Rare Antagonistic Leptin Variants and Severe, Early-Onset Obesity.

Hormone absence or inactivity is common in congenital disease, but hormone antagonism remains controversial. Here, we characterize two novel homozygous leptin variants that yielded antagonistic proteins in two unrelated children with intense hyperphagia, severe obesity, and high circulating levels of leptin. Both variants bind to the leptin receptor but trigger marginal, if any, signaling. In the presence of nonvariant leptin, the variants act as competitive antagonists. Thus, treatment with recombinant leptin was initiated at high doses, which were gradually lowered. Both patients eventually attained near-normal weight. Antidrug antibodies developed in the patients, although they had no apparent effect on efficacy. No severe adverse events were observed. (Funded by the German Research Foundation and others.).

Design, synthesis, biological evaluation and molecular docking studies of quinoline-anthranilic acid hybrids as potent anti-inflammatory drugs.

Despite the high global prevalence, rheumatoid arthritis lacks a satisfactory treatment. Hence, the present study is undertaken to design and synthesize novel anti-inflammatory compounds. For this, quinoline and anthranilic acid, two medicinally-privileged moieties, were linked by pharmacophore hybridization, and following their computational assessments, three hybrids 5a-c were synthesized in good over all yields. The in vitro and in vivo anti-inflammatory potential of these hybrids was determined by anti-denaturation and anti-proteinase, and carrageenan-induced paw edema models. The computational studies of these hybrids revealed their drug-likeness, optimum pharmacokinetics, and less toxicity. Moreover, they demonstrated high binding affinity (-9.4 to -10.6 kcal mol-1) and suitable binding interactions for TNF-α, FLAP, and COX-II. A three-step synthetic route resulted in the hybrids 5a-c with 83-86% yield of final step. At 50 µg mL-1, the antiprotease and anti-denaturation activity of compound 5b was significantly higher than 5a and 5c. Furthermore, 5b significantly reduced the edema in the right paw of the rats that received carrageenan. The results of this study indicate the medicinal worth of the novel hybrids in treating inflammatory disorders such as rheumatoid arthritis.

Unveiling the significance of foliar-applied silicon, selenium and phosphorus for the management and remediation of arsenic in two different rice genotypes.

Under paddy soil conditions, rice plants are vulnerable to arsenic (As) accumulation, thus causing potential threat to human health. Here we investigated the influence of foliar-applied phosphorus (P: 10 and 20 mg L-1), silicon (Si: 0.6 and 1.5 g L-1) and selenium (Se: 5 and 10 mg L-1) on As accumulation, morphological and physiological attributes of two contrasting rice genotypes (KSK-133 and Super Basmati) under As stress (25 mg kg-1 as arsenate). Silicon foliar dressing significantly (p < 0.05) reduced grain As uptake (up to 67%) and improved rice growth and chlorophyll content (28-66%) in both rice genotypes over their controls. Phosphorus foliar application resulted in a notable decrease (17%) in grain As uptake of coarse rice genotype (KSK-133), while it slightly increased grain As uptake in the fine one (Super Basmati; 6%) compared to controls. However, foliar-applied Se did not show significant effects on rice plants growth attributes and As uptake in both genotypes. Similarly, biochemical and enzymatic attributes (i.e., lipid peroxidation, electrolyte leakage, peroxidase and catalase) were improved with Si application in rice plants, except for P treatment that was only effective for coarse one. Foliar-applied Si also resulted in reduced cancer risk and hazard quotient (< 0.10) for both rice genotypes. This study advances our understanding on critical role of different foliar-applied nutrients and rice genotypes, which is imperative to develop effective As remediation and management strategies in coarse and fine rice genotypes and protect human health.

This study provided new insights on the significance of foliar-applied phosphorus, silicon and selenium for the management and remediation of arsenic in fine (Super Basmati) and coarse (KSK-133) rice genotypes. Foliar-applied silicon was the most promising strategy to mitigate arsenic uptake and minimizing health risk in rice grain of both genotypes, while phosphorus was effective only for coarse one, thus showing a genotype dependent response. Interestingly, selenium foliar application had no significant effect on arsenic accumulation in both rice genotypes.

Cushions serve as conservation refuges for the Himalayan alpine plant diversity: Implications for nature-based environmental management.

Globally, rapid climate and land-use changes in alpine environments are posing severe risks to their bountiful biodiversity and ecosystem services. Currently, nature-based solutions are fast-emerging as the preferred approach to address the challenges of environmental sustainability. In alpine environments, cushion plants owing to their unique architecture and adaptability offer a potential nature-based system to plan biodiversity conservation and habitat restoration strategies. Here, we employed an analytical framework to test whether and how the cushion plants facilitate the sustenance of alpine plant diversity in Kashmir Himalaya. We specifically aimed to answer: what are the effects of the cushion plants on the patterns of alpine species richness and phylogenetic diversity, and whether these effects vary across spatial scales (local versus landscape), cushion types, and changing elevation. We randomly selected pairs of cushion and neighbouring non-cushion plots (size 100 m2) across 34 different alpine sites in the study region. Within each plot, we randomly laid three 5 m2 quadrats for vegetation sampling, and sampled a total of 204 quadrats in 68 plots with seven cushion types along elevation ranging from 3100 to 3850 m. Our results revealed positive effects of the cushions by supporting a higher community species richness (SR) and phylogenetic diversity (PD). The effects were consistent both at the local (i.e., quadrat) and landscape (i.e., plot) scales, but varied significantly with the cushion type. Interestingly, SR and PD showed an increasing trend with increase in elevation in cushion communities, thereby supporting stress gradient hypothesis. Along the elevational gradient, the cushion communities showed phylogenetic overdispersion, but clustering by non-cushions. Overall, our study provides empirical evidence to reinforce the role of the cushions as conservation refugia for an imperilled alpine plant diversity in the Himalaya. Looking ahead, we highlight the far-reaching implications of our findings in guiding the nature-based environmental management of alpine ecosystems worldwide.

Whole genome analysis of Bacillus amyloliquefaciens TA-1, a promising biocontrol agent against Cercospora arachidicola pathogen of early leaf spot in Arachis hypogaea L.

BACKGROUND: Early leaf spot disease, caused by Cercospora arachidicola, is a devastating peanut disease that has severely impacted peanut production and quality. Chemical fungicides pollute the environment; however, Bacillus bacteria can be used as an environmentally friendly alternative to chemical fungicides. To understand the novel bacterial strain and unravel its molecular mechanism, De novo whole-genome sequencing emerges as a rapid and efficient omics approach. RESULTS: In the current study, we identified an antagonistic strain, Bacillus amyloliquefaciens TA-1. In-vitro assay showed that the TA-1 strain was a strong antagonist against C. arachidicola, with an inhibition zone of 88.9 mm. In a greenhouse assay, results showed that the TA-1 strain had a significant biocontrol effect of 95% on peanut early leaf spot disease. De novo whole-genome sequencing analysis, shows that strain TA-1 has a single circular chromosome with 4172 protein-coding genes and a 45.91% guanine and cytosine (GC) content. Gene function was annotated using non-redundant proteins from the National Center for Biotechnology Information (NCBI), Swiss-Prot, the Kyoto Encyclopedia of Genes and Genomes (KEGG), clusters of orthologous groups of proteins, gene ontology, pathogen-host interactions, and carbohydrate-active enZYmes. antiSMASH analysis predicted that strain TA-1 can produce the secondary metabolites siderophore, tailcyclized peptide, myxochelin, bacillibactin, paenibactin, myxochelin, griseobactin, benarthin, tailcyclized, and samylocyclicin. CONCLUSION: The strain TA-1 had a significant biological control effect against peanut early leaf spot disease in-vitro and in greenhouse assays. Whole genome analysis revealed that, TA-1 strain belongs to B. amyloliquefaciens and could produce the antifungal secondary metabolites.

Unravelling diversity, drivers, and indicators of soil microbiome of Trillium govanianum, an endangered plant species of the Himalaya.

In an era of global environmental change, conservation of threatened biodiversity and ecosystem restoration are formidable ecological challenges. The forest understory strata and the belowground soil environment including rhizospheric microbial communities, which are crucial for ecosystem functioning and overall forest biodiversity maintenance, have remained understudied. Here, we investigate the soil microbiome of Trillium govanianum - an endangered Himalayan Forest herb, to unravel the underground diversity, drivers, and potential indicators of the microbial community. We collected rhizospheric and bulk soil samples for microbiome and physicochemical analysis at three sites along an elevation gradient (2500-3300 m) in Kashmir Himalaya. Amplicon sequencing of 16 S rRNA and ITS was used to identify the bacterial and fungal soil microorganisms. We found significant differences in the structure and diversity of microbial community (bacterial and fungal) between the rhizosphere and bulk soil along the altitudinal gradient, and noticeable shifts in the nutrient level in dominant microbial phyla associated with T. govanianum. A significant difference between soil physicochemical parameters and increasing altitude suggests that microbial community structure is determined by altitude and soil type. Similarly, the microbial communities showed a significant (P < 0.05) correlation with soil physicochemical variables along the altitudinal gradient. The moisture content in bacterial and total organic carbon in fungal communities showed the most substantial impact on the physiochemical drivers. We also identify potential bacterial and fungal plant growth promoter indicator species in the soil microbiome of T. govanianum. Overall, our findings provide novel research insights that can be pivotal in designing integrated species recovery programs and long-term restoration plans for T. govanianum, with learnings for biodiversity conservation elsewhere.

An Adaptive Distributed Denial of Service Attack Prevention Technique in a Distributed Environment.

Cyberattacks in the modern world are sophisticated and can be undetected in a dispersed setting. In a distributed setting, DoS and DDoS attacks cause resource unavailability. This has motivated the scientific community to suggest effective approaches in distributed contexts as a means of mitigating such attacks. Syn Flood is the most common sort of DDoS assault, up from 76% to 81% in Q2, according to Kaspersky's Q3 report. Direct and indirect approaches are also available for launching DDoS attacks. While in a DDoS attack, controlled traffic is transmitted indirectly through zombies to reflectors to compromise the target host, in a direct attack, controlled traffic is sent directly to zombies in order to assault the victim host. Reflectors are uncompromised systems that only send replies in response to a request. To mitigate such assaults, traffic shaping and pushback methods are utilised. The SYN Flood Attack Detection and Mitigation Technique (SFaDMT) is an adaptive heuristic-based method we employ to identify DDoS SYN flood assaults. This study suggested an effective strategy to identify and resist the SYN assault. A decision support mechanism served as the foundation for the suggested (SFaDMT) approach. The suggested model was simulated, analysed, and compared to the most recent method using the OMNET simulator. The outcome demonstrates how the suggested fix improved detection.

Self-harm and suicidal ideation in Pakistani youth amid COVID-19 pandemic: findings of a large, cross-sectional study.

Suicide is the fourth leading cause of death among young people. COVID-19 pandemic has exacerbated various factors which could lead to suicidal ideation. Therefore, this study was aimed to assess self-harm and suicidal ideation among university students in Pakistan. We conducted an online, cross-sectional study among students of four major Pakistani universities. The generalized anxiety scale and patient health questionnaire were used to screen students for anxiety, depression and suicidal ideation/self-harm. Suicidal ideation/self-harm was determined from the ninth-item (score ≥1) of the patient health questionnaire. Brief-COPE was used to assess coping methods. This study included 1134 respondents (age 21.76 ± 3.48 years; female 70.5%). Around 32% students reported having thoughts of death and/or self-harm in the past 2 weeks (several days 14.8%, over half the days 7.1%, and nearly every day 10.2%). Moreover, these thoughts were equally prevalent among the demographics. Suicidal ideation/self-harm was found to be increased by the severity of generalized anxiety and depression (p < 0.001). In conclusion, the rate of suicidal ideation/self-harm is alarmingly high in Pakistani university students during the COVID-19 pandemic. There is a dire need to initiate the psychological measures to prevent suicidal behaviors in Pakistani youth. Addressing mental health disparities and preparing support systems to mitigate mental health consequences as the pandemic evolves will continue to be needed urgently.

Functional Characterization of Novel MC4R Variants Identified in Two Unrelated Patients with Morbid Obesity in Qatar.

The leptin-melanocortin pathway is pivotal in appetite and energy homeostasis. Pathogenic variants in genes involved in this pathway lead to severe early-onset monogenic obesity (MO). The MC4R gene plays a central role in leptin-melanocortin signaling, and heterozygous variants in this gene are the most common cause of MO. A targeted gene panel consisting of 52 obesity-related genes was used to screen for variants associated with obesity. Variants were analyzed and filtered to identify potential disease-causing activity and validated using Sanger sequencing. We identified two novel heterozygous variants, c.253A>G p.Ser85Gly and c.802T>C p.Tyr268His, in the MC4R gene in two unrelated patients with morbid obesity and evaluated the functional impact of these variants. The impact of the variants on the MC4R gene was assessed using in silico prediction tools and molecular dynamics simulation. To further study the pathogenicity of the identified variants, GT1-7 cells were transfected with plasmid DNA encoding either wild-type or mutant MC4R variants. The effects of allelic variations in the MC4R gene on cAMP synthesis, MC4R protein level, and activation of PKA, ERB, and CREB signaling pathways in both stimulated and unstimulated ɑ-MSH paradigms were determined for their functional implications. In silico analysis suggested that the variants destabilized the MC4R structure and affected the overall dynamics of the MC4R protein, possibly leading to intracellular receptor retention. In vitro analysis of the functional impact of these variants showed a significant reduction in cell surface receptor expression and impaired extracellular ligand binding activity, leading to reduced cAMP production. Our analysis shows that the variants do not affect total protein expression; however, they are predicted to affect the post-translational localization of the MC4R protein to the cell surface and impair downstream signaling cascades such as PKA, ERK, and CREB signaling pathways. This finding might help our patients to benefit from the novel therapeutic advances for monogenic forms of obesity.

The role of various ameliorants on geochemical arsenic distribution and CO2-carbon efflux under paddy soil conditions.

Climate change is a global challenge that is accelerated by contamination with hazardous substances like arsenic (As), posing threat to the agriculture, ecosystem and human health. Here, we explored the impact of various ameliorants on geochemical distribution of As in two soils with contrasting textures (sandy clay loam (Khudpur Village) and clay loam (Mattital Village)) under paddy soil conditions and their influence on the CO2-carbon efflux. The exchangeable As pool in clay loam soil increased as: lignite (0.4%) < biogas slurry (6%) < cow dung (9%), and < biochar (20%). However, in the sandy clay loam soil exchangeable soil As pool was found to be maximum with farmyard manure followed by biogas slurry, biochar and cow dung (17%, 14%, 13% and 7%, respectively). Interestingly, in the sandy clay loam soil the percentage As distribution in organic fraction was: biochar (38%) > cow dung (33%) > biogas slurry (23%) > sugarcane bagasse (22%) > farmyard manure (21%) that was higher compared to the clay loam soil (< 6% for all the amendments). In addition to the highest As immobilization by biochar in sandy clay loam soil, it also led to the lowest CO2-carbon efflux (1470 CO2-C mg kg-1) among all the organic/inorganic amendments. Overall, the current study advances our understanding on the pivotal role of organic amendments, notably biochar, in immobilizing As under paddy soil conditions with low (CO2) carbon loss, albeit it is dependent on soil and ameliorant types.

Glycemic outcomes of Advanced Hybrid Closed Loop system in children and adolescents with Type 1 Diabetes, previously treated with Multiple Daily Injections (MiniMed 780G system in T1D individuals, previously treated with MDI).

BACKGROUND: The objective of this study was to evaluate the glycemic outcomes in children and adolescents with Type 1 Diabetes (T1D) previously treated with Multiple Daily Injections (MDI) using a structured initiation protocol for the Advanced Hybrid Closed Loop (AHCL) Minimed 780G insulin pump system. METHODS: In this prospective open label single-arm, single-center, clinical investigation, we recruited children and adolescents (aged 7-17 years) with T1D on MDI therapy and HbA1c below 12.5%. All participants followed a 10-day structured initiation protocol which included 4 steps: step 1: AHCL system assessment; step 2: AHCL system training; step 3: Sensor augmented pump therapy (SAP) for 3 days; step 4: AHCL system use for 12 weeks, successfully completing the training from MDI to AHCL in 10 days. The primary outcome of the study was the change in the time spent in the target in range (TIR) of 70-180 mg/dl and HbA1c from baseline (MDI + CGM, 1 week) to study phase (AHCL, 12 weeks). The paired student t-test was used for statistical analysis and a value < 0.05 was considered statistically significant. RESULTS: Thirty-four participants were recruited and all completed the 12 weeks study. TIR increased from 42.1 ± 18.7% at baseline to 78.8 ± 6.1% in the study phase (p < 0.001). HbA1c decreased from 8.6 ± 1.7% (70 ± 18.6 mmol/mol) at baseline, to 6.5 ± 0.7% (48 ± 7.7 mmol/mol) at the end of the study (p = 0.001). No episodes of severe hypoglycemia or DKA were reported. CONCLUSION: Children and adolescents with T1D on MDI therapy who initiated the AHCL system following a 10-days structured protocol achieved the internationally recommended goals of glycemic control with TIR > 70% and a HbA1c of < 7%.

Prediction of CYP-mediated silybin A-losartan pharmacokinetic interactions using physiological based pharmacokinetic modeling.

The concomitant use of herbal products and synthetic drugs necessitates the assessment of their interaction potentials. The herbal hepatoprotective medicine, silybin A inhibits cytochrome P450 (CYP) 2C9 and 3A4 enzymes, thus, may interact with the drugs that are substrates of CYP2C9 and 3A4, such as losartan. The three most prominent genotypes, expressed by CYP2C9 are the CYP2C9*1/*1, CYP2C9*1/*2 and CYP2C9*1/*3. This study aimed to assess silybin A-losartan interaction in different CYP2C9 genotypes using physiological-based pharmacokinetic (PBPK) model approach. The individual PBPK models for silybin A and losartan were developed using PK-Sim®. Losartan pharmacokinetics was predicted with or without co-administration of silybin A in individuals of different CYP2C9 genotypes to find herbal-drug interaction. The predicted drug plasma curves and pharmacokinetic parameters were optimized using parameter identification tool and were compared with reported pharmacokinetic parameters from the published clinical studies for model validation. The silybin-losartan interactions were predicted by change in area under the curve (AUC) and peak systemic concentration (Cmax). The co-treatment of silybin A, 420 mg/24 h (140 mg/8 h) with losartan 50 mg/24 h, exhibited a genotype-dependent change in the losartan's AUC and Cmax. In CYP 2C9*1/*1 genotype, AUC and Cmax of losartan were increased 1.16 and 1.37 folds, respectively falling in a range stipulated for negligible interaction. Increase in AUC and Cmax by 0.873 and 0.294 folds, respectively in CYP2C9*1/*3 after co-administration of silybin A exhibited a minor interaction with losartan. However, in individuals with CYP2C9*1/*2 genotype, the losartan's AUC and Cmax were decreased by 0.01 folds, manifesting a moderate interaction. Hence, in CYP2C9*1/*1 and CYP2C9*1/*3 genotypes, silybin A is a weak CYP inhibitor for losartan while in CYP2C9*1/*2 genotype, the co-administration of silybin consequents into a moderate pharmacokinetic interaction with losartan.

Diospyros malabarica (Desr.) Kostel fruits extract attenuated acute and chronic inflammation through modulation of the expression of pro- and anti-inflammatory biomarkers in rat models.

Rheumatoid arthritis is a chronic inflammatory disorder of polyarticular tissues, characterised by progressive synovitis. Its prolonged treatment imparts a huge burden on the healthcare system and results in toxicity, which necessitates the search for safe, efficacious and cost-effective therapies. Diospyros malabarica (Desr.) Kostel is traditionally used for anti-inflammatory purposes; however, to the best of our knowledge, there is no detailed study reporting the in vivo anti-inflammatory potential of this plant. Therefore, in the current study, the methanol extract of D. malabarica (Desr.) Kostel fruit (mDMF) was evaluated for its antioxidant, anti-inflammatory and anti-arthritic potentials, along with its underlying mechanisms. The antioxidant activity was evaluated by DPPH assay. Total phenolic and flavonoid contents were estimated via colorimetric and high-performance liquid chromatography (HPLC) methods. Different doses (250, 500 and 750 mg/kg) of mDMF were used to evaluate the anti-inflammatory and anti-arthritis actions in acute inflammatory (carrageenan and histamine-induced paw oedema) and Freund's complete adjuvant (FCA)-induced arthritis rat models. Levels of various pro- and anti-inflammatory biomarkers were estimated using ELISA and RT-PCR techniques. Paw samples were used for different histopathological and radiographic studies. Qualitative phytochemical and HPLC analyses indicated the presence of various polyphenolic compounds in mDMF, which exhibited marked antioxidant activity in the DPPH assay. mDMF showed time-dependent anti-inflammatory and anti-arthritic effects in in vivo models. ELISA assay data showed significant (p < 0.05) reduction in the serum levels of C-reactive protein and rheumatoid factor in the mDMF treatment groups. RT-PCR data showed significant (p < 0.05) down-regulation of various pro-inflammatory markers (TNF-α, NF-κB, COX-2, IL-1ß and IL-6) and up-regulation of anti-inflammatory markers (IκB, IL-4 and IL-10) in serum samples of rats treated with mDMF. The histopathology of the ankle joints showed reduced pannus formation, joint swelling and synovial hyperplasia in mDMF-treated animals when compared with the untreated disease control group. Overall, it may be concluded that the antioxidant, anti-inflammatory and anti-arthritis properties of mDMF are due to its flavonoid and phenolic constituents. Further studies using a stable oral dosage form of D. malabarica (Desr.) Kostel fruits extract are warranted to explore its effects in other inflammatory disorders, including irritable bowel syndrome, appendicitis and hepatitis.

Anti-diabetic and anti-lipidepmic effect of Albizia lebbeck seeds against HepG2 cells.

Albizia lebbeck has been a medicinally important plant for its pharmacological potential. This study aims to determine the in vitro antioxidant, anti-diabetic and anti-lipidemic potential of A. lebbeck seeds. The seed extracts were prepared in petroleum ether, chloroform and methanol. Crude methanolic extract (ME ext) was subjected further to sequential fractionation in increasing polarity based solvents. Extracts and fractions were analyzed for their antioxidant, anti-diabetic and anti-lipidemic potentials using hepatic cell line, HepG2. Results showed that crude extracts of A. lebbeck seeds specifically, ME ext are rich in polyphenols and flavonoids. ME ext has also shown highly significant antioxidant and alpha-amylase inhibition potential compared to petroleum ether and chloroform extracts. In vitro assays using different fractions of methanolic extract further highlighted the ethyl acetate and chloroform fractions exhibiting significant antioxidant and anti-diabetic potentials. Alpha-amylase inhibition coupled with enhanced glucose uptake of cells treated with ME ext and ethyl acetate fraction emphasized on significant anti-diabetic potential of the plant. Expression alteration of genes and reduced level of cholesterol suggested the lipid synthesis mediated anti-diabetic activity of the plant. It is therefore, concluded that A. lebbeck seed has significant antioxidant, anti-diabetic and anti-lipidemic potentials.

In silico, in vivo enzymatic, non-enzymatic toxicity and antioxidant activity of a heterocyclic compound: 5-Benzyl -1, 3, 4-oxadiazole-2-thiol, a potential drug candidate.

The present study aimed to investigate the enzymetic, non-enzymetic toxicity and antioxidant potential of a drug candidate 5-Benzyl-1,3,4-Oxadiazole-2-Thiol(OXPA) using computational tools and in vivo models. The binding pattern of it, with different toxicity/oxidative enzymes was predicted using software pkCSM, Protox- II, LAZAR, Mcule 1-Click Docking 3D-Ligand binding Site and best score obtained used as an evaluating criterion. After acute oral toxicity, in vivo. antioxidant and hepato protective activity was investigated on male wistar rats, segregated into four groups as control (NS), toxic (INH-RIF), standard (Silymerin) and sample (OXPA, 100mg/Kg) for 21days. Level of antioxidant enzymes / histopathology and serum biochemical parameters in liver and blood of treated rats was assessed by using scientific tools. In silico study reveal no profound toxicity parameters however, LD50 found to be 560mg/Kg while in vivo study declared it safe till 1000mg/Kg, as having no toxicity symptoms. Molecular interaction score with GTH reductase, s-transferase and significant in vivo antioxidant effect on catalase, SOD, TBARS enzymes and histopathological assessment, declare OXPA a good antioxidant having significant (P< 0.05) hepato protective activity. Results of in silico, in vivo studies declare the propensity of 5-Benzyl-1, 3, 4-oxadiazole-2-thiol as potential antioxidant, for further investigations as a drug.

Microscopic, phytochemical and antibacterial studies of Zizyphus oxyphylla Edgew. leaves.

Microscopic, phytochemical and pharmacological profiles are required for correct identification of a plant material to ensure consistent efficacy and safety. But such data are not available for the leaf of an important medicinal plant, Zizyphus oxyphylla Edgew. (Family: Rhamnaceae). Therefore, the current study aimed to investigate leaves of the plant for microscopic, phytochemical and antibacterial studies. Powdered material was subjected to microscopy, proximate analyses and estimation of total primary metabolites. Then, different types of extracts prepared using various solvents in order of increasing polarity were screened for antibacterial activity against seven standard strains. The most active extract was hydrolyzed and aglycone enriched fraction was extracted and screened for antibacterial activity. The powder microscopy indicated the presence of vascular bundles filled with cuboidal calcium oxalate crystals, anisocytic stomata and xylary vessels with reticulate and scalariform thickenings. Proximate features and primary metabolites provided characteristic identifying patterns. The most active extract (methanol) upon acidic hydrolysis exhibited higher activity against B. bronchiseptica (26.01±0.01 mm), S. aureus (26.00±0.00 mm), P. aeruginosa (24.03±0.02 mm) and M. luteus (24.02± 0.00 mm). The results of the current study provide identifying microscopic and phytochemical profiles that may be useful for correct identification of leaves of the plant. Aglycone enriched extract is having remarkable antibacterial activity hence may be used for activity-guided isolation.

Functional assessment of variants associated with Wolfram syndrome.

Wolfram syndrome (WS) is a heterogeneous multisystem neurodegenerative disorder with two allelic variations in addition to a separate subtype known as WS type 2. The wide phenotypic spectrum of WS includes diabetes mellitus and optic atrophy which is often accompanied by diabetes insipidus, deafness, urological and neurological complications in combination or in isolation. To date, the understanding of the genotype-phenotype relationship in this complex syndrome remains poorly understood. In this study, we identified and explored the functionality of rare and novel variants in the two causative WS genes WFS1 and CISD2 by assessing the effects of the mutations on the encoded proteins Wolframin and ERIS, in a cohort of 12 patients with autosomal recessive WS, dominant WS and WS type 2. The identified pathogenic variants included missense changes, frameshift deletions and insertions in WFS1 and an exonic deletion in CISD2 which all altered the respective encoded protein in a manner that did not correlate to the phenome previously described. These observations suggest the lack of genotype-phenotype correlation in this complex syndrome and the need to explore other molecular genetic mechanisms. Additionally, our findings highlight the importance of functionally assessing variants for their pathogenicity to tackle the problem of increasing variants of unknown significance in the public genetic databases.

A Novel SEMA3G Mutation in Two Siblings Affected by Syndromic GnRH Deficiency.

INTRODUCTION: Gonadotropin-releasing hormone (GnRH) deficiency causes hypogonadotropic hypogonadism (HH), a rare genetic disorder that impairs sexual reproduction. HH can be due to defective GnRH-secreting neuron development or function and may be associated with other clinical signs in overlapping genetic syndromes. With most of the cases being idiopathic, genetics underlying HH is still largely unknown. OBJECTIVE: To assess the contribution of mutated Semaphorin 3G (SEMA3G) in the onset of a syndromic form of HH, characterized by intellectual disability and facial dysmorphic features. METHOD: By combining homozygosity mapping with exome sequencing, we identified a novel variant in the SEMA3G gene. We then applied mouse as a model organism to examine SEMA3Gexpression and its functional requirement in vivo. Further, we applied homology modelling in silico and cell culture assays in vitro to validate the pathogenicity of the identified gene variant. RESULTS: We found that (i) SEMA3G is expressed along the migratory route of GnRH neurons and in the developing pituitary, (ii) SEMA3G affects GnRH neuron development, but is redundant in the adult hypothalamic-pituitary-gonadal axis, and (iii) mutated SEMA3G alters binding properties in silico and in vitro to its PlexinA receptors and attenuates its effect on the migration of immortalized GnRH neurons. CONCLUSION: In silico, in vitro, and in vivo models revealed that SEMA3G regulates GnRH neuron migration and that its mutation affecting receptor selectivity may be responsible for the HH-related defects.

Clinical and genetic characteristics of patients with congenital hyperinsulinism in 21 non-consanguineous families from Serbia.

Persistent hypoglycaemia in newborns and infants is most commonly caused by congenital hyperinsulinism (CHI). Most CHI studies report outcomes in children from both consanguineous and non-consanguineous families which can affect the phenotype-genotype analysis. The aim of this study was to analyze characteristics of patients with CHI in 21 non-consanguineous families from Serbia. This retrospective cohort study included a total of 21 patients with CHI treated in the Mother and Child Healthcare Institute of Serbia during the past 20 years. The prevalence of macrosomia at birth was very low in our cohort (4.8%). Median age at presentation was 6 days, with seizures as the presenting symptom in 76% of patients. Only four patients (19%) were diazoxide unresponsive, and eventually underwent pancreatectomy. Genetic testing was performed in 15 patients and genetic diagnosis was confirmed in 60%, with all patients being heterozygous for detected mutations. The ABCC8 gene mutations were detected in 55.6%, GLUD1 in three patients (33.3%) with HIHA syndrome and one patient had HNF4A gene mutation and unusual prolonged hyperglycaemia lasting 6 days after diazoxide cessation. Neurodevelopmental deficits persisted in 33% of patients.Conclusion: This is the first study regarding CHI patients in Serbia. It suggests that in countries with low consanguinity rate, majority of CHI patients are diazoxide responsive. The most common mutations were heterozygous ABCC8, followed by GLUD1 and HNF4A mutations, suggesting the potential benefit of population-tailored genetic analysis approach, targeting the mutations causing CHI via dominant inheritance model in regions with low consanguinity rates. What is Known: • Persistent hypoglycaemia during infancy and early childhood is most commonly caused by congenital hyperinsulinism (CHI). • Consanguinity is a very important factor regarding the genetics and phenotype of CHI, increasing the risk of autosomal recessive genetic disorders, including the severe, diazoxide-unresponsive forms caused by recessive inactivating mutations in ABCC8 and KCNJ11. What is New: • Results of the present study which included CHI patients from 21 non-consanguineous families suggest that in countries with low consanguinity rates, majority of CHI patients can be diazoxide responsive, with most common mutations being heterozygous ABCC8, followed by GLUD1 and HNF4A mutations. • Unusually prolonged hyperglycaemic reaction to diazoxide treatment in a patient with HNF4A mutation was also described in the present study.