In vivo versus in silico assessment of potentially pathogenic missense variants in human reproductive genes.

Infertility is a heterogeneous condition, with genetic causes thought to underlie a substantial fraction of cases. Genome sequencing is becoming increasingly important for genetic diagnosis of diseases including idiopathic infertility; however, most rare or minor alleles identified in patients are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of these variants in key fertility genes, we functionally evaluated 11 missense variants in the genes ANKRD31, BRDT, DMC1, EXO1, FKBP6, MCM9, M1AP, MEI1, MSH4 and SEPT12 by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction (PPI) in the yeast two hybrid (Y2H) assay. Though these genes are essential for normal meiosis or spermiogenesis in mice, only one variant, observed in the MCM9 gene of a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely used algorithms to 1) those annotated in ClinVar and 2) those evaluated in mice. All the algorithms performed poorly in terms of predicting the effects of human missense variants modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms and emphasize the need for alternative and efficient in vitro or in vivo functional validation models for more effective and accurate VUS description to either pathogenic or benign categories.

Design, synthesis, and biological evaluation of 3-benzenesulfonamide-linked 3-hydrazinoisatin derivatives as carbonic anhydrase inhibitors.

A new series of isatin-linked benzenesulfonamide derivatives (9a-w) were synthesized using the tail approach and assayed for their inhibitory potency against four different human carbonic anhydrase (hCA) isoforms, hCA I, II, IX, and XII. Most of these synthesized compounds exhibited interesting inhibition potency against isoforms hCA I, IX, and XII in the nanomolar range and by taking the standard drug acetazolamide. The most potent compounds in the case of hCA I were 9c (435.8 nM) and 9s (956.4 nM), for hCA IX, 9a (60.5 nM), 9d (95.6 nM), 9g (92.1 nM), and 9k (75.4 nM), and for hCA XII, 9p (84.5 nM). However, these compounds showed more selectivity toward hCA IX over hCA I, II, and XII. Thus, these compounds can be further developed as potential lead molecules for the development of isoform-selective hCA IX inhibitors with further structural modifications.

Role of Adipose-Derived Mesenchymal Stem Cells in Bone Regeneration.

Bone regeneration involves multiple factors such as tissue interactions, an inflammatory response, and vessel formation. In the event of diseases, old age, lifestyle, or trauma, bone regeneration can be impaired which could result in a prolonged healing duration or requiring an external intervention for repair. Currently, bone grafts hold the golden standard for bone regeneration. However, several limitations hinder its clinical applications, e.g., donor site morbidity, an insufficient tissue volume, and uncertain post-operative outcomes. Bone tissue engineering, involving stem cells seeded onto scaffolds, has thus been a promising treatment alternative for bone regeneration. Adipose-derived mesenchymal stem cells (AD-MSCs) are known to hold therapeutic value for the treatment of various clinical conditions and have displayed feasibility and significant effectiveness due to their ease of isolation, non-invasive, abundance in quantity, and osteogenic capacity. Notably, in vitro studies showed AD-MSCs holding a high proliferation capacity, multi-differentiation potential through the release of a variety of factors, and extracellular vesicles, allowing them to repair damaged tissues. In vivo and clinical studies showed AD-MSCs favoring better vascularization and the integration of the scaffolds, while the presence of scaffolds has enhanced the osteogenesis potential of AD-MSCs, thus yielding optimal bone formation outcomes. Effective bone regeneration requires the interplay of both AD-MSCs and scaffolds (material, pore size) to improve the osteogenic and vasculogenic capacity. This review presents the advances and applications of AD-MSCs for bone regeneration and bone tissue engineering, focusing on the in vitro, in vivo, and clinical studies involving AD-MSCs for bone tissue engineering.

Assessing the Effect of Dopants on the C-H Activation Activity of γ-Al2 O3 using First-Principles Calculations.

In recent years, the high availability of methane in the shale gas reserves has raised significant interest in its conversion to high-value chemicals but this process is still not commercially viable. Metal oxides, due to their surface heterogeneity and the presence of Lewis acidic and basic site pairs are known to facilitate the activation of C-H bonds of methane. In this work, we investigate the C-H bond activation of methane on pristine and doped γ-Al2 O3 clusters using density functional theory (DFT) calculations. Our results demonstrate that the polar pathway is energetically preferred over the radical pathway on these systems. We found that the metal dopants (boron and gallium) not only alter the catalytic activity of dopant sites but this effect is more pronounced on some of the adjacent sites (non-local). Among the selected dopants, gallium greatly improves the catalytic activity on most of the site pairs (including most active and least active) of pristine γ-Al2 O3 . Additionally, we identified a correlation between H2 binding energies and the C-H activation free energies on Ga-doped γ-Al2 O3 .

