10-(6-Plastoquinonyl) decyltriphenylphosphonium imparts anti-colitogenic protection through recovery of mitochondrial dysfunction in ulcerated murine colon: Implications in ulcerative colitis.

AIMS: To elucidate the impact of 10-(6-plastoquinonyl) decyltriphenylphosphonium (SkQ1) as an anti-colitogenic agent for maintenance of colon epithelial tract in ulcerated mice through recovery of mitochondrial dysfunction and mitochondrial stress by virtue of its free radical scavenging properties. MAIN METHODS: DSS induced ulcerated BALB/c mice were treated with SkQ1 for 14 days @ 30 nmol/kg/body wt./day/mice. Post-treatment, isolated colonic mitochondria were utilized for spectrophotometric and spectrofluorometric biochemical analysis of various mitochondrial functional variables including individual mitochondrial respiratory enzyme complexes. Confocal microscopy was utilized for measuring mitochondrial membrane potential in vivo. ELISA technique was adapted for measuring colonic nitrite and 3-nitrotyrosine (3-NT) content. Finally in vitro cell line study was carried out to substantiate in vivo findings and elucidate the involvement of free radicals in UC using antioxidant/free radical scavenging regimen. KEY FINDINGS: Treatment with SkQ1 in vivo reduced histopathological severity of colitis, induced recovery of mitochondrial respiratory complex activities and associated functional variables, improved oxidative stress indices and normalized mitochondrial cardiolipin content. Importantly, SkQ1 lowered nitrite concentration and 3-nitrotyrosine formation in vivo. In vitro SkQ1 restored mitochondrial functions wherein the efficacy of SkQ1 proved equal or better compared to SOD and DMSO indicating predominant involvement of O2- and OH in UC. However, NO and ONOO- also seemed to play a secondary role as MEG and L-NAME provided lesser protection as compared to SOD and DMSO. SIGNIFICANCE: SkQ1 can be considered as a potent anti-colitogenic agent by virtue of its free radical scavenging properties in treating UC.

Haematological, biochemical, enzymological changes and mitochondrial dysfunction of liver in freshwater climbing perch Anabas testudineus during their acute and chronic exposure to sodium fluoride.

Anthropogenic activities are increasing fluoride concentration in watercourses. The present study focuses on the sublethal toxicity of sodium fluoride during sub-chronic and chronic time periods in the freshwater fish Anabas testudineus. The 96-hour LC50 value for fluoride was found to be 616.50 mg/L. Excessive mucous production and hyper excitability, followed by loss of balance, were seen in fish under acute fluoride exposure. Significant reduction in yield and specific growth rate of fish were assessed at 15, 30 and 45-days exposure intervals. Different bio-indicators like Hepatosomatic-index, Gonadosomatic-index and fecundity were reduced significantly in fish exposed to 10% (61.6 mg/L) and 20% (123.2 mg/L) of 96 h of LC50 values of fluoride in comparison to control. Toxicant concentrations directly correlated with parameter lowering. Fluoride exposure increased plasma glucose, creatinine, AST, and ALT and reduced total RBC, haemoglobin content, Hct (%), plasma protein, and cholesterol. Moreover, fluoride exposure significantly reduces the mitochondrial membrane potential in liver. This may result in metabolic depression, haematological, biochemical, and enzymological stress. The in-silico structural analysis predicts that fluoride may impede cytochrome c oxidase of the electron transport system, hence inhibiting mitochondrial functionality. These findings collectively highlight the urgent need for stringent regulation and monitoring of fluoride levels in freshwater ecosystems, as the subchronic and chronic effects observed in A. testudineus may have broader implications for aquatic ecosystems.

Polymorphism in ADAM33 gene associated with asthmatics in West Bengal, India - An investigation by in-silico analysis.

Introduction: Asthma is one of the common chronic polygenic inflammatory diseases. Genome wide association studies have identified ADAM33 as an asthma candidate gene. The present study investigated possible association of rs2280090 (T1), rs2280091 (T2) and rs3918396 (S1) single nucleotide polymorphisms (SNPs) of ADAM33 with aeroallergen induced asthma in West Bengal population, India. In addition, in-silico analysis was performed to find out changes in protein function. Methods: Forced expiratory volume in 1 second (FEV1)/Forced vital capacity (FVC), peak expiratory flow rate (PEFR) were assessed using spirometry in 1039 participants. Allergic sensitivity of 619 spirometry positive asthma patients was assessed by skin prick test (SPT) against 22 aeroallergens. For genotyping of T1, T2, and S1 SNPs in 540 allergic asthma patient and 420 control subjects, polymerase chain reaction-based restriction fragment length polymorphism was performed. Total Immunoglobulin-E (IgE) level was measured in both patients and controls. ADAM333 haplotype blocks were constructed using Haploview software v.4.2. Structural model of transmembrane and cytoplasmic domains of ADAM33 was generated using RaptorX. Protein-protein interaction was analysed using the STRING server. Results: Highest number of patient sensitivity was observed towards Cocos nusifera (n = 215) and Dermatophagoides farinae (n = 229). Significant difference in sensitivity was observed between child and late adult (P = 0.03), child and early adult (P = 0.02), adolescent and late adult (P = 0.02) and adolescent and early adult (P = 0.01). Genotypic frequencies differed significantly between patients and controls (P < 0.05). rs2280090 GG, rs2280091GG and AG genotype, and rs3918396 AA carried significant risk for asthma (P = 0.02, P = 0.008, P = 0.04, P = 0.01 respectively). ADAM33 T1, T2, and S1 polymorphisms were in high Linkage Disequilibrium (D = 0.98). Haplotype consisting of rs2280090G, rs2280091G and rs3918396A alleles were found significantly higher in patient population in comparison with controls (OR = 2.03). IgE level differed significantly among different genotypes for T1, T2, and S1 SNPs analysed in pair (P < 0.0001). FEV1/FVC ratio differed significantly among different genotypes for T1, T2 and S1 SNPs analysed in pair (P < 0.0001). Significant difference of FEV1/FVC was also found between GGA and AAG haplotype (P < 0.0001). In-silico analysis revealed T1 and T2 polymorphisms are located in cytoplasmic domain of ADAM33 may cause bronchial smooth muscle cell mobility and cellular hyperplasia as well as cytoskeletal remodelling by altered interaction with different cytoplasmic proteins found by string analysis. Conclusion: Present study showed significant association of T1, T2, and S1 polymorphisms of ADAM33 with aeroallergen-induced asthma in West Bengal, India. These polymorphisms may be used as prognostic markers and possible targets for therapeutics in future.

Triazophos-induced Respiratory and Behavioral Effects and Development of Adverse Outcome Pathway (AOP) for short-term Exposed Freshwater Snail, Bellamya Bengalensis.

The physiological effects of triazophos were examined using respiratory and behavioral endpoints in Bellamya bengalensis under a 96-hour acute exposure regime. Physiological manifestation of respiratory stress was measured using the rate of oxygen consumption while behavioral toxicity was measured using crawling reflexes, touch response, and mucus production. The threshold effect values for LOEC (Lowest Observed Effect Concentration), NOEC (No Observed Effect Concentration), and MATC (Maximum Acceptable Toxicant Concentration) at 96 h were 0.40, 0.60, and 0.075 mg/l, respectively. Definitive 96 h acute exposures for both respiratory and behavioral endpoints tests were determined using a control group and concentrations ranging from 0.40 to 1.60 mg/l monitored for 24, 48, 72, and 96 h. Test organisms irrespective of exposure concentration demonstrated an initial rise in oxygen consumption rate after 24 h, followed by a progressive decrease in toxicant concentration and exposure period. The in silico structural analysis presents triazophos as having an electrophilic toxic structure similar to choline esterase inhibitors, and also capable of inducing oxidative stress. The AOP highlighted neurotoxicity and oxidative stress as plausible pathways of triazophos toxicity in mollusk species.

In silico analysis of single nucleotide polymorphism (rs34377097) of TBXA2R gene and pollen induced bronchial asthma susceptibility in West Bengal population, India.

Introduction: Prevalence of asthma is increasing steadily among general population in developing countries over past two decades. One of the causative agents of broncho-constriction in asthma is thromboxane A2 receptor (TBXA2R). However few studies of TBXA2R polymorphism were performed so far. The present study aimed to assess potential association of TBXA2R rs34377097 polymorphism causing missense substitution of Arginine to Leucine (R60L) among 482 patients diagnosed with pollen-induced asthma and 122 control participants from West Bengal, India. Also we performed in-silico analysis of mutated TBXA2R protein (R60L) using homology modeling. Methods: Clinical parameters like Forced expiratory volume in 1 second (FEV1), FEV1/Forced vital capacity (FVC) and Peak expiratory flow rate (PEFR) were assessed using spirometry. Patients' sensitivity was measured by skin prick test (SPT) against 16 pollen allergens. Polymerase chain reaction-based Restriction fragment length polymorphism was done for genotyping. Structural model of wild type and homology model of polymorphic TBXA2R was generated using AlphaFold2 and MODELLER respectively. Electrostatic surface potential was calculated using APBS plugin in PyMol. Results: Genotype frequencies differed significantly between the study groups (P=0.03). There was no significant deviation from Hardy-Weinberg equilibrium in control population (χ2=1.56). Asthmatic patients have significantly higher frequency of rs34377097TT genotype than control subjects (P=0.03). SPT of patients showed maximum sensitivity in A. indica (87.68%) followed by C. nusifera (83.29%) and C. pulcherima (74.94%). Significant difference existed for pollen sensitivity in adolescent and young adult (P=0.01) and between young and old adult (P=0.0003). Significant negative correlation was found between FEV1/FVC ratio and intensity of SPT reactions (P<0.0001). Significant association of FEV1, FEV1/FVC and PEFR was observed with pollen-induced asthma. Furthermore, risk allele T was found to be clinically correlated with lower FEV1/FVC ratio (P=0.015) in patients. Our data showed R60L polymorphism, which was conserved across mammals, significantly reduced positive electrostatic charge of polymorphic protein in cytoplasmic domain thus altered downstream pathway and induced asthma response. Discussion: The present in-silico study is the first one to report association of TBXA2R rs34377097 polymorphism in an Indian population. It may be used as prognostic marker of clinical response to asthma in West Bengal and possible target of therapeutics in future.

Behavioral toxicity, histopathological alterations and oxidative stress in Tubifex tubifex exposed to aromatic carboxylic acids- acetic acid and benzoic acid: A comparative time-dependent toxicity assessment.

This study evaluated Acetic acid (AA) and Benzoic acid's (BA) acute and sublethal toxicity by observing mortality, behavioral responses, and changes in the levels of oxidative stress enzymes in Tubifex tubifex. Exposure-induced changes in antioxidant activity (Catalase, Superoxide dismutase), oxidative stress (Malondialdehyde concentrations), and histopathological alterations in the tubificid worms were also noted across exposure intervals. The 96 h LC50 values of AA and BA to T. tubifex were 74.99 and 37.15 mg/l, respectively. Severity in behavioral alterations (including increased mucus production, wrinkling, and reduction in clumping) and autotomy showed concentration-dependent trends for both toxicants. Although histopathological effects also showed marked degeneration in the alimentary and integumentary systems in highest exposure groups (worms exposed to 14.99 mg/l for AA and 7.42 mg/l for BA) for both toxicants. Antioxidant enzymes (catalase and superoxide dismutase) also showed a marked increase of up to 8-fold and 10-fold for the highest exposure group of AA and BA respectively. While species sensitivity distribution analysis revealed T. tubifex as most sensitive to AA and BA compared to other freshwater vertebrates and invertebrates, General Unified Threshold model of Survival (GUTS) predicted individual tolerance effects (GUTS-IT), with slower potential for toxicodynamic recovery, as a more likely pathway for population mortality. Study findings demonstrate BA with greater potential for ecological effects compared to AA within 24 h of exposure. Furthermore, ecological risks to critical detritus feeders like T. tubifex may have severe implications for ecosystem services and nutrient availability within freshwater habitats.

Deltamethrin-Induced Respiratory and Behavioral Effects and Adverse Outcome Pathways (AOP) in Short-Term Exposed Mozambique Tilapia, Oreochromis mossambicus.

Disrupted behavior and respiratory distress effects of 96-h acute deltamethrin exposures in adult Mozambique tilapia, Oreochromis mossambicus, were investigated using behavioral indices and opercular movement, respectively. Deltamethrin concentrations were found to be associated with toxicological (lethal and sublethal) responses. At 24, 48, 72, and 96 h, the LC50 values and 95% confidence limits were 12.290 (11.174-14.411 µg/L), 12.671 (11.334-15.649 µg/L), 10.172 (9.310-11.193 µg/L), and 8.639 (7.860-9.417 µg/L), respectively. The GUTS-model analysis showed that GUTS-SD (stochastic death) with a narrow tolerance distribution in deltamethrin exposed O. mossambicus populations was more sensitive than the GUTS-IT (individual tolerance) model. Prior to death, exposed fish demonstrated concentration-dependent mortality and disturbed behavioral responses, including uncoordinated swim motions, increased mucus secretion, unbalanced and unpredictable swimming patterns, and inactivity. The altered behavioral patterns and increased opercular movement with increased deltamethrin levels and exposure time are strongly suggestive of neurotoxicity and respiratory distress, respectively. Adverse Outcome Pathways (AOPs), describing biological mechanisms and plausible pathways, highlighted oxidative stress and cholinergic effects as intermediate steps linked to respiratory distress and behavioral toxicity.

Mechanistic insights to lactic and formic acid toxicity on benthic oligochaete worm Tubifex tubifex.

Lactic and formic acid are two commonly found monocarboxylic organic acids. Lactic acid is discharged into the water bodies as acidic industrial effluent from the food, cosmetic, chemical, and pharmaceutical industries, whereas formic acid is discharged from various paper, leather tanning, and textile processing industries. The present study investigated the toxicity of both organic acids upon the benthic oligochaete worm Tubifex tubifex. The 96-h median lethal concentration (LC50) values for lactic and formic acid are determined as 143.81 mg/l and 57.99 mg/l respectively. The effects of two sublethal concentrations (10% and 30% of 96 h LC50) of these acids on differential expression of oxidative stress enzymes are investigated. The comparative analysis of acute toxicity demonstrates that formic acid exposure is more detrimental to T. tubifex than lactic acid. The in silico structural analysis predicts that formic acid can interact with cytochrome c oxidase of the electron transport system and thereby inhibits its functionality and induces reactive oxygen species production. Integrated biomarker response (IBR) analysis illustrates that overall oxidative stress of formic acid to T. tubifex is significantly higher than that of lactic acid, which supports the structural analysis. It is concluded from this study that toxicokinetic-toxicodynamic and species sensitivity distributions studies are helpful for ecological risk management of environmental toxicants.

Contributions and Limitations of Mitochondria-Targeted and Non-Targeted Antioxidants in the Treatment of Parkinsonism: an Updated Review.

As conventional therapeutics can only treat the symptoms of Parkinson's disease (PD), major focus of research in recent times is to slow down or prevent the progression of neuronal degeneration in PD. Non-targeted antioxidants have been an integral part of the conventional therapeutics regimen; however, their importance have lessened over time because of their controversial outcomes in clinical PD trials. Inability to permeate and localize within the mitochondria remains the main drawback on the part of non-targeted antioxidants inspite of possessing free radical scavenging properties. In contrast, mitochondrial-targeted antioxidants (MTAs), a special class of compounds have emerged having high advantages over non-targeted antioxidants by virtue of efficient pharmacokinetics and better absorption rate with capability to localize many fold inside the mitochondrial matrix. Preclinical experimentations indicate that MTAs have the potential to act as better alternatives compared to conventional non-targeted antioxidants in treating PD; however, sufficient clinical trials have not been conducted to investigate the efficacies of MTAs in treating PD. Controversial clinical outcomes on the part of non-targeted antioxidants and lack of clinical trials involving MTAs have made it difficult to go ahead with a direct comparison and in turn have slowed down the progress of development of safer and better alternate strategies in treating PD. This review provides an insight on the roles MTAs and non-targeted antioxidants have played in the treatment of PD till date in preclinical and clinical settings and discusses about the limitations of mitochondria-targeted and non-targeted antioxidants that can be resolved for developing effective strategies in treating Parkinsonism.

The antiactivator FleN uses an allosteric mechanism to regulate σ54-dependent expression of flagellar genes in Pseudomonas aeruginosa.

Diverse sigma factors associate with the RNA polymerase (RNAP) core enzyme to initiate transcription of specific target genes in bacteria. σ54-Mediated transcription uses AAA+ activators that utilize their ATPase activity for transcription initiation. FleQ is a σ54-dependent master transcriptional regulator of flagellar genes in Pseudomonas aeruginosa. The ATPase activity of FleQ is regulated via a P-loop ATPase, FleN, through protein-protein interaction. We report a high-resolution crystal structure of the AAA+ domain of FleQ in complex with antiactivator FleN. The data reveal that FleN allosterically prevents ATP binding to FleQ. Furthermore, FleN remodels the region of FleQ essential for engagement with σ54 for transcription initiation. Disruption of the conserved protein-protein interface, by mutation, shows motility and transcription defects in vivo and multiflagellate phenotype. Our study provides a detailed mechanism used by monoflagellate bacteria to fine-tune the expression of flagellar genes to form and maintain a single flagellum.

Molecular and structural facets of c-di-GMP signalling associated with biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen and is the primary cause of nosocomial infections. Biofilm formation by this organism results in chronic and hard to eradicate infections. The intracellular signalling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a secondary messenger in bacterial cells crucial for motile to sessile transition. The signalling pathway components encompass two classes of enzymes with antagonistic activities, the diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that regulate the cellular levels of c-di-GMP at distinct stages of biofilm initiation, maturation and dispersion. This review summarizes the structural analysis and functional studies of the DGCs and PDEs involved in biofilm regulation in P. aeruginosa. In addition, we also describe the effector proteins that sense the perturbations in c-di-GMP levels to elicit a functional output. Finally, we discuss possible mechanisms that allow the dynamic levels of c-di-GMP to regulate cognate cellular response. Uncovering the details of the regulation of the c-di-GMP signalling pathway is vital for understanding the behaviour of the pathogen and characterization of novel targets for anti-biofilm interventions.

Effector mining from the Erysiphe pisi haustorial transcriptome identifies novel candidates involved in pea powdery mildew pathogenesis.

Pea powdery mildew (PM) is an important fungal disease caused by an obligate biotroph, Erysiphe pisi (Ep), which significantly impacts pea production worldwide. The phytopathogen secretes a plethora of effectors, primarily through specialized infection structures termed haustoria, to establish a dynamic relationship with its host. To identify Ep effector candidates, a cDNA library of enriched haustoria from Ep-infected pea leaves was sequenced. The Ep transcriptome encodes 622 Ep candidate secreted proteins (CSPs), of which 167 were predicted to be candidate secreted effector proteins (CSEPs). Phylogenetic analysis indicates that Ep CSEPs are highly diverse, but, unlike cereal PM CSEPs, exhibit extensive sequence similarity with effectors from other PMs. Quantitative real-time PCR of a subset of EpCSEP/CSPs revealed that the majority are preferentially expressed in haustoria and exhibit infection stage-specific expression patterns. The functional roles of EpCSEP001, EpCSEP009 and EpCSP083 were probed by host-induced gene silencing (HIGS) via a double-stranded (ds) RNA-mediated RNAi approach. Foliar application of individual EpCSEP/CSP dsRNAs resulted in a marked reduction in PM disease symptoms. These findings were consistent with microscopic and molecular studies, suggesting that these Ep CSEP/CSPs play important roles in pea PM pathogenesis. Homology modelling revealed that EpCSEP001 and EpCSEP009 are analogous to fungal ribonucleases and belong to the RALPH family of effectors. This is the first study to identify and functionally validate candidate effectors from the agriculturally relevant pea PM, and highlights the utility of transcriptomics and HIGS to elucidate the key proteins associated with Ep pathogenesis.

Sensor I Regulated ATPase Activity of FleQ Is Essential for Motility to Biofilm Transition in Pseudomonas aeruginosa.

Members of the AAA+ (ATPase associated with various cellular activities) family of ATPases couple chemical energy derived from ATP hydrolysis for generation of mechanical force, resulting in conformational changes. The hydrolysis is brought about by highly conserved domains and motifs. The sensor I motif is critical for sensing and hydrolysis of the nucleotide. Pseudomonas aeruginosa FleQ is an ATPase that is a positive regulator of flagellar gene expression. We have determined the crystal structures of the ATPase domain of wild-type FleQ and sensor I mutants H287N and H287A in complex with ATPγS and Mg2+ to 2.4, 1.95, and 2.25 Šresolution, respectively. The structural data highlight the role of sensor I in regulating the ATPase activity. The in vitro and in vivo data demonstrate that the moderate ATPase activity of FleQ due to the presence of histidine in sensor I is essential for maintaining the monotrichous phenotype and for the rapid motility to biofilm transition.

Ancestral Variations of the PCDHG Gene Cluster Predispose to Dyslexia in a Multiplex Family.

Dyslexia is a heritable neurodevelopmental disorder characterized by difficulties in reading and writing. In this study, we describe the identification of a set of 17 polymorphisms located across 1.9Mb region on chromosome 5q31.3, encompassing genes of the PCDHG cluster, TAF7, PCDH1 and ARHGAP26, dominantly inherited with dyslexia in a multi-incident family. Strikingly, the non-risk form of seven variations of the PCDHG cluster, are preponderant in the human lineage, while risk alleles are ancestral and conserved across Neanderthals to non-human primates. Four of these seven ancestral variations (c.460A>C [p.Ile154Leu], c.541G>A [p.Ala181Thr], c.2036G>C [p.Arg679Pro] and c.2059A>G [p.Lys687Glu]) result in amino acid alterations. p.Ile154Leu and p.Ala181Thr are present at EC2: EC3 interacting interface of γA3-PCDH and γA4-PCDH respectively might affect trans-homophilic interaction and hence neuronal connectivity. p.Arg679Pro and p.Lys687Glu are present within the linker region connecting trans-membrane to extracellular domain. Sequence analysis indicated the importance of p.Ile154, p.Arg679 and p.Lys687 in maintaining class specificity. Thus the observed association of PCDHG genes encoding neural adhesion proteins reinforces the hypothesis of aberrant neuronal connectivity in the pathophysiology of dyslexia. Additionally, the striking conservation of the identified variants indicates a role of PCDHG in the evolution of highly specialized cognitive skills critical to reading.

ATP-Induced Structural Remodeling in the Antiactivator FleN Enables Formation of the Functional Dimeric Form.

FleN, a P loop ATPase is vital for maintaining a monotrichous phenotype in Pseudomonas aeruginosa. FleN exhibits antagonistic activity against FleQ, the master transcriptional regulator of flagellar genes. Crystal structures of FleN in the apo form (1.66 Å) and in complex with ß,γ-imidoadenosine 5'-triphosphate (1.55 Å) reveal that it undergoes drastic conformational changes on ATP binding to attain a structure capable of dimerization. Mutations of the residues that stabilize the binding of ATP were defective in their ability to dimerize and do not inhibit ATP hydrolysis by FleQ. Conversely, the catalytic mutant of FleN, was an efficient inhibitor. These observations posit that the dimer is the functional form of FleN and it is nucleotide binding and not hydrolysis by FleN that is necessary to exert an antagonistic effect against FleQ. Our study shows that ATP-induced dimerization may be a strategy to achieve reversible inhibition of FleQ to fine-tune the function of this activator to an optimal level.