A Retrospective Analysis of BCR-ABL1 Kinase Domain Mutations in the Frontline Drug Intolerant or Resistant Chronic Myeloid Leukemia Patients: An Indian Experience from a High-End Referral Laboratory.

Atreye MajumdarSambit K. MohantyObjective This article identifies and evaluates the frequency of mutations in the BCR-ABL1 kinase domain (KD) of chronic myeloid leukemia (CML) patients who showed suboptimal response to their current tyrosine kinase inhibitor (TKI) regime and assesses their clinical value in further treatment decisions. Materials and Methods Peripheral and/or bone marrow were collected from 791 CML patients. Ribonucleic acid was extracted, reverse transcribed, and Sanger sequencing method was utilized to detect single-nucleotide variants (SNVs) in BCR-ABL1 KD. Results Thirty-eight different SNVs were identified in 29.8% ( n = 236/791) patients. T315I, E255K, and M244V were among the most frequent mutations detected. In addition, one patient harbored a novel L298P mutation. A subset of patients from the abovementioned harbored compound mutations (13.3%, n = 33/236). Follow-up data was available in 28 patients that demonstrated the efficacy of TKIs in correlation with mutation analysis and BCR-ABL1 quantitation. Molecular response was attained in 50% patients following an appropriate TKI shift. A dismal survival rate of 40% was observed in T315I-harboring patients on follow-up. Conclusion This study shows the incidence and pattern of mutations in one of the largest sets of Indian CML patients. In addition, our findings strengthen the prognostic value of KD mutation analysis among strategies to overcome TKI resistance.

Short-chain fatty acids abrogate Japanese encephalitis virus-induced inflammation in microglial cells via miR-200a-3p/ZBTB20/IKßα axis.

Japanese encephalitis virus (JEV), a member of the Flaviviridae family, is a leading cause of viral encephalitis in humans. Survivors of this infection often develop lifelong neurological sequelae. Short-chain fatty acids (SCFAs) produced in the gut are vital mediators of the gut-brain axis. We aimed to study microRNA-based mechanisms of SCFAs in an in vitro model of JEV infection. N9 microglial cells were pretreated with SCFA cocktail before JEV infection. Cytokine bead analysis, immunoblotting, and PCR were performed to analyze relevant inflammatory markers. microRNA sequencing was performed using Illumina Hiseq, and bioinformatics tools were used for differentially expressed (DE) miRNAs and weighted gene co-expression network analysis (WGCNA). microRNA mimic/inhibitor experiments and luciferase assay were performed to study miRNA-target interaction. A significant reduction in monocyte chemoattractant protein (MCP1) and tumor necrosis factor alpha (TNFα) along with reduced expression of phospho-nuclear factor kappa B (phospho-NF-κB) was observed in SCFA conditions. Significant attenuation of histone deacetylase activity and protein expression was recorded. miRNA sequencing revealed 160 DE miRNAs in SCFA + JEV-treated cells at 6 h post-infection. WGCNA revealed miR-200a-3p, a hub miRNA significantly upregulated in SCFA conditions. Transcription factor ZBTB20 was bioinformatically predicted and validated as a gene target for miR-200a-3p. Further miRNA mimic/inhibitor assay demonstrated that miR-200-3p regulated ZBTB20 along with Iκßα that possibly dampened NF-κB signal activation downstream. IMPORTANCE: The gut-brain axis plays a pivotal role in the physiological state of an organism. Gut microbiota-derived metabolites are known to play a role in brain disorders including neuroviral infections. Short-chain fatty acids (SCFAs) appear to quench inflammatory markers in Japanese encephalitis virus-infected microglial cells in vitro. Mechanistically, we demonstrate the interaction between miR-200a-3p and ZBTB20 in regulating the canonical nuclear factor kappa B (NF-κB) signaling pathway via transcriptional regulation of Iκßα. Findings of this study pave the way to a better understanding of SCFA mechanisms that can be used to develop strategies against viral neuroinflammation.

Prophylactic Administration of Gut Microbiome Metabolites Abrogated Microglial Activation and Subsequent Neuroinflammation in an Experimental Model of Japanese Encephalitis.

Short-chain fatty acids (SCFAs) are gut microbial metabolic derivatives produced during the fermentation of ingested complex carbohydrates. SCFAs have been widely regarded to have a potent anti-inflammatory and neuro-protective role and have implications in several disease conditions, such as, inflammatory bowel disease, type-2 diabetes, and neurodegenerative disorders. Japanese encephalitis virus (JEV), a neurotropic flavivirus, is associated with life threatening neuro-inflammation and neurological sequelae in infected hosts. In this study, we hypothesize that SCFAs have potential in mitigating JEV pathogenesis. Postnatal day 10 BALB/c mice were intraperitoneally injected with either a SCFA mixture (acetate, propionate, and butyrate) or PBS for a period of 7 days, followed by JEV infection. All mice were observed for onset and progression of symptoms. The brain tissue was collected upon reaching terminal illness for further analysis. SCFA-supplemented JEV-infected mice (SCFA + JEV) showed a delayed onset of symptoms, lower hindlimb clasping score, and decreased weight loss and increased survival by 3 days (p < 0.0001) upon infection as opposed to the PBS-treated JEV-infected animals (JEV). Significant downregulation of inflammatory cytokines TNF-α, MCP-1, IL-6, and IFN-Υ in the SCFA + JEV group relative to the JEV-infected control group was observed. Inflammatory mediators, phospho-NF-kB (P-NF-kB) and iba1, showed 2.08 ± 0.1 and 3.132 ± 0.43-fold upregulation in JEV versus 1.19 ± 0.11 and 1.31 ± 0.11-fold in the SCFA + JEV group, respectively. Tissue section analysis exhibited reduced glial activation (JEV group─42 ± 2.15 microglia/ROI; SCFA + JEV group─27.07 ± 1.8 microglia/ROI) in animals that received SCFA supplementation prior to infection as seen from the astrocytic and microglial morphometric analysis. Caspase-3 immunoblotting showed 4.08 ± 1.3-fold upregulation in JEV as compared to 1.03 ± 0.14-fold in the SCFA + JEV group and TUNEL assay showed a reduced cellular death post-JEV infection (JEV-6.4 ± 1.5 cells/ROI and SCFA + JEV-3.7 ± 0.73 cells/ROI). Our study critically contributes to the increasing evidence in support of SCFAs as an anti-inflammatory and neuro-protective agent, we further expand its scope as a potential supplementary intervention in JEV-mediated neuroinflammation.

Regulation of Onecut2 by miR-9-5p in Japanese encephalitis virus infected neural stem/progenitor cells.

Japanese encephalitis virus (JEV) is one of the major neurotropic viral infections that is known to dysregulate the homeostasis of neural stem/progenitor cells (NSPCs) and depletes the stem cell pool. NSPCs are multipotent stem cell population of the central nervous system (CNS) which are known to play an important role in the repair of the CNS during insults/injury caused by several factors such as ischemia, neurological disorders, CNS infections, and so on. Viruses have evolved to utilize host factors for their own benefit and during JEV infection, host factors, including the non-coding RNAs such as miRNAs, are reported to be affected, thereby cellular processes regulated by the miRNAs exhibit perturbed functionality. Previous studies from our laboratory have demonstrated the role of JEV infection in dysregulating the function of neural stem cells (NSCs) by altering the cell fate and depleting the stem cell pool leading to a decline in stem cell function in CNS repair mechanism post-infection. JEV-induced alteration in miRNA expression in the NSCs is one of the major interest to us. In prior studies, we have observed an altered expression pattern of certain miRNAs following JEV infection. In this study, we have validated the role of JEV infection in NSCs in altering the expression of miR-9-5p, which is a known regulator of neurogenesis in NSCs. Furthermore, we have validated the interaction of this miRNA with its target, Onecut2 (OC2), in primary NSCs utilizing miRNA mimic and inhibitor transfection experiments. Our findings indicate a possible role of JEV mediated dysregulated interaction between miR-9-5p and its putative target OC2 in NSPCs. IMPORTANCE: MicroRNAs have emerged as key disease pathogenic markers and potential therapeutic targets. In this study, we solidify this concept by studying a key miRNA, miR-9-5p, in Japanese encephalitis virus infection of neural stem/progenitor cells. miRNA target Onecut2 has a possible role in stem cell pool biology. Here, we show a possible mechanistic axis worth investing in neurotropic viral biology.