A novel function for CDK2 activity at meiotic crossover sites.

Genetic diversity in offspring is induced by meiotic recombination, which is initiated between homologs at >200 sites originating from meiotic double-strand breaks (DSBs). Of this initial pool, only 1-2 DSBs per homolog pair will be designated to form meiotic crossovers (COs), where reciprocal genetic exchange occurs between parental chromosomes. Cyclin-dependent kinase 2 (CDK2) is known to localize to so-called "late recombination nodules" (LRNs) marking incipient CO sites. However, the role of CDK2 kinase activity in the process of CO formation remains uncertain. Here, we describe the phenotype of 2 Cdk2 point mutants with elevated or decreased activity, respectively. Elevated CDK2 activity was associated with increased numbers of LRN-associated proteins, including CDK2 itself and the MutL homolog 1 (MLH1) component of the MutLγ complex, but did not lead to increased numbers of COs. In contrast, reduced CDK2 activity leads to the complete absence of CO formation during meiotic prophase I. Our data suggest an important role for CDK2 in regulating MLH1 focus numbers and that the activity of this kinase is a key regulatory factor in the formation of meiotic COs.

Biparental contributions of the H2A.B histone variant control embryonic development in mice.

Histone variants expand chromatin functions in eukaryote genomes. H2A.B genes are testis-expressed short histone H2A variants that arose in placental mammals. Their biological functions remain largely unknown. To investigate their function, we generated a knockout (KO) model that disrupts all 3 H2A.B genes in mice. We show that H2A.B KO males have globally altered chromatin structure in postmeiotic germ cells. Yet, they do not show impaired spermatogenesis or testis function. Instead, we find that H2A.B plays a crucial role postfertilization. Crosses between H2A.B KO males and females yield embryos with lower viability and reduced size. Using a series of genetic crosses that separate parental and zygotic contributions, we show that the H2A.B status of both the father and mother, but not of the zygote, affects embryonic viability and growth during gestation. We conclude that H2A.B is a novel parental-effect gene, establishing a role for short H2A histone variants in mammalian development. We posit that parental antagonism over embryonic growth drove the origin and ongoing diversification of short histone H2A variants in placental mammals.

Opportunities and Challenges in the Development of Layered Positive Electrode Materials for High-Energy Sodium Ion Batteries: A Computational Perspective.

In recent years, high-energy-density sodium ion batteries (SIBs) have attracted enormous attention as a potential replacement for LIBs due to the chemical similarity between Li and Na, high natural abundance, and low cost of Na. Despite the promise of high energy, SIBs with layered cathode materials face several challenges including irreversible capacity loss, voltage hysteresis, voltage decay, irreversible TM migrations that lead to fast capacity fading, and structural degradation. However, their electrochemical performance can be improved by introducing reversible anionic redox along with conventional cationic redox. This Perspective systematically summarizes different factors that trigger the irreversible anionic redox in Na-based cathode materials. Additionally, this Perspective highlights the mechanistic understanding and key challenges for reversible anionic redox and proposes plausible solutions to overcome these limitations. The overview of various existing experimental and theoretical approaches presented here could provide a futuristic pathway to design Na-based cathode materials for high-energy-density SIBs.

PET radiotracers and fluorescent probes for imaging human carbonic anhydrase IX and XII in hypoxic tumors.

Positron emission tomography (PET) and fluorescent imaging play a pivotal role in medical diagnosis, biomedical oncologic research, and drug development process, which include identification of target location, target engagement, but also prove on mechanism of action or pharmacokinetics of new drug candidates. PET estimates physiological changes at the molecular level using specific radiotracers containing a short-lived positron emitting radionuclide such as fluorine-18 or carbon-11, whereas fluorescent imaging techniques use fluorescent probes labeled with suitable drug candidates for detection at the molecular level. The human carbonic anhydrase (hCA) isoforms IX and XII are overexpressed in hypoxic cancer cells, promoting tumor growth by regulating extra/intracellular pH, ferroptosis, and metabolism, being recognized as promising targets for anticancer theranostic agents. In this review, we have focused on PET radiotracers as well as fluorescent probes for diagnosis and treatment of tumors expressing hCA IX and hCA XII.

Isoxazole carboxylic acid methyl ester-based urea and thiourea derivatives as promising antitubercular agents.

In our continued efforts to find potential chemotherapeutics active against drug-resistant (DR) Mycobacterium tuberculosis (Mtb), causative agent of Tuberculosis (TB) and to curb the current burdensome treatment regimen, herein we describe the synthesis and biological evaluation of urea and thiourea variants of 5-phenyl-3-isoxazolecarboxylic acid methyl esters as promising anti-TB agent. Majority of the tested compounds displayed potent in vitro activity not only against drug-susceptible (DS) Mtb H37Rv but also against drug-resistant (DR) Mtb. Cell viability test against Vero cells deemed these compounds devoid of significant toxicity. 3,4-Dichlorophenyl derivative (MIC 0.25 µg/mL) and 4-chlorophenyl congener (MIC 1 µg/mL) among urea and thiourea libraries respectively exhibited optimum potency. Lead optimization resulted in the identification of 1,4-linked analogue of 3,4-dichlorophenyl urea derivative demonstrating improved selectivity. Further, in silico study complemented with previously proposed prodrug like attributes of isoxazole esters. Taken together, this molecular hybridization approach presents a new chemotype having potential to be translated into an alternate anti-Mtb agent.

Meditation-A Slippery Slope for Psychosis: A Case Series With Review of Evidence.

ABSTRACT: The practice of meditation has been traditionally viewed as a self-regulatory approach that aids in psychological well-being. Over the last decade, mindfulness-based meditation has gained a separate therapeutic significance in various mental health conditions. There has also been considerable understanding of the adverse effects of meditation over the years. Despite this, there is still underreporting of the altered behavioral presentations arising possibly because of intensive and unguided meditation practices. We present two cases of meditation-related psychosis with different clinical presentations. The write-up highlights the need for tailoring the meditation practices after evaluation of mental state of the individuals and early detection of "at-risk" mental states. There should be regular screening of emergence of any unwanted effects during the course of meditation and the guides should be able to detect the early warning signs of psychosis.

Testis formation in XX individuals resulting from novel pathogenic variants in Wilms' tumor 1 (WT1) gene.

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10-6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10-4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor ß-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

Variants in RABL2A causing male infertility and ciliopathy.

Approximately 7% of men worldwide suffer from infertility, with sperm abnormalities being the most common defect. Though genetic causes are thought to underlie a substantial fraction of idiopathic cases, the actual molecular bases are usually undetermined. Because the consequences of most genetic variants in populations are unknown, this complicates genetic diagnosis even after genome sequencing of patients. Some patients with ciliopathies, including primary ciliary dyskinesia and Bardet-Biedl syndrome, also suffer from infertility because cilia and sperm flagella share several characteristics. Here, we identified two deleterious alleles of RABL2A, a gene essential for normal function of cilia and flagella. Our in silico predictions and in vitro assays suggest that both alleles destabilize the protein. We constructed and analyzed mice homozygous for these two single-nucleotide polymorphisms, Rabl2L119F (rs80006029) and Rabl2V158F (rs200121688), and found that they exhibit ciliopathy-associated disorders including male infertility, early growth retardation, excessive weight gain in adulthood, heterotaxia, pre-axial polydactyly, neural tube defects and hydrocephalus. Our study provides a paradigm for triaging candidate infertility variants in the population for in vivo functional validation, using computational, in vitro and in vivo approaches.

CDK2 kinase activity is a regulator of male germ cell fate.

The ability of men to remain fertile throughout their lives depends upon establishment of a spermatogonial stem cell (SSC) pool from gonocyte progenitors, and thereafter balancing SSC renewal versus terminal differentiation. Here, we report that precise regulation of the cell cycle is crucial for this balance. Whereas cyclin-dependent kinase 2 (Cdk2) is not necessary for mouse viability or gametogenesis stages prior to meiotic prophase I, mice bearing a deregulated allele (Cdk2Y15S ) are severely deficient in spermatogonial differentiation. This allele disrupts an inhibitory phosphorylation site (Tyr15) for the kinase WEE1. Remarkably, Cdk2Y15S/Y15S mice possess abnormal clusters of mitotically active SSC-like cells, but these are eventually removed by apoptosis after failing to differentiate properly. Analyses of lineage markers, germ cell proliferation over time, and single cell RNA-seq data revealed delayed and defective differentiation of gonocytes into SSCs. Biochemical and genetic data demonstrated that Cdk2Y15S is a gain-of-function allele causing elevated kinase activity, which underlies these differentiation defects. Our results demonstrate that precise regulation of CDK2 kinase activity in male germ cell development is crucial for the gonocyte-to-spermatogonia transition and long-term spermatogenic homeostasis.

Synthesis of a new series of quinoline/pyridine indole-3-sulfonamide hybrids as selective carbonic anhydrase IX inhibitors.

In this manuscript, design, rational, synthesisand carbonic anhydrases (CAs) inhibitory profile of the quinoline/pyridine linked indole-3-sulfonamide derivatives were reported. The library of 29newly quinoline/pyridine indole-3-sulfonamide derivatives have been generated and examined against the panel of four physiological relevant human CA isoforms, namely, the cytosolic isoforms hCA I and hCA II and the transmembrane tumor associated isoforms hCA IX and hCA XII. Pyridine indole-3-sulfonamide hybrids are selective inhibit transmembrane tumor associated isoforms hCA IX and hCA XII. However, all synthesized quinoline indole-3-sulfonamide hybrids have inhibitory effect on hCA IX isoforms, whereas few have shown inhibitory activity against hCA II and hCA XII as well. However, among all synthesized compound 6q and6p having good inhibitory activity against hCA IX with Ki 1.47 µM and 1.57 µM respectively.These quinoline/pyridine indole-3-sulfonamide conjugatesmay be regarded as potential leads for hCA IXselective inhibitors as anti-cancer agents.

Unraveling the Mechanistic Details of Ru-Bis(pyridyl)borate Complex Catalyst for the Dehydrogenation of Ammonia Borane.

Ru-Bis(pyridyl)borate complex (CAT) is an efficient catalyst for ammonia borane (AB) dehydrogenation. Although the mechanistic pathway of this catalyst has been theoretically investigated previously, the gap between the experimental findings and the computational results could not be bridged thus far. In our study, using density functional theory calculations, we elucidate the mechanism of AB dehydrogenation of CAT at a variable degree of ligand hydrogenation. Our results confirm that the acetonitrile ligands get reduced in the presence of AB and remain hydrogenated. Moreover, in line with experiments, we find that AB dehydrogenation on CAT proceeds via a concerted mechanism (with the free energy energetic span between 25.4 and 32.5 kcal/mol). We find that the ligand reduction alters the electronic structure and activity of CAT and the highest activity of the catalyst is expected at the fifth degree of hydrogenation of ligands with an energetic span of 25.4 kcal/mol. Additionally, the mechanism for the removal of molecular H2 from the catalysts also alters with the degree of ligand hydrogenation. Furthermore, our results show that optimal H2 binding free energy calculations can be used as a descriptor to identify the most active sites. Finally, this work demonstrates that ligand reduction improves the activity of the catalyst. These results highlight the importance of ligand hydrogenation in probing the activity and operating mechanism of the Ru-bis(pyridyl)borate complexes for AB dehydrogenation. Further, we identify a plausible dimer structure and rationalized experimental observation that the deactivation chemistry of this catalyst is different from the Shvo's catalyst.

Design, synthesis, SAR, and biological evaluation of saccharin-based hybrids as carbonic anhydrase inhibitors.

Saccharin is a cyclic secondary sulfonamide, which is a selective inhibitor of the tumor-associated carbonic anhydrase (CA; EC 4.2.1.1) enzymes CA IX and CA XII compared to many primary sulfonamides. In this study, new saccharin-1,2,3-triazole and saccharin-1,2,4-oxadiazole hybrids were synthesized. All the newly synthesized molecules were screened for their CA-inhibitory activity against four important human CA (hCA) isoforms: hCA I, hCA II, hCA IX, and hCA XII. Compounds 8a and 8f emerged as potent hCA II inhibitors (Ki = 3 µM). Compounds 6d, 6e and 7a, 7b were highly selective against hCA IX (6d, 6e) and hCA II (7a, 7b), with moderate inhibitory activity. The activity of these compounds was further confirmed by performing in silico docking studies against hCA II and hCA IX.

Design and development of novel series of indole-3-sulfonamide ureido derivatives as selective carbonic anhydrase II inhibitors.

Indole is a privileged moiety with a wide range of bioactivities, making it a popular scaffold in drug design and development studies as well as in synthetic chemistry. Here, novel urea derivatives of indole, containing sulfonamide at position-3 of indole, were synthesized using a well-known tail approach, as carbonic anhydrases (CAs; EC 4.2.1.1) inhibitors. All the newly synthesized molecules were screened for their CA-inhibitory activity against four clinically relevant isoforms of human-origin carbonic anhydrase (hCA), that is, hCA I, hCA II, hCA IX, and hCA XII. These compounds were specifically active against hCA II, more than against hCA I, hCA IX, and hCA XII. Derivative 6l was found to be most active, with a Ki value of 7.7 µM against hCA II.

Design, Synthesis and Biological Assessment of Rhodanine-Linked Benzenesulfonamide Derivatives as Selective and Potent Human Carbonic Anhydrase Inhibitors.

A novel series of twenty-five rhodamine-linked benzenesulfonamide derivatives (7a-u and 9a-d) were synthesized and screened for their inhibitory action against four physiologically relevant human (h) carbonic anhydrase (CA) isoforms, namely hCA I, hCA II, hCA IX, and hCA XII. All the synthesized molecules showed good to excellent inhibition against all the tested isoforms in the nanomolar range due to the presence of the sulfonamide as a zinc binding group. The target compounds were developed from indol-3-ylchalcone-linked benzenesulfonamide where the indol-3-ylchalcone moiety was replaced with rhodanine-linked aldehydes or isatins to improve the inhibition. Interestingly, the molecules were slightly more selective towards hCA IX and XII compared to hCA I and II. The most potent and efficient ones against hCA I were 7h (KI 22.4 nM) and 9d (KI 35.8 nM) compared to the standard drug AAZ (KI 250.0 nM), whereas in case of hCA II inhibition, the derivatives containing the isatin nucleus as a tail were preferred. Collectively, all compounds were endowed with better inhibition against hCA IX compared to AAZ (KI 25.8 nM) as well as strong potency against hCA XII. Finally, these newly synthesized molecules could be taken as potential leads for the development of isoform selective hCA IX and XII inhibitors.

3-Functionalised benzenesulphonamide based 1,3,4-oxadiazoles as selective carbonic anhydrase XIII inhibitors: Design, synthesis and biological evaluation.

A new series of benzenesulphonamide linked-1,3,4-oxadiazole hybrids (6a-s) has been synthesized and tested for their carbonic anhydrase inhibition against human (h) carbonic anhydrase (CA) isoforms hCA I, II, IX, and XIII. Fluorescence properties of some of the synthesized molecules were studied. Most of the molecules exhibited significant inhibitory power, comparable or better than the standard drug acetazolamide (AAZ) on hCA XIII. Out of 19 tested molecules, compound 6e (75.8 nM) was 3 times more potent than AAZ (250.0 nM) against hCA I, whereas compound 6e (15.4 nM), 6g (16.2 nM), 6h (16.4 nM) and 6i (17.0 nM) were found to be more potent than AAZ (17.0 nM) against isoform hCA XIII. It is anticipated that these compounds could be taken as the potential leads for the development of selective hCA XIII isoform inhibitors with improved potency.

Discovery of a novel series of indolylchalcone-benzenesulfonamide hybrids acting as selective carbonic anhydrase II inhibitors.

The primary sulfonamide group is one of the most efficient zinc binding group (ZBG) for designing carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. In the present study primary sulfonamide linked with indolylchalcone were designed. The newly synthesized molecules (5a-r) were examined against four human (h) CA isoforms (hCA I, hCA II, hCA IX and hCA XIII). These sulfonamides showed good inhibition activity against isoforms hCA I, hCA II and hCA XIII. Compound 5i (2.3 nM), 5m (2.4 nM), 5o (3.6 nM) and 5q (7.0 nM) were more potent than standard drug AAZ (12.1 nM) against isoform hCA II, respectively. Most of the other compounds in the present series inhibited hCA XIII and hCA IX in the range of 50 nM - 100 nM.