👤 Gouranga Biswas

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains the leading cause of death from a single infectious agent worldwide. Drug-resistant TB (DR-TB) poses a major challenge. Bedaquiline (BDQ) is central to multidrug resistant tuberculosis/extensively drug-resistant TB (MDR/XDR-TB) treatment, yet emerging resistance prompted this study to assess its prevalence. This prospective observational study was conducted in the TB culture and drug susceptibility testing (DST) laboratory of a tertiary care hospital over 9 months. Sputum samples from presumptive DR-TB cases were included, whereas extrapulmonary and nontuberculous mycobacteria samples were excluded from the study. A total of 1190 samples were subjected to the first-line probe assay (LPA) for drug resistance detection in MTB. MDR-TB was most common, followed by mono-isoniazid and monorifampicin resistance. Of 512 DR-TB samples tested by second-line probe assay (SL-LPA), 25 yielded invalid results. Fluoroquinolone (FQ) resistance was highest (47.7%), whereas second-line injectable drug (SLID) resistance was rare (1.4%); combined FQ + SLID resistance occurred in 10.7% samples, whereas 35.4% samples were sensitive. Among 380 isolates subjected to liquid culture DST, resistance was detected to moxifloxacin (7.4%), linezolid (2.1%), and BDQ (0.78%). BDQ resistance is low but emerging; routine DST, rational drug use, and robust surveillance are vital to preserve BDQ efficacy and ensure effective DR-TB management. Show less

Movement of lipoprotein lipase (LPL) from myocytes or adipocytes to the capillary lumen is essential for intravascular lipolysis and plasma triglyceride homeostasis-low LPL activity in the capillary lumen causes hypertriglyceridemia. The trans-endothelial transport of LPL depends on ionic interactions with GPIHBP1's intrinsically disordered N-terminal tail, which harbors two acidic clusters at positions 5-12 and 19-30. This polyanionic tail provides a molecular switch that controls LPL detachment from heparan sulfate proteoglycans (HSPGs) by competitive displacement. When the acidic tail was neutralized in gene-edited mice, LPL remained trapped in the sub-endothelial spaces triggering hypertriglyceridemia. Due to its disordered state, the crystal structure of LPL•GPIHBP1 provided no information on these electrostatic interactions between LPL and GPIHBP1 acidic tail. In the current study, we positioned the acidic tail on LPL using zero-length crosslinking. Acidic residues at positions 19-30 in GPIHBP1 mapped to Lys Show less

Poly(ADP-ribose) (PAR) polymerase-1 (PARP1) has been implicated in DNA damage responses and neuroinflammation in Alzheimer's disease (AD), yet its role in amyloid-β (Aβ) pathology remains unclear. Here, we show that PARP1 activation drives Aβ pathology and neurodegeneration. Using a sensitive ELISA, we observed significantly elevated PAR levels in the cerebrospinal fluid (CSF) of patients with mild cognitive impairment (MCI) and AD compared to controls. Show less

Hyperlipidemia, a key risk factor for cardiovascular disease, is characterized by elevated low-density lipoprotein cholesterol (LDL-C), triglycerides, and reduced high-density lipoprotein cholesterol (HDL-C). Cholesteryl ester transfer protein (CETP) inhibitors, such as anacetrapib, obicetrapib, evacetrapib, dalcetrapib, and torcetrapib, aim to improve lipid profiles by increasing HDL-C and reducing LDL-C, but their comparative efficacy remains unclear. This systematic review and frequentist network meta-analysis, conducted per PRISMA-NMA guidelines, included 33 randomized controlled trials (RCTs) involving 120,292 adults with hyperlipidemia. We compared CETP inhibitors, alone or with statins, against placebo or other lipid-lowering therapies. Primary outcome was LDL-C reduction; secondary outcomes included HDL-C, triglycerides, and total cholesterol changes. Random-effects models calculated mean differences (MD) with 95% confidence intervals (CI), and P-scores ranked interventions. Atorvastatin + obicetrapib showed the largest reduction in LDL-C levels (MD: -69.00, 95% CI: -95.96 to -42.04, p < 0.0001), followed by rosuvastatin + obicetrapib (MD: -60.70, 95% CI: -99.28 to -22.12, p = 0.0020). Atorvastatin + obicetrapib yielded highly significant increase in HDL-C levels (MD: 149.90, 95% CI: 121.70 to 178.10, p < 0.0001), but rosuvastatin + obicetrapib showed the greatest increase (MD: 158.90, 95% CI: 118.59 to 199.21, p < 0.0001) and obicetrapib monotherapy (MD: 139.00, 95% CI: 129.05 to 148.96, p < 0.0001), while rosuvastatin + evacetrapib led triglyceride reductions (MD: -31.70 mg/dL). Rosuvastatin was most effective for total cholesterol (MD: -31.60 mg/dL). CETP inhibitors, particularly anacetrapib and obicetrapib combined with statins, significantly improve lipid profiles, offering potential therapeutic benefits for hyperlipidemia management and cardiovascular risk reduction. The study was registered with PROSPERO to ensure transparency and adherence to methodological rigor (Registration ID: CRD420250652666). Show less

Background Metabolic syndrome (MetS) is a major public-health concern that substantially increases the risk of cardiovascular disease and type 2 diabetes. Traditional lipid profiles may not fully capture the atherogenic burden in MetS, prompting investigation of novel lipid biomarkers, such as lipoprotein(a) (Lp(a)), apolipoproteins, and the Comprehensive Lipid Tetrad Index (CLTI). This study evaluated the association of these biomarkers with MetS in an Indian outpatient population. Methods In a cross-sectional study of 707 adults aged 25-75 years at a tertiary care hospital in Jamnagar, Gujarat, MetS was diagnosed using NCEP-ATP III criteria. Serum Lp(a), apolipoprotein A-I (Apo A-I), apolipoprotein B (Apo B), and CLTI were measured using standard methods. Associations were tested using Chi-square analyses and logistic regression, and diagnostic performance was assessed by receiver operating characteristic (ROC) curve analysis. Results MetS was present in 397/707 (56.15%) participants, occurring in 197/332 (59.34%) of females and 200/375 (53.33%) of males. Elevated Lp(a), elevated Apo B, elevated CLTI, and reduced Apo A-I were all significantly associated with MetS (p < 0.001 for each). ROC analysis demonstrated the highest diagnostic accuracy for CLTI (area under the curve, or AUC = 0.835, 95% CI 0.806-0.862), followed by Lp(a) (AUC = 0.760, 95% CI 0.730-0.794), Apo B (AUC = 0.700, 95% CI 0.665-0.734), and Apo A-I (AUC = 0.620, 95% CI 0.584-0.657). Multivariable logistic regression identified elevated blood pressure, low high-density lipoprotein cholesterol, and elevated triglycerides as significant predictors of abnormal biomarker levels. Conclusion In this Indian outpatient cohort, CLTI and Lp(a) showed strong predictive value for MetS, and outperformed Apo A-I and Apo B when used alone. Incorporating CLTI and Lp(a) into clinical assessment may improve early detection and risk stratification in individuals at risk of MetS. Show less

Current conventions, partly derived from self-reported data, typically equate 1 minute of vigorous physical activity (VPA) to 2 minutes of moderate physical activity (MPA). Using accelerometer-derived intensity classification in 73,485 UK Biobank participants (mean follow-up: 8.0 [1.0] years), we assess the equivalence of light activity (LPA) and MPA to 1 minute of VPA for all-cause (ACM) and cardiovascular (CVD) mortality, major adverse cardiovascular events (MACE), type 2 diabetes, and cancer outcomes. For a standardised 5%-35% risk reduction, the median MPA equivalent per minute of VPA is 4.1 (ACM, 95% CI: 4.1-4.2), 7.8 (CVD mortality, 7.7-8.0), 5.4 (MACE, 5.3-5.5), 9.4 (type 2 diabetes, 9.3-9.6), and 3.5 (cancer mortality, 3.4-3.5) minutes. For non-cancer outcomes, the median LPA equivalent per 1 minute of VPA ranges from 53 (ACM) to 94 minutes (type 2 diabetes), reflecting generally weaker dose-response curves of LPA with all outcomes. These findings indicate a substantial departure from self-reported estimates and support integrating device-based equivalence into guidelines and wearables. Show less

The stem cell microenvironment has been evidenced to robustly affect its biological functions and clinical grade. Natural or synthetic growth factors, especially, are essential for modulating stem cell proliferation, metabolism, and differentiation via the interaction with specific extracellular receptors. Fibroblast growth factor-2 (FGF-2) possesses pleiotropic functions in various tissues and organs. It interacts with the FGF receptor (FGFR) and activates FGFR signaling pathways, which involve numerous biological functions, such as angiogenesis, wound healing, cell proliferation, and differentiation. Here, we aim to explore the molecular functions, mode of action, and therapeutic activity of yet undetermined function, FGF-2-derived peptide, FP2 (44-ERGVVSIKGV-53) in promoting the proliferation, differentiation, and therapeutic application of human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) in comparison to other test peptides, canofin1 (FP1), hexafin2 (FP3), and canofin3 (FP4) with known functions. The immobilization of test peptides that are fused with mussel adhesive proteins (MAP) on the culture plate was carried out via EDC/NHS chemistry. Cell Proliferation assay, colony-forming unit, western blotting analysis, gene expression analysis, RNA-Seq. analysis, osteogenic, and chondrogenic differentiation capacity were applied to test the activity of the test peptides. We additionally utilized three-dimensional (3D) structural analysis and artificial intelligence (AI)-based AlphaFold2 and CABS-dock programs for receptor interaction prediction of the peptide receptor. We also verified the in vivo therapeutic capacity of FP2-cultured hWJ-MSCs using an osteoarthritis mice model. Culture of hWJ-MSC onto an FP2-immobilized culture plate showed a significant increase in cell proliferation (n = 3; *p < 0.05, **p < 0.01) and the colony-forming unit (n = 3; *p < 0.05, **p < 0.01) compared with the test peptides. FP2 showed a significantly upregulated phosphorylation of FRS2α and FGFR1 and activated the AKT and ERK signaling pathways (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Interestingly, we detected efficient FP2 receptor binding that was predicted using AI-based tools. Treatment with an AKT inhibitor significantly abrogated the FP2-mediated enhancement of cell differentiation (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Intra-articular injection of FP2-cultured MSCs significantly mitigated arthritis symptoms in an osteoarthritis mouse model, as shown through the functional tests (n = 10; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), modulation of the expression level of the pro-inflammatory and anti-inflammatory genes, and improved osteochondral regeneration as demonstrated by tissue sections. Our study identified the FGF-2-derived peptide FP2 as a promising candidate peptide to improve the therapeutic potential of hWJ-MSCs, especially in bone and cartilage regeneration. Show less

Primary cilia (PC) are cellular organelles that regulate the cellular homeostasis. They are the seats of many oncogenic pathways and indirectly regulate the epithelial-mesenchymal transition (EMT) and extracellular matrix, both critical for the tumor microenvironment (TME). Though there are a few studies highlighting the alteration of PC in the tumor cells of various malignancies, none depict the PC in the stromal cells in the urothelial carcinoma of the urinary bladder (UC), the stromal cells being an essential component of TME. Therefore, we intend to evaluate the PC in the stromal cells at the tumor-stromal interface in UC. Immunohistochemistry for acetylated-α-tubulin (for PC), Ki67, E-cadherin, and SNAI1 was performed in 141 cases of UC and 5 normal controls, and primary cilium: nucleus (C:N) ratio was counted in the stromal cells at the tumor-stromal interface. The C:N ratio was correlated with various clinical and histopathological parameters. The C:N ratio showed significant diminution from normal control (mean=0.75) to low-grade UC (mean=0.24) ( P =0.001) to high-grade UC (mean value=0.17) ( P =0.001). There was a significant diminution of the C:N ratio from the noninvasive to invasive UC ( P =0.025). The C:N ratio did not show any correlation with EMT although negatively correlated with the Ki67 index ( r =-0.32; P =0.001), and a higher ratio showed a trend with a higher recurrence-free survival ( P =0.07). The diminution of the PC in the stromal cells at the tumor-stromal interface is an early event and correlates with an aggressive tumor biology of UC. Show less

Many drugs are being administered to tackle coronavirus disease 2019 (COVID-19) pandemic situations without establishing clinical effectiveness or tailoring safety. A repurposing strategy might be more effective and successful if pharmacogenetic interventions are being considered in future clinical studies/trials. Although it is very unlikely that there are almost no pharmacogenetic data for COVID-19 drugs, however, from inferring the pharmacokinetic (PK)/pharmacodynamic(PD) properties and some pharmacogenetic evidence in other diseases/clinical conditions, it is highly likely that pharmacogenetic associations are also feasible in at least some COVID-19 drugs. We strongly mandate to undertake a pharmacogenetic assessment for at least these drug-gene pairs (atazanavir- Show less

Soleris® Direct Yeast and Mold (DYM) is a growth-based, automated method for detection of yeast and mold in select foods and other sample types including nutraceuticals and cosmetics. The Soleris method is used in a "dilute-to-specification" or threshold manner in which a result is scored as positive or negative around a predetermined cutoff (in CFU/g) established by the dilution and volume of sample homogenate tested. The objective of this study was to validate the method for testing of dried cannabis flower. The validation was conducted under the Emergency Response Validation program of the AOAC Research Institute. The study included inclusivity and exclusivity testing, in particular testing of yeast and mold species associated with cannabis, and a matrix study in which Soleris method presumptive results were compared with Soleris confirmed results using Dichloran Rose-Bengal Chloramphenicol (DRBC) agar for confirmation. Samples at four different levels of natural yeast and mold contamination were tested at two test thresholds. In inclusivity testing, all 63 yeast and mold strains tested produced positive results within the specified test duration of 72 h. In exclusivity testing, 36 of 37 strains tested produced no detection within 72 h. In matrix testing, there were no significant differences between Soleris presumptive and confirmed results for any contamination level or test threshold as determined by probability of detection analysis. Results indicate that the Soleris method is an effective procedure for detection of yeast and mold in dried cannabis flower. With the Soleris method, results are available within 72 h compared with the 5-7 days required for microbiological culture methods. Show less

Parkinson's disease (PD) is a genetically heterogeneous neurodegenerative disease with poorly defined environmental influences. Genomic studies of PD patients have identified disease-relevant monogenic genes, rare variants of significance, and polygenic risk-associated variants. In this study, whole genome sequencing data from 90 young onset Parkinson's disease (YOPD) individuals are analyzed for both monogenic and polygenic risk. The genetic variant analysis identifies pathogenic/likely pathogenic variants in eight of the 90 individuals (8.8%). It includes large homozygous coding exon deletions in PRKN and SNV/InDels in VPS13C, PLA2G6, PINK1, SYNJ1, and GCH1. Eleven rare heterozygous GBA coding variants are also identified in 13 (14.4%) individuals. In 34 (56.6%) individuals, one or more variants of uncertain significance (VUS) in PD/PD-relevant genes are observed. Though YOPD patients with a prioritized pathogenic variant show a low polygenic risk score (PRS), patients with prioritized VUS or no significant rare variants show an increased PRS odds ratio for PD. This study suggests that both significant rare variants and polygenic risk from common variants together may contribute to the genesis of PD. Further validation using a larger cohort of patients will confirm the interplay between monogenic and polygenic variants and their use in routine genetic PD diagnosis and risk assessment. Show less

Triple-negative breast cancers (TNBCs) are characterized by poor survival, prognosis, and gradual resistance to cytotoxic chemotherapeutics, like doxorubicin (DOX). The clinical utility of DOX is limited by its cardiotoxic and chemoresistant effects that manifest over time. To induce chemoresistance, TNBC rewires oncogenic gene expression and cell signaling pathways. Recent studies have demonstrated that reprogramming of branched-chain amino acids (BCAAs) metabolism facilitates tumor growth and survival. Branched-chain ketoacid dehydrogenase kinase (BCKDK), a regulatory kinase of the rate-limiting enzyme of the BCAA catabolic pathway, is reported to activate RAS/RAF/MEK/ERK signaling to promote tumor cell proliferation. However, it remains unexplored if BCKDK action remodels TNBC proliferation and survival per se and influences susceptibility to DOX-induced genotoxic stress. TNBC cells treated with DOX exhibited reduced BCKDK expression and intracellular BCKAs. Genetic and pharmacological inhibition of BCKDK in TNBC cell lines also showed a similar reduction in intracellular and secreted BCKAs. BCKDK silencing in TNBC cells downregulated mitochondrial metabolism genes, reduced electron complex protein expression, oxygen consumption, and ATP production. Transcriptome analysis of BCKDK silenced cells confirmed dysregulation of mitochondrial metabolic networks and upregulation of the apoptotic signaling pathway. Furthermore, BCKDK inhibition with concurrent DOX treatment exacerbated apoptosis, caspase activity, and loss of TNBC proliferation. Inhibition of BCKDK in TNBC also upregulated sestrin 2 and concurrently decreased mTORC1 signaling and protein synthesis. Overall, loss of BCKDK action in TNBC remodels BCAA flux, reduces protein translation triggering cell death, ATP insufficiency, and susceptibility to genotoxic stress. Show less

Protocadherin-19 belongs to the cadherin family of cell surface receptors and has been shown to play essential roles in the development of the vertebrate nervous system. Mutations in human Protocadherin-19 (PCDH19) lead to PCDH19 Female-limited epilepsy (PCDH19 FLE) in humans, characterized by the early onset of epileptic seizures in children and a range of cognitive and behavioral problems in adults. Despite being considered the second most prevalent gene in epilepsy, very little is known about the intercellular pathways in which it participates. In order to characterize the protein complexes within which Pcdh19 functions, we generated Pcdh19-BioID fusion proteins and utilized proximity-dependent biotinylation to identify neighboring proteins. Proteomic identification and analysis revealed that the Pcdh19 interactome is enriched in proteins that regulate Rho family GTPases, microtubule binding proteins and proteins that regulate cell divisions. We cloned the centrosomal protein Nedd1 and the RacGEF Dock7 and verified their interactions with Pcdh19 in vitro. Our findings provide the first comprehensive insights into the interactome of Pcdh19, and provide a platform for future investigations into the cellular and molecular biology of this protein critical to the proper development of the nervous system. Show less

Dissecting the genetic mechanism underlying a complex disease hinges on discovering gene-environment interactions (GXE). However, detecting GXE is a challenging problem especially when the genetic variants under study are rare. Haplotype-based tests have several advantages over the so-called collapsing tests for detecting rare variants as highlighted in recent literature. Thus, it is of practical interest to compare haplotype-based tests for detecting GXE including the recent ones developed specifically for rare haplotypes. We compare the following methods: haplo.glm, hapassoc, HapReg, Bayesian hierarchical generalized linear model (BhGLM) and logistic Bayesian LASSO (LBL). We simulate data under different types of association scenarios and levels of gene-environment dependence. We find that when the type I error rates are controlled to be the same for all methods, LBL is the most powerful method for detecting GXE. We applied the methods to a lung cancer data set, in particular, in region 15q25.1 as it has been suggested in the literature that it interacts with smoking to affect the lung cancer susceptibility and that it is associated with smoking behavior. LBL and BhGLM were able to detect a rare haplotype-smoking interaction in this region. We also analyzed the sequence data from the Dallas Heart Study, a population-based multi-ethnic study. Specifically, we considered haplotype blocks in the gene ANGPTL4 for association with trait serum triglyceride and used ethnicity as a covariate. Only LBL found interactions of haplotypes with race (Hispanic). Thus, in general, LBL seems to be the best method for detecting GXE among the ones we studied here. Nonetheless, it requires the most computation time. Show less

Branched-chain α-keto acids (BCKAs) are catabolites of branched-chain amino acids (BCAAs). Intracellular BCKAs are cleared by branched-chain ketoacid dehydrogenase (BCKDH), which is sensitive to inhibitory phosphorylation by BCKD kinase (BCKDK). Accumulation of BCKAs is an indicator of defective BCAA catabolism and has been correlated with glucose intolerance and cardiac dysfunction. However, it is unclear whether BCKAs directly alter insulin signaling and function in the skeletal and cardiac muscle cell. Furthermore, the role of excess fatty acids (FAs) in perturbing BCAA catabolism and BCKA availability merits investigation. By using immunoblotting and ultra-performance liquid chromatography MS/MS to analyze the hearts of fasted mice, we observed decreased BCAA-catabolizing enzyme expression and increased circulating BCKAs, but not BCAAs. In mice subjected to diet-induced obesity (DIO), we observed similar increases in circulating BCKAs with concomitant changes in BCAA-catabolizing enzyme expression only in the skeletal muscle. Effects of DIO were recapitulated by simulating lipotoxicity in skeletal muscle cells treated with saturated FA, palmitate. Exposure of muscle cells to high concentrations of BCKAs resulted in inhibition of insulin-induced AKT phosphorylation, decreased glucose uptake, and mitochondrial oxygen consumption. Altering intracellular clearance of BCKAs by genetic modulation of BCKDK and BCKDHA expression showed similar effects on AKT phosphorylation. BCKAs increased protein translation and mTORC1 activation. Pretreating cells with mTORC1 inhibitor rapamycin restored BCKA's effect on insulin-induced AKT phosphorylation. This study provides evidence for FA-mediated regulation of BCAA-catabolizing enzymes and BCKA content and highlights the biological role of BCKAs in regulating muscle insulin signaling and function. Show less

Neuronal ceroid lipofuscinoses are a group of neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigments in neuronal cells. As a result of storage material in the brain and retina, clinical manifestations include speech delay, cognitive dysfunction, motor regression, epilepsy, vision loss, and early death. At present, 14 different ceroid lipofuscinosis (CLN) genes are known. Recently, the FDA approved the use of recombinant human proenzyme of tripeptidyl-peptidase 1 for CLN2 disease, while phase I/IIa clinical trials for gene therapy in CLN3 and CLN6 are ongoing. Early diagnosis is, therefore, key to initiating treatment and arresting disease progression. Neuroimaging features of CLN1, CLN2, CLN3, and CLN5 diseases are well-described, with sparse literature on other subtypes. We aimed to investigate and expand the MR imaging features of genetically proved neuronal ceroid lipofuscinoses subtypes at our institution and also to report the time interval between the age of disease onset and the diagnosis of neuronal ceroid lipofuscinoses. We investigated and analyzed the age of disease onset and neuroimaging findings (signal intensity in periventricular, deep, and subcortical white matter, thalami, basal ganglia, posterior limb of the internal capsule, insular/subinsular regions, and ventral pons; and the presence or absence of supratentorial and/or infratentorial atrophy) of patients with genetically proved neuronal ceroid lipofuscinoses at our institution. This group consisted of 24 patients who underwent 40 brain MR imaging investigations between 1993 and 2019, with a male preponderance (male/female ratio = 15:9). The mean ages of disease onset, first brain MR imaging, and diagnosis of neuronal ceroid lipofuscinoses were 4.70 ± 3.48 years, 6.76 ± 4.49 years, and 7.27 ± 4.78 years, respectively. Findings on initial brain MR imaging included T2/FLAIR hypointensity in the thalami ( We identified reported classic neuroimaging features in all except 1 patient with neuronal ceroid lipofuscinoses in our study. CLN2, CLN5, and CLN7 diseases showed predominant cerebellar-over-cerebral atrophy. We demonstrate that abnormal signal intensity in the deep white matter, posterior limb of the internal capsule, and ventral pons is more common than previously reported in the literature. We report abnormal signal intensity in the insular/subinsular region for the first time. The difference in the median time from disease onset and diagnosis was 1.5 years. Show less

Recognition of the non-self signature of invading pathogens is a crucial step for the initiation of the innate immune mechanisms of the host. The host response to viral and bacterial infection involves sets of pattern recognition receptors (PRRs), which bind evolutionarily conserved pathogen structures, known as pathogen-associated molecular patterns (PAMPs). Recent advances in the identification of different types of PRRs in teleost fish revealed a number of cytosolic sensors for recognition of viral and bacterial nucleic acids. These are DExD/H-box RNA helicases including a group of well-characterized retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and non-RLR DExD/H-box RNA helicases (e.g., DDX1, DDX3, DHX9, DDX21, DHX36 and DDX41) both involved in recognition of viral RNAs. Another group of PRRs includes cytosolic DNA sensors (CDSs), such as cGAS and LSm14A involved in recognition of viral and intracellular bacterial dsDNAs. Moreover, dsRNA-sensing protein kinase R (PKR), which has a role in antiviral immune responses in higher vertebrates, has been identified in fish. Additionally, fish possess a novel PKR-like protein kinase containing Z-DNA binding domain, known as PKZ. Here, we review the current knowledge concerning cytosolic sensors for recognition of viral and bacterial nucleic acids in teleosts. Show less

To identify the molecular basis of inherited retinal degeneration (IRD) in a familial case of Pakistani origin using whole-exome sequencing. A thorough ophthalmic examination was completed, and genomic DNA was extracted using standard protocols. Whole exome(s) were captured with Agilent V5 + UTRs probes and sequenced on Illumina HiSeq genome analyzer. The exomeSuite software was used to filter variants, and the candidate causal variants were prioritized, examining their allele frequency and PolyPhen2, SIFT, and MutationTaster predictions. Sanger dideoxy sequencing was performed to confirm the segregation with disease phenotype and absence in ethnicity-matched control chromosomes. Ophthalmic examination confirmed retinal degeneration in all affected individuals that segregated as an autosomal recessive trait in the family. Whole-exome sequencing identified two homozygous missense variants: c.1304G > A; p.Arg435Gln in ZNF408 (NM₀₂₄₇₄₁₎ and c.902G > A; p.Gly301Asp in C1QTNF4 (NM₀₃₁₉₀₉₎. Both variants segregated with the retinal phenotype in the PKRD320 and were absent in ethnically matched control chromosomes. Whole-exome sequencing coupled with bioinformatics analysis identified potential novel variants that might be responsible for IRD. Show less

The chronic inflammatory nature of chronic rhinosinusitis (CRS) makes it a morbid condition for individuals with the disease and one whose pathogenesis is poorly understood. To date, proteomic approaches have been applied successfully in a handful of CRS studies. In this study we use a multifaceted approach, including proteomics (iTRAQ labeling) and microbiome (bacterial 16S rRNA gene sequencing) analyses of middle meatus swabs, as well as immune cell analysis of the underlying tissue, to investigate the host-microbe interaction in individuals with CRS ( Show less

To establish the key insulin receptor substrate 1 (IRS-1) structural elements required in this insulin regulatory pathway, we investigated the effects of substituting alanine for serine 307 in IRS-1 on the ability of tumor necrosis factor-α (TNF-α) and a related mediator, suppressor of cytokine signaling 3 (SOCS3), to phosphorylate IRS-1 and regulate insulin signaling in the rat retinal Müller cell (rMC-1) cell line. rMC-1 cells were grown in normal (5 mM) or high (25 mM) glucose medium and transfected with either normal IRS-1(Ser307)plasmid or a mutated IRS-1(Ser307Ala) plasmid. Cells were also treated with recombinant TNF-α or SOCS3 to induce increased levels of these proteins. In cells with IRS-1(Ser307Ala), TNF-α and SOCS3 failed to phosphorylate IRS-1. Likewise, resulting downstream effects, including changes in phosphorylation of insulin receptor(Tyr960), antiapoptotic Akt phosphorylation, and proapoptotic cleavage of caspase 3 were also blocked. We also report for the first time that SOCS3 and TNF-α are reciprocally stimulatory leading to a mutual enhancement of levels of both factors, thus forming a potential positive feedback loop that contributes to insulin receptor resistance. Increases in TNF-α and SOCS3 are triggered by high glucose and through reciprocal stimulation of expression of these two factors, which in turn could be major drivers of insulin resistance and related cell death. The demonstration that a single phosphorylation site is key for these pathways suggests that drugs targeted to this site might be effective in protecting against diabetic damage to the retina. Show less

Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development. Show less

Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6(nclf/nclf) cerebellar cells and compared them to wild-type and CbCln3(Δex7/8/Δex7/8) cerebellar cells. CbCln6(nclf/nclf) cells and CbCln3(Δex7/8/Δex7/8) cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6(nclf/nclf) cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6(nclf/nclf) and CbCln3(Δex7/8/Δex7/8) cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3(Δex7/8) and Cln6(nclf) mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival. Show less