Nestorone (segesterone acetate) effects on neuroregeneration.

Nestorone® (segesterone acetate) is a progestin with a chemical structure closely related to progesterone with high affinity and selectivity for the progesterone receptor without significant interaction with other steroid receptors. It has been developed for female and male contraception and is FDA-approved in a first long-acting contraceptive vaginal system for female contraception. Its safety has been extensively demonstrated in both preclinical and clinical studies for contraceptive indications. Nestorone was found to display neuroprotective and neuroregenerative activity in animal models of various central nervous system diseases, including multiple sclerosis, stroke, and amyotrophic lateral sclerosis. Reviewed herein are neuroprotective and myelin- regenerating properties of Nestorone in various animal models and its translational potential as a therapeutic agent for debilitating neurological diseases for which limited therapeutic options are available (Table 1).

Balancing growth amidst salt stress - lifestyle perspectives from the extremophyte model Schrenkiella parvula.

Schrenkiella parvula, a leading extremophyte model in Brassicaceae, can grow and complete its lifecycle under multiple environmental stresses, including high salinity. Yet, the key physiological and structural traits underlying its stress-adapted lifestyle are unknown along with trade-offs when surviving salt stress at the expense of growth and reproduction. We aimed to identify the influential adaptive trait responses that lead to stress-resilient and uncompromised growth across developmental stages when treated with salt at levels known to inhibit growth in Arabidopsis and most crops. Its resilient growth was promoted by traits that synergistically allowed primary root growth in seedlings, the expansion of xylem vessels across the root-shoot continuum, and a high capacity to maintain tissue water levels by developing thicker succulent leaves while enabling photosynthesis during salt stress. A successful transition from vegetative to reproductive phase was initiated by salt-induced early flowering, resulting in viable seeds. Self-fertilization in salt-induced early flowering was dependent upon filament elongation in flowers otherwise aborted in the absence of salt during comparable plant ages. The maintenance of leaf water status promoting growth, and early flowering to ensure reproductive success in a changing environment, were among the most influential traits that contributed to the extremophytic lifestyle of S. parvula.

A dimeric proteomimetic prevents SARS-CoV-2 infection by dimerizing the spike protein.

Protein tertiary structure mimetics are valuable tools to target large protein-protein interaction interfaces. Here, we demonstrate a strategy for designing dimeric helix-hairpin motifs from a previously reported three-helix-bundle miniprotein that targets the receptor-binding domain (RBD) of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Through truncation of the third helix and optimization of the interhelical loop residues of the miniprotein, we developed a thermostable dimeric helix-hairpin. The dimeric four-helix bundle competes with the human angiotensin-converting enzyme 2 (ACE2) in binding to RBD with 2:2 stoichiometry. Cryogenic-electron microscopy revealed the formation of dimeric spike ectodomain trimer by the four-helix bundle, where all the three RBDs from either spike protein are attached head-to-head in an open conformation, revealing a novel mechanism for virus neutralization. The proteomimetic protects hamsters from high dose viral challenge with replicative SARS-CoV-2 viruses, demonstrating the promise of this class of peptides that inhibit protein-protein interaction through target dimerization.

Unearthing the genotype-inhibitor phenotype association in severe haemophilia A: A north Indian cohort study.

INTRODUCTION: Various risk factors for inhibitor development in haemophilia A (HA) have been described but Indian data remains scanty. AIM: We aimed to evaluate the genetic changes in Indian HA-patients that are associated with the development of inhibitors. METHODS: All HA-patients with inhibitors who availed coagulation-laboratory services from January-2015 till December-2021 and had their samples preserved for DNA extraction were included in this study. An equal number of severity-matched HA patients without inhibitors were also included as controls. Intron 22 and intron 1 inversions in Factor VIII gene were identified using inverse-shifting-PCR. Inversion-negative patients were further assessed by targeted NGS, MLPA. RESULTS: Thirty HA-patients with inhibitors were identified. All had severe-HA. Thirty severe-HA-patients without inhibitors were also included as controls. Intron 22 inversion (63.3%) and large deletions (15%) were the commonest variants identified. There was no difference in genetic variants in patients with low and high titre inhibitors. A3, A2 and C2 were the most common domains involved in inversion-negative patients with inhibitors. However, there was no significant difference in domain involvement among inversion-negative patients with and without inhibitors. Seven novel-variants were identified, including three large deletions, one large duplication and two nonsense variants in inhibitor-positive patients, and one frameshift variant in inhibitor-negative patient. After adjusting for clinical risk-factors, large deletions were independently associated with the presence of inhibitors [aOR:6.1 (1.41-56.3)]. CONCLUSION: Intron 22 inversions are the commonest variant in Indian patients with severe-HA. Large deletions predispose to inhibitor development independent of clinical risk factors.

Pancreatic Cancer and Venous Thromboembolism.

Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of all pancreatic cancers and is the most fatal of all cancers. The treatment response from combination chemotherapies is far from satisfactory and surgery remains the mainstay of curative strategies. These challenges warrant identifying effective treatments for combating this deadly cancer. PDAC tumor progression is associated with the robust activation of the coagulation system. Notably, cancer-associated thrombosis (CAT) is a significant risk factor in PDAC. CAT is a concept whereby cancer cells promote thromboembolism, primarily venous thromboembolism (VTE). Of all cancer types, PDAC is associated with the highest risk of developing VTE. Hypoxia in a PDAC tumor microenvironment also elevates thrombotic risk. Direct oral anticoagulants (DOACs) or low-molecular-weight heparin (LMWH) are used only as thromboprophylaxis in PDAC. However, a precision medicine approach is recommended to determine the precise dose and duration of thromboprophylaxis in clinical setting.

Polymorphism of growth hormone (GH) gene and its association with performance and body conformation of Harnali sheep.

The present study was carried out to study the polymorphism in the GH gene and its association with various performance and body conformation traits, viz., birth weight (B-WT), weaning weight (W-WT), six-month body weight (6 M-WT), one-year body weight (Y-WT), annual greasy fleece weight (AGFW), body length (BL), body height (BH), heart girth (HG) and paunch girth (PG) in 138 Harnali sheep. PCR-RFLP was performed to identify polymorphism in the targeted region of the GH gene. The PCR product of 422 bp size of the GH gene was amplified encompassing partial exon 2 and inton 3 in Harnali sheep. The PCR product was digested with HaeIII restriction enzyme for the detection of Single nucleotide polymorphism (SNP). The digested products revealed the presence of two genotypes, i.e. AA and AB in the studied population. A > G mutation (A781G) was observed in our resource population. The AA genotype was found to be the predominant genotype (0.62). Chi square value revealed that resource population was not under Hardy-Weinberg equilibrium with respect to target locus. Period of birth was found to have significant effect on W-WT, Y-WT, BL, BH and PG. Sex of animal was found to have significant (P < 0.05) effect on W-WT and highly significant (P < 0.01) effect on 6 M-WT, Y-WT and AGFW in Harnali sheep. The effect of genotype was found to be significant (P < 0.05) on annual greasy fleece weight. AB genotype was found to be associated with higher annual greasy fleece weight and can be used as a potential candidate marker in selection criteria for improving greasy fleece weight in Harnali sheep.

The NIN-LIKE PROTEIN 7 transcription factor modulates auxin pathways to regulate root cap development in Arabidopsis.

The root cap is a small tissue located at the tip of the root with critical functions for root growth. Present in nearly all vascular plants, the root cap protects the root meristem, influences soil penetration, and perceives and transmits environmental signals that are critical for root branching patterns. To perform these functions, the root cap must remain relatively stable in size and must integrate endogenous developmental pathways with environmental signals, yet the mechanism is not clear. We previously showed that low pH conditions altered root cap development, and these changes are mediated by the NIN LIKE PROTEIN 7 (NLP7) transcription factor, a master regulator of nitrate signaling. Here we show that in Arabidopsis NLP7 integrates nitrate signaling with auxin pathways to regulate root cap development. We found that low nitrate conditions promote aberrant release of root cap cells. Nitrate deficiency impacts auxin pathways in the last layer of the root cap, and this is mediated in part by NLP7. Mutations in NLP7 abolish the auxin minimum in the last layer of the root cap and alter root cap expression of the auxin carriers PIN-LIKES 3 (PILS3) and PIN-FORMED 7 (PIN7) as well as transcription factors that regulate PIN expression. Together, our data reveal NLP7 as a link between endogenous auxin pathways and nitrate signaling in the root cap.

Molecular spectrum of inherited FVII deficiency in North India revealed a recurrent variant with a founder effect.

INTRODUCTION: Inherited Factor VII (FVII) deficiency is commonest among the rare bleeding disorders. A small number of patients present in infancy with severe bleeding, and many may remain asymptomatic but detected before surgery/invasive procedures. Genetic testing may be helpful in predictive testing/prenatal diagnosis in severe cases. AIM: Characterisation of clinical and genotypic spectrum of patients with inherited FVII deficiency. METHODS: Retro-prospectively, 35 cases with prolonged prothrombin time and FVII activity (FVII:C) <50 IU/dl were subjected to targeted resequencing. After in-silico analysis, variant/s were validated by Sanger sequencing in index cases and family members. Haplotype analysis was done for F7 polymorphisms. RESULTS: Severe FVII deficiency was found in 50% of patients (FVII:C ≤1 IU/dl), and 42.9% were asymptomatic. Clinical severity assessment revealed 17% severe, 17% moderate and 22.9% patients with mild bleeds. FVII levels ranged from .3 to 38 IU/dl. Molecular analysis revealed variants in 30/35 cases, of which 17 were homozygous, 10 were compound heterozygous and 3 were heterozygous. Twelve genetic variants were identified, one promoter variant c.-30A>C; seven missense (c.215C>G, c.244T>C, c.253G>C, c.904G>A, c.961C>T, c.1109G>T, c.1211G>A), two deletions (c.21delG, c.868_870delATC), and one each of nonsense c.634C>T and splice-site variant c.316+1G>A. Recurrent variants c.1109G>T and c.215C>G were found in 17 and 8 cases, 12 of the former cases were homozygous. They had the same haplotype, indicating the founder effect in North Indians. CONCLUSION: This is the largest cohort of FVII genotyping from India, confirming heterogeneity in terms of clinical manifestations, FVII activity and zygosity of the variants with a limited genotypic phenotypic correlation.

Cherenkov Radiation in Optical Fibres as a Versatile Machine Protection System in Particle Accelerators.

Machine protection systems in high power particle accelerators are crucial. They can detect, prevent, and respond to events which would otherwise cause damage and significant downtime to accelerator infrastructure. Current systems are often resource heavy and operationally expensive, reacting after an event has begun to cause damage; this leads to facilities only covering certain operational modes and setting lower limits on machine performance. Presented here is a new type of machine protection system based upon optical fibres, which would be complementary to existing systems, elevating existing performance. These fibres are laid along an accelerator beam line in lengths of ∼100 m, providing continuous coverage over this distance. When relativistic particles pass through these fibres, they generate Cherenkov radiation in the optical spectrum. This radiation propagates in both directions along the fibre and can be detected at both ends. A calibration based technique allows the location of the Cherenkov radiation source to be pinpointed to within 0.5 m with a resolution of 1 m. This measurement mechanism, from a single device, has multiple applications within an accelerator facility. These include beam loss location monitoring, RF breakdown prediction, and quench prevention. Detailed here are the application processes and results from measurements, which provide proof of concept for this device for both beam loss monitoring and RF breakdown detection. Furthermore, highlighted are the current challenges for future innovation.

COVID-19 pandemic and care of transfusion-dependent patients of thalassaemia: Experience from a paediatric centre in North India.

AIM: COVID-19 has presented an unprecedented challenge to health services and has significantly affected the management of non-Covid illnesses, like thalassemia. The present study documents the impact of Covid-associated restrictions and disruptions on working of the pediatric thalassemia day care centre (TDCC), and measures taken by TDCC and blood transfusion services to adapt to and mitigate the negative impact of Covid pandemic and associated lockdown on patient care. METHODS: Pre-transfusion haemoglobin and packed cell transfusion requirement were compared across three time periods, namely pre-lockdown, lockdown and post-lockdown in paediatric transfusion-dependent thalassaemia (TDT) patients. Caregivers were interviewed to document any problems faced by them. RESULTS: The study involved 181 TDT patients. There was a significant reduction in mean pre-transfusion haemoglobin and red cells transfused during lockdown phase as compared to pre-lockdown phase. Regular care was interrupted in 45% of patients and 76% of patients getting blood from outside could not get leukoreduced red cells. Investigations, monitoring and continuity of iron chelation were also affected. Blood centre faced 30.5% reduction in blood supply during lockdown. TDCC and blood centre took several steps, including prolongation of service hours and staggering of transfusions to ensure maximum transfusions while ensuring social distancing. CONCLUSION: The COVID-19 pandemic imposed many unprecedented challenges to the routine care of thalassaemic patients; however, some of them could be dealt with by a proactive approach and micro-planning at the institution level. Other similar resource-limited settings could learn from experiences for continued quality care for chronic medical conditions during pandemic like situations.

The CDK Inhibitor SIAMESE Targets Both CDKA;1 and CDKB1 Complexes to Establish Endoreplication in Trichomes.

Endoreplication, also known as endoreduplication, is a modified cell cycle in which DNA is replicated without subsequent cell division. Endoreplication plays important roles in both normal plant development and in stress responses. The SIAMESE (SIM) gene of Arabidopsis (Arabidopsis thaliana) encodes a cyclin-dependent kinase (CDK) inhibitor that plays a central role in establishing endoreplication, and is the founding member of the SIAMESE-RELATED (SMR) family of plant-specific CDK inhibitor genes. However, there has been conflicting evidence regarding which specific cyclin/CDK complexes are inhibited by SIM in vivo. In this work, we use genetic evidence to show that SIM likely inhibits both CDKA;1- and CDKB1-containing CDK complexes in vivo, thus promoting endoreplication in developing Arabidopsis trichomes. We also show that SIM interacts with CYCA2;3, a binding partner of CDKB1;1, via SIM motif A, which we previously identified as a CDK-binding motif. By contrast, SIM motif C, which has been indicated as a cyclin binding motif in other contexts, appears to be relatively unimportant for interaction between SIM and CYCA2;3. Together with earlier results, our work suggests that SIM and other SMRs likely have a multivalent interaction with CYC/CDK complexes.

A Case of Novel GATA-1 Mutation-positive Transient Abnormal Myelopoiesis With Life-threatening Complications in a Neonate With Down Syndrome.

Transient abnormal myelopoiesis is a transient myeloproliferative disorder seen in ∼15% to 20% of infants with Down syndrome. These infants are usually asymptomatic, requiring only monitoring, but they can have variable severity of symptoms up to multisystemic dysfunction requiring chemotherapy. GATA-1 somatic mutations acquired in utero are pathognomic of this entity and present nearly in all cases. Herein, we present a case of Down syndrome in a neonate who presented within her first week of life with life-threatening features of transient abnormal myelopoiesis requiring chemotherapy support. In addition, next-generation sequencing revealed a small mutant clone (8%) positive for a novel frameshift GATA-1 mutation.

Evaluation of a fully automated bioinformatics tool to predict antibiotic resistance from MRSA genomes.

OBJECTIVES: The genetic prediction of phenotypic antibiotic resistance based on analysis of WGS data is becoming increasingly feasible, but a major barrier to its introduction into routine use is the lack of fully automated interpretation tools. Here, we report the findings of a large evaluation of the Next Gen Diagnostics (NGD) automated bioinformatics analysis tool to predict the phenotypic resistance of MRSA. METHODS: MRSA-positive patients were identified in a clinical microbiology laboratory in England between January and November 2018. One MRSA isolate per patient together with all blood culture isolates (total n = 778) were sequenced on the Illumina MiniSeq instrument in batches of 21 clinical MRSA isolates and three controls. RESULTS: The NGD system activated post-sequencing and processed the sequences to determine susceptible/resistant predictions for 11 antibiotics, taking around 11 minutes to analyse 24 isolates sequenced on a single sequencing run. NGD results were compared with phenotypic susceptibility testing performed by the clinical laboratory using the disc diffusion method and EUCAST breakpoints. Following retesting of discrepant results, concordance between phenotypic results and NGD genetic predictions was 99.69%. Further investigation of 22 isolate genomes associated with persistent discrepancies revealed a range of reasons in 12 cases, but no cause could be found for the remainder. Genetic predictions generated by the NGD tool were compared with predictions generated by an independent research-based informatics approach, which demonstrated an overall concordance between the two methods of 99.97%. CONCLUSIONS: We conclude that the NGD system provides rapid and accurate prediction of the antibiotic susceptibility of MRSA.

Screening for paroxysmal nocturnal hemoglobinuria (PNH) in patients presenting with cerebral sinovenous thrombosis (CSVT): Results of a FLAER based flowcytometry study in Indian patients.

Patients with paroxysmal nocturnal hemoglobinuria (PNH) may present with thrombosis at unusual sites, of which cerebral sinovenous thrombosis (CSVT) is one and screening for PNH is recommended in this condition. Though many patients diagnosed with PNH develop CSVT, it is unclear how many patients with PNH would present for the first time with thrombosis. We analysed the results of screening for PNH by flowcytometry in our patients with CSVT. The laboratory data of patients referred for thrombophilia and PNH testing in CSVT was examined to assess the frequency of PNH at presentation in these patients. FLAER and CD24 on granulocytes and FLAER and CD14 on monocytes respectively were used to screen the leucocytes for PNH by flowcytometry. The data for Protein C, S and Antithrombin deficiency, antiphospholipid antibodies and the Factor V Leiden mutation was examined and circumstantial risk factors were also assessed. Of the 180 cases of CSVT screened by flowcytometry for PNH, not a single case tested positive. Positivity for anti-phospholipid antibodies was the most common thrombophilic risk factor (5%). Pregnancy was the most common circumstantial risk factor. Our data on FLAER based flowcytometry in the North Indian population with CSVT suggests that PNH is not a common risk factor in our patients with thrombosis at this unusual site.

Cross-border spread of blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae: a European collaborative analysis of whole genome sequencing and epidemiological data, 2014 to 2019.

Analysis of sequencing data for 143 blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae isolates from 13 European national collections and the public domain resulted in the identification of 15 previously undetected multi-country transmission clusters. For 10 clusters, cases had prior travel/hospitalisation history in countries outside of the European Union including Egypt, Iran, Morocco, Russia, Serbia, Tunisia and Turkey. These findings highlight the benefit of European whole genome sequencing-based surveillance and data sharing for control of antimicrobial resistance.

Pilot Evaluation of a Fully Automated Bioinformatics System for Analysis of Methicillin-Resistant Staphylococcus aureus Genomes and Detection of Outbreaks.

Genomic surveillance that combines bacterial sequencing and epidemiological information will become the gold standard for outbreak detection, but its clinical translation is hampered by the lack of automated interpretation tools. We performed a prospective pilot study to evaluate the analysis of methicillin-resistant Staphylococcus aureus (MRSA) genomes using the Next Gen Diagnostics (NGD) automated bioinformatics system. Seventeen unselected MRSA-positive patients were identified in a clinical microbiology laboratory in England over a period of 2 weeks in 2018, and 1 MRSA isolate per case was sequenced on the Illumina MiniSeq instrument. The NGD system automatically activated after sequencing and processed fastq folders to determine species, multilocus sequence type, the presence of a mec gene, antibiotic susceptibility predictions, and genetic relatedness based on mapping to a reference MRSA genome and detection of pairwise core genome single-nucleotide polymorphisms. The NGD system required 90 s per sample to automatically analyze data from each run, the results of which were automatically displayed. The same data were independently analyzed using a research-based approach. There was full concordance between the two analysis methods regarding species (S. aureus), detection of mecA, sequence type assignment, and detection of genetic determinants of resistance. Both analysis methods identified two MRSA clusters based on relatedness, one of which contained 3 cases that were involved in an outbreak linked to a clinic and ward associated with diabetic patient care. We conclude that, in this pilot study, the NGD system provided rapid and accurate data that could support infection control practices.

Functional Analysis of Short Linear Motifs in the Plant Cyclin-Dependent Kinase Inhibitor SIAMESE.

Endoreplication, a modified cell cycle in which DNA is replicated without subsequent cell division, plays an important but poorly understood role in plant growth and in plant responses to biotic and abiotic stress. The Arabidopsis (Arabidopsis thaliana) SIAMESE (SIM) gene encodes the first identified member of the SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors. SIM controls endoreplication during trichome development, and sim mutant trichomes divide several times instead of endoreplicating their DNA. The SMR family is defined by several short linear amino acid sequence motifs of largely unknown function, and family members have little sequence similarity to any known protein functional domains. Here, we investigated the roