knockdown screen named 'iBeetle.' All these 74 candidate genes were functionally characterized by RNAi-mediated gene knockdown. Therefore, they were selected for a subsequent gas chromatography-mass spectrometry (GC-MS) analysis of secretion volatiles in respective RNAi knockdown glands. 33 of them were observed to alter the phenotype of stink gland. In the GC-MS analysis, 7 candidate genes were noted to display a strongly altered gland, in terms of secretion color and chemical composition, upon knockdown, showing their key role in the biosynthesis of gland secretion. Morphologically altered stink glands were found for odorant receptor and protein kinase superfamily. Subsequent GC-MS analysis of secretion volatiles revealed reduced benzoquinone levels in LIM domain, PDZ domain, PBP/GOBP family knockdowns and a complete lack of benzoquinones in the knockdown of sulfatase-modifying factor enzyme 1, sulfate transporter family. Based on stink gland transcriptome data, we analyzed the function of sulfatase-modifying factor enzyme 1 and sulfate transporter family via RNAi-mediated gene knockdowns, GC-MS, in situ hybridization, and enzymatic activity assays. Morphologically altered stink glands were noted in knockdown of both these genes. Furthermore, GC-MS analysis of secretion volatiles showed a complete lack of benzoquinones in the knockdown of these two genes. In situ hybridization showed that these two genes are expressed around the vesicle of certain subgroup of secretory stink gland cells. Enzymatic activity assays on stink gland tissue showed that these genes are involved in p-benzoquinone biosynthesis. These results suggest that sulfatase-modifying factor enzyme 1 and sulfate transporter family play a role specifically in benzoquinone biosynthesis in red flour beetles.

Keywords: Red Flour Beetle, defensive stink gland, benzoquinones, sulfate transporter, sulfatase-modifying factor enzyme 1

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Authors: Suliman Al-Fayoumi, Sherri Amberg, Huafeng Zhou, Jack W. Singer, James P. Dean

Abstract:

Pacritinib is an oral kinase inhibitor with specificity for JAK2, FLT3, IRAK1, and CSF1R. In clinical studies, pacritinib was well tolerated with clinical activity in patients with myelofibrosis. The most frequent adverse events (AEs) observed with pacritinib are gastrointestinal (diarrhea, nausea, and vomiting; mostly grade 1-2 in severity) and typically resolve within 2 weeks. A human ADME mass balance study demonstrated that pacritinib is predominantly cleared via hepatic metabolism and biliary excretion (>85% of administered dose). The major hepatic metabolite identified, M1, is not thought to materially contribute to the pharmacological activity of pacritinib. Hepatic diseases are known to impair hepatic blood flow, drug-metabolizing enzymes, and biliary transport systems and may affect drug absorption, disposition, efficacy, and toxicity. This phase 1 study evaluated the pharmacokinetics (PK) and safety of pacritinib and the M1 metabolite in study subjects with mild, moderate, or severe hepatic impairment (HI) and matched healthy subjects with normal liver function to determine if pacritinib dosage adjustments are necessary for patients with varying degrees of hepatic insufficiency. Study participants (aged 18-85 y) were enrolled into 4 groups based on their degree of HI as defined by Child-Pugh Clinical Assessment Score: mild (n=8), moderate (n=8), severe (n=4), and healthy volunteers (n=8) matched for age, BMI, and sex. Individuals with concomitant renal dysfunction or progressive liver disease were excluded. A single 400 mg dose of pacritinib was administered to all participants. Blood samples were obtained for PK evaluation predose and at multiple time points postdose through 168 h. Key PK parameters evaluated included maximum plasma concentration (Cmax), time to Cmax (Tmax), area under the plasma concentration time curve (AUC) from hour zero to last measurable concentration (AUC0-t), AUC extrapolated to infinity (AUC0-∞), and apparent terminal elimination half-life (t1/2). Following treatment, pacritinib was quantifiable for all study participants at 1 h through 168 h postdose. Systemic pacritinib exposure was similar between healthy volunteers and individuals with mild HI. However, there was a significant difference between those with moderate and severe HI and healthy volunteers with respect to peak concentration (Cmax) and plasma exposure (AUC0-t, AUC0-∞). Mean Cmax decreased by 47% and 57% respectively in participants with moderate and severe HI vs matched healthy volunteers. Similarly, mean AUC0-t decreased by 36% and 45% and mean AUC0-∞ decreased by 46% and 48%, respectively in individuals with moderate and severe HI vs healthy volunteers. Mean t1/2 ranged from 51.5 to 74.9 h across all groups. The variability on exposure ranged from 17.8% to 51.8% across all groups. Systemic exposure of M1 was also significantly decreased in study participants with moderate or severe HI vs. healthy participants and individuals with mild HI. These changes were not significantly dissimilar from the inter-patient variability in these parameters observed in healthy volunteers. All AEs were grade 1-2 in severity. Diarrhea and headache were the only AEs reported in >1 participant (n=4 each). Based on these observations, it is unlikely that dosage adjustments would be warranted in patients with mild, moderate, or severe HI treated with pacritinib.

Keywords: pacritinib, myelofibrosis, hepatic impairment, pharmacokinetics

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Authors: Bianca Peterson, Tomasz J. Sańko, Carlos C. Bezuidenhout, Johnnie Van Den Berg

Abstract:

Busseola fusca (Fuller) (Lepidoptera: Noctuidae), the maize stem borer, is a major pest in sub-Saharan Africa. It causes economic damage to maize and sorghum crops and has evolved non-recessive resistance to genetically modified (GM) maize expressing the Cry1Ab insecticidal toxin. Since B. fusca is a non-model organism, very little genomic information is publicly available, and is limited to some cytochrome c oxidase I, cytochrome b, and microsatellite data. The biology of B. fusca is well-described, but still poorly understood. This, in combination with its larval-specific behavior, may pose problems for limiting the spread of current resistant B. fusca populations or preventing resistance evolution in other susceptible populations. As part of on-going research into resistance evolution, B. fusca larvae were collected from Bt and non-Bt maize in South Africa, followed by RNA isolation (15 specimens) and sequencing on the Illumina HiSeq 2500 platform. Quality of reads was assessed with FastQC, after which Trimmomatic was used to trim adapters and remove low quality, short reads. Trinity was used for the de novo assembly, whereas TransRate was used for assembly quality assessment. Transcript identification employed BLAST (BLASTn, BLASTp, and tBLASTx comparisons), for which two libraries (nucleotide and protein) were created from 3.27 million lepidopteran sequences. Several transcripts that have previously been implicated in Cry toxin resistance was identified for B. fusca. These included aminopeptidase N, cadherin, alkaline phosphatase, ATP-binding cassette transporter proteins, and mitogen-activated protein kinase. MEGA7 was used to align these transcripts to reference sequences from Lepidoptera to detect mutations that might potentially be contributing to Cry toxin resistance in this pest. RSEM and Bioconductor were used to perform differential gene expression analysis on groups of B. fusca larvae challenged and unchallenged with the Cry1Ab toxin. Pairwise expression comparisons of transcripts that were at least 16-fold expressed at a false-discovery corrected statistical significance (p) ≤ 0.001 were extracted and visualized in a hierarchically clustered heatmap using R. A total of 329,194 transcripts with an N50 of 1,019 bp were generated from the over 167.5 million high-quality paired-end reads. Furthermore, 110 transcripts were over 10 kbp long, of which the largest one was 29,395 bp. BLAST comparisons resulted in identification of 157,099 (47.72%) transcripts, among which only 3,718 (2.37%) were identified as Cry toxin receptors from lepidopteran insects. According to transcript expression profiles, transcripts were grouped into three subclusters according to the similarity of their expression patterns. Several immune-related transcripts (pathogen recognition receptors, antimicrobial peptides, and inhibitors) were up-regulated in the larvae feeding on Bt maize, indicating an enhanced immune status in response to toxin exposure. Above all, extremely up-regulated arylphorin genes suggest that enhanced epithelial healing is one of the resistance mechanisms employed by B. fusca larvae against the Cry1Ab toxin. This study is the first to provide a resource base and some insights into a potential mechanism of Cry1Ab toxin resistance in B. fusca. Transcriptomic data generated in this study allows identification of genes that can be targeted by biotechnological improvements of GM crops.

Keywords: epithelial healing, Lepidoptera, resistance, transcriptome

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Authors: Adenike Adesanya, Nonthaphat Wong, Xiang-Yun Lan, Shea Ping Yip, Chien-Ling Huang

Abstract:

Background: The most challenging mutation of the oncokinase BCR-ABL protein T315I, which is commonly known as the “gatekeeper” mutation and is notorious for its strong resistance to almost all tyrosine kinase inhibitors (TKIs), especially imatinib. Therefore, this study aims to identify T315I-dependent downstream microRNA (miRNA) pathways associated with drug resistance in chronic myeloid leukemia (CML) for prognostic and therapeutic purposes. Methods: T315I-carrying K562 cell clones (K562-T315I) were generated by the CRISPR-Cas9 system. Imatinib-treated K562-T315I cells were subjected to small RNA library preparation and next-generation sequencing. Putative lncRNA-miRNA-mRNA networks were analyzed with (i) DESeq2 to extract differentially expressed miRNAs, using Padj value of 0.05 as cut-off, (ii) STarMir to obtain potential miRNA response element (MRE) binding sites of selected miRNAs on lncRNA H19, (iii) miRDB, miRTarbase, and TargetScan to predict mRNA targets of selected miRNAs, (iv) IntaRNA to obtain putative interactions between H19 and the predicted mRNAs, (v) Cytoscape to visualize putative networks, and (vi) several pathway analysis platforms – Enrichr, PANTHER and ShinyGO for pathway enrichment analysis. Moreover, mitochondria isolation and transcript quantification were adopted to determine the new mechanism involved in T315I-mediated resistance of CML treatment. Results: Verification of the CRISPR-mediated mutagenesis with digital droplet PCR detected the mutation abundance of ≥80%. Further validation showed the viability of ≥90% by cell viability assay, and intense phosphorylated CRKL protein band being detected with no observable change for BCR-ABL and c-ABL protein expressions by Western blot. As reported by several investigations into hematological malignancies, we determined a 7-fold increase of H19 expression in K562-T315I cells. After imatinib treatment, a 9-fold increment was observed. DESeq2 revealed 171 miRNAs were differentially expressed K562-T315I, 112 out of these miRNAs were identified to have MRE binding regions on H19, and 26 out of the 112 miRNAs were significantly downregulated. Adopting the seed-sequence analysis of these identified miRNAs, we obtained 167 mRNAs. 6 hub miRNAs (hsa-let-7b-5p, hsa-let-7e-5p, hsa-miR-125a-5p, hsa-miR-129-5p, and hsa-miR-372-3p) and 25 predicted genes were identified after constructing hub miRNA-target gene network. These targets demonstrated putative interactions with H19 lncRNA and were mostly enriched in pathways related to cell proliferation, senescence, gene silencing, and pluripotency of stem cells. Further experimental findings have also shown the up-regulation of mitochondrial transcript and lncRNA MALAT1 contributing to the lncRNA-miRNA-mRNA networks induced by BCR-ABL T315I mutation. Conclusions: Our results have indicated that lncRNA-miRNA regulators play a crucial role not only in leukemogenesis but also in drug resistance, considering the significant dysregulation and interactions in the K562-T315I cell model generated by CRISPR-Cas9. In silico analysis has further shown that lncRNAs H19 and MALAT1 bear several complementary miRNA sites. This implies that they could serve as a sponge, hence sequestering the activity of the target miRNAs.

Keywords: chronic myeloid leukemia, imatinib resistance, lncRNA-miRNA-mRNA, T315I mutation

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Authors: Mohammadjavad Sotoudeheian, Navid Farahmandian

Abstract:

Kawasaki disease (KD) is the primary cause of acquired pediatric heart disease as an acute vasculitis. In children with prolonged fever, rash, and inflammation of the mucosa KD must be considered as a clinical diagnosis. There is a persuasive suggestion of immune-mediated damage as the pathophysiologic cascade of KD. For example, the invasion of cytotoxic T-cells supports a viral etiology and the inflammasome of the innate immune system is a critical component in the vasculitis formation in KD. Animal models of KD propose the cytokine profiles, such as increased IL-1 and GM-CSF, which cause vascular damage. CRP and IFN-γ elevated expression and the upregulation of IL-6, and IL-10 production are also described in previous studies. Untreated KD is a critical risk factor for coronary artery diseases and myocardial infarction. Vascular damage may encompass amplified T-cell activity. SMAD3 is an essential molecule in down-regulating T-cells and increasing expression of FoxP3. It has a critical effect in the differentiation of regulatory T-cells. The discrepancy of regulatory T-cells and pro-inflammatory Th17 has been studied in acute coronary syndrome during KD. However in the coronary artery damaged lymphocytes and IgA plasma cells are seen at the lesion locations, the major immune cells in the coronary lesions are monocytes/macrophages and neutrophils. These cells secrete TNF-α, and activates matrix metalloprotease (MMP)-9, reducing the integrity of vessels and prompting patients to arise aneurysm. MMPs can break down the components of the extracellular matrix and assist immune cell movement. IVIG as an effective form of treatment clarified the role of the immune system, which may target pathogenic antigens and regulate cytokine production. Several reports have revealed that in the coronary arteries, high expression of MMP-9 in monocyte/macrophage results in pathologic cascades. Curcumin is a potent antioxidant and anti-inflammatory molecule. Curcumin decreases the production of reactive oxygen and nitrogen species and inhibits transcription factors like AP-1 and NF-κB. Curcumin also contains the characteristics of inhibitory effects on MMPs, especially MMP-9. The upregulation of MMP-9 is an important cellular response. Curcumin treatment caused a reverse effect and down-regulates MMP-9 gene expression which may fund the anti-inflammatory effect. Curcumin inhibits MMP-9 expression via PKC and AMPK-dependent pathways in Human monocytes cells. Elevated expression and activity of MMP-9 are correlated with advanced vascular lesions. AMPK controls lipid metabolism and oxidation, and protein synthesis. AMPK is also necessary for the MMP-9 activity and THP-1 cell adhesion to endothelial cells. Curcumin was shown to inhibit the activation of AMPKα. Compound C (AMPK inhibitor) inhibits MMP-9 expression level. Therefore, through inactivating AMPKs and PKC, curcumin decreases the MMP-9 level, which results in inhibiting monocyte/macrophage differentiation. Compound C also suppress the phosphorylation of three major classes of MAP kinase signaling, suggesting that curcumin may suppress MMP-9 level by inactivation of MAPK pathways. MAPK cascades are activated to induce the expression of MMP-9. Curcumin inhibits MAPKs phosphorylation, which contributes to the down-regulation of MMP-9. This study demonstrated that the potential inhibitory properties of curcumin over MMP-9 lead to a therapeutic strategy to reduce the risk of coronary artery involvement during KD.

Keywords: MMP-9, coronary artery aneurysm, Kawasaki’s disease, curcumin, AMPK, immune system, NF-κB, MAPK

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Authors: Merrick Lopez, Aashish Abraham, Melissa Egge, Marissa Hood, Jui Shah

Abstract:

A 3 year old male with unknown medical history presented initially with encephalopathy, intubated for respiratory failure, and admitted to the pediatric intensive care unit (PICU) with refractory shock. During resuscitation in the emergency department, he was found to be in severe metabolic acidosis with a pH of 7.03 and escalated on vasopressor drips for hypotension. His initial sodium was 174. He was noted to have burn injuries to his scalp, forehead, right axilla, bilateral arm creases and lower legs. He had rhabdomyolysis (initial creatinine kinase 5,430 U/L with peak levels of 62,340 normal <335 U/L), cardiac injury (initial troponin 88 ng/L with peak at 145 ng/L, normal <15ng/L), hypernatremia (peak 174, normal 140), hypocalcemia, liver injury, acute kidney injury, and neuronal loss on magnetic resonance imaging (MRI). Soft restraints and a shock collar were found in the home. He was critically ill for 8 days, but was gradually weaned off drips, extubated, and started on feeds. Discussion Electrical injury, specifically lightning injury is an uncommon but devastating cause of injury in pediatric patients. This patient with suspected abusive use of a dog shock collar presented similar to a lightning strike. Common entrance points include the hands and head, similar to our patient with linear wounds on his forehead. When current enters, it passes through tissues with the least resistance. Nerves, blood vessels, and muscles, have high fluid and electrolyte content and are commonly affected. Exit points are extremities: our child who had circumferential burns around his arm creases and ankles. Linear burns preferentially follow areas of high sweat concentration, and are thought to be due to vaporization of water on the skin’s surface. The most common cause of death from a lightning strike is due to cardiopulmonary arrest. The massive depolarization of the myocardium can result in arrhythmias and myocardial necrosis. The patient presented in cardiogenic shock with evident cardiac damage. Electricity going through vessels can lead to vaporization of intravascular water. This can explain his severe hypernatremia. He also sustained other internal organ injuries (adrenal glands, pancreas, liver, and kidney). Electrical discharge also leads to direct skeletal muscle injury in addition to prolonged muscular spasm. Rhabdomyolysis, the acute damage of muscle, leads to release of potentially toxic components into the circulation which could lead to acute renal failure. The patient had severe rhabdomyolysis and renal injury. Early hypocalcemia has been consistently demonstrated in patients with rhabdomyolysis. This was present in the patient and led to increased vasopressor needs. Central nervous system injuries are also common which can include encephalopathy, hypoxic injury, and cerebral infarction. The patient had evidence of brain injury as seen on MRI. Conclusion Electrical injuries due to lightning strikes and abusive use of a dog shock collar are rare, but can both present in similar ways with respiratory failure, shock, hypernatremia, rhabdomyolysis, brain injury, and multiorgan damage. Although rare, it is essential for early identification and prompt management for acute and chronic complications in these children.

Keywords: cardiogenic shock, dog shock collar, lightning strike, rhabdomyolysis

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Authors: Kuladip Jana, Bhaswati Banerjee, Parimal C. Sen

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Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems along with carcinogenesis in skin, prostate, ovary, lung and mammary glands. Our study was focused on elucidating the molecular mechanism of B(a)P induced male reproductive toxicity and its prevention with phytochemical like resveratrol. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of Wistar rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased Reactive Oxygen Species (ROS), altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38 mitogen activated protein kinase (p38MAPK), cytosolic translocation of cytochrome-c, upregulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of poly ADP ribose polymerase (PARP) and down regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like steroidogenic acute regulatory protein (StAR), cytochrome P450 IIA1 (CYPIIA1), 3β hydroxy steroid dehydrogenase (3β HSD), 17β hydroxy steroid dehydrogenase (17β HSD) showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis. Next we investigated the role of resveratrol on B(a)P induced male reproductive toxicity. Our study highlighted that resveratrol co-treatment with B(a)P maintained testicular redox potential, increased serum testosterone level and prevented steroidogenic dysfunction with enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3β HSD,17β HSD) relative to treatment with B(a)P only. Resveratrol suppressed B(a)P-induced testicular activation of p38 MAPK, ATF2, iNOS and ROS production; cytosolic translocation of Cytochome c and Caspase 3 activation thereby prevented oxidative stress of testis and inhibited apoptosis. Resveratrol co-treatment also decreased B(a)P-induced AhR protein level, its nuclear translocation and subsequent CYP1A1 promoter activation, thereby decreased protein and mRNA levels of testicular cytochrome P4501A1 (CYP1A1) and prevented BPDE-DNA adduct formation. Our findings cumulatively suggest that resveratrol prevents activation of B(a)P by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.

Keywords: benzo(a)pyrene, resveratrol, testis, apoptosis, cytochrome P450 1A1 (CYP1A1), aryl hydrocarbon receptor (AhR), p38 MAPK/ATF2/iNOS

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Authors: Aastha Arora, Vikas Bhuria, Saurabh Singh, Uma Pathak, Shweta Mathur, Puja P. Hazari, Rajat Sandhir, Ravi Soni, Anant N. Bhatt, Bilikere S. Dwarakanath

Abstract:

Radiotherapy is an effective curative and palliative option for patients with thoracic malignancies. However, lung injury, comprising of pneumonitis and fibrosis, remains a significant clin¬ical complication of thoracic radiation, thus making it a dose-limiting factor. Also, injury to the lung is often reported as part of multi-organ failure in victims of accidental radiation exposures. Radiation induced inflammatory response in the lung, characterized by leukocyte infiltration and vascular changes, is an important contributing factor for the injury. Therefore, countermeasure agents to attenuate radiation induced inflammatory response are considered as an important approach to prevent chronic lung damage. Although Amifostine, the widely used, FDA approved radio-protector, has been found to reduce the radiation induced pneumonitis during radiation therapy of non-small cell lung carcinoma, its application during mass and field exposure is limited due to associated toxicity and ineffectiveness with the oral administration. The amifostine analogue (DRDE-30) overcomes this limitation as it is orally effective in reducing the mortality of whole body irradiated mice. The current study was undertaken to investigate the potential of DRDE-30 to ameliorate radiation induced lung damage. DRDE-30 was administered intra-peritoneally, 30 minutes prior to 13.5 Gy thoracic (60Co-gamma) radiation in C57BL/6 mice. Broncheo- alveolar lavage fluid (BALF) and lung tissues were harvested at 12 and 24 weeks post irradiation for studying inflammatory and fibrotic markers. Lactate dehydrogenase (LDH) leakage, leukocyte count and protein content in BALF were used as parameters to evaluate lung vascular permeability. Inflammatory cell signaling (p38 phosphorylation) and anti-oxidant status (MnSOD and Catalase level) was assessed by Western blot, while X-ray CT scan, H & E staining and trichrome staining were done to study the lung architecture and collagen deposition. Irradiation of the lung increased the total protein content, LDH leakage and total leukocyte count in the BALF, reflecting endothelial barrier dysfunction. These disruptive effects were significantly abolished by DRDE-30, which appear to be linked to the DRDE-30 mediated abrogation of activation of the redox-sensitive pro- inflammatory signaling cascade, the MAPK pathway. Concurrent administration of DRDE-30 with radiation inhibited radiation-induced oxidative stress by strengthening the anti-oxidant defense system and abrogated p38 mitogen-activated protein kinase activation, which was associated with reduced vascular leak and macrophage recruitment to the lungs. Histopathological examination (by H & E staining) of the lung showed radiation-induced inflammation of the lungs, characterized by cellular infiltration, interstitial oedema, alveolar wall thickening, perivascular fibrosis and obstruction of alveolar spaces, which were all reduced by pre-administration of DRDE-30. Structural analysis with X-ray CT indicated lung architecture (linked to the degree of opacity) comparable to un-irradiated mice that correlated well with the lung morphology and reduced collagen deposition. Reduction in the radiation-induced inflammation and fibrosis brought about by DRDE-30 resulted in a profound increase in animal survival (72 % in the combination vs 24% with radiation) observed at the end of 24 weeks following irradiation. These findings establish the potential of the Amifostine analogue, DRDE-30, in reducing radiation induced pulmonary injury by attenuating the inflammatory and fibrotic responses.

Keywords: amifostine, fibrosis, inflammation, lung injury radiation

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Authors: Claire Harrison, Raajit K. Rampal, Vikas Gupta, Srdan Verstovsek, Moshe Talpaz, Jean-Jacques Kiladjian, Ruben Mesa, Andrew Kuykendall, Alessandro Vannucchi, Francesca Palandri, Sebastian Grosicki, Timothy Devos, Eric Jourdan, Marielle J. Wondergem, Haifa Kathrin Al-Ali, Veronika Buxhofer-Ausch, Alberto Alvarez-Larrán, Sanjay Akhani, Rafael Muñoz-Carerras, Yury Sheykin, Gozde Colak, Morgan Harris, John Mascarenhas

Abstract:

Myelofibrosis (MF) is characterized by bone marrow fibrosis, anemia, splenomegaly and constitutional symptoms. Progressive bone marrow fibrosis results from aberrant megakaryopoeisis and expression of proinflammatory cytokines, both of which are heavily influenced by bromodomain and extraterminal domain (BET)-mediated gene regulation and lead to myeloproliferation and cytopenias. Pelabresib (CPI-0610) is an oral small-molecule investigational inhibitor of BET protein bromodomains currently being developed for the treatment of patients with MF. It is designed to downregulate BET target genes and modify nuclear factor kappa B (NF-κB) signaling. MANIFEST-2 was initiated based on data from Arm 3 of the ongoing Phase 2 MANIFEST study (NCT02158858), which is evaluating the combination of pelabresib and ruxolitinib in Janus kinase inhibitor (JAKi) treatment-naïve patients with MF. Primary endpoint analyses showed splenic and symptom responses in 68% and 56% of 84 enrolled patients, respectively. MANIFEST-2 (NCT04603495) is a global, Phase 3, randomized, double-blind, active-control study of pelabresib and ruxolitinib versus placebo and ruxolitinib in JAKi treatment-naïve patients with primary MF, post-polycythemia vera MF or post-essential thrombocythemia MF. The aim of this study is to evaluate the efficacy and safety of pelabresib in combination with ruxolitinib. Here we report updates from a recent protocol amendment. The MANIFEST-2 study schema is shown in Figure 1. Key eligibility criteria include a Dynamic International Prognostic Scoring System (DIPSS) score of Intermediate-1 or higher, platelet count ≥100 × 10^9/L, spleen volume ≥450 cc by computerized tomography or magnetic resonance imaging, ≥2 symptoms with an average score ≥3 or a Total Symptom Score (TSS) of ≥10 using the Myelofibrosis Symptom Assessment Form v4.0, peripheral blast count <5% and Eastern Cooperative Oncology Group performance status ≤2. Patient randomization will be stratified by DIPSS risk category (Intermediate-1 vs Intermediate-2 vs High), platelet count (>200 × 10^9/L vs 100–200 × 10^9/L) and spleen volume (≥1800 cm^3 vs <1800 cm^3). Double-blind treatment (pelabresib or matching placebo) will be administered once daily for 14 consecutive days, followed by a 7 day break, which is considered one cycle of treatment. Ruxolitinib will be administered twice daily for all 21 days of the cycle. The primary endpoint is SVR35 response (≥35% reduction in spleen volume from baseline) at Week 24, and the key secondary endpoint is TSS50 response (≥50% reduction in TSS from baseline) at Week 24. Other secondary endpoints include safety, pharmacokinetics, changes in bone marrow fibrosis, duration of SVR35 response, duration of TSS50 response, progression-free survival, overall survival, conversion from transfusion dependence to independence and rate of red blood cell transfusion for the first 24 weeks. Study recruitment is ongoing; 400 patients (200 per arm) from North America, Europe, Asia and Australia will be enrolled. The study opened for enrollment in November 2020. MANIFEST-2 was initiated based on data from the ongoing Phase 2 MANIFEST study with the aim of assessing the efficacy and safety of pelabresib and ruxolitinib in JAKi treatment-naïve patients with MF. MANIFEST-2 is currently open for enrollment.

Keywords: CPI-0610, JAKi treatment-naïve, MANIFEST-2, myelofibrosis, pelabresib

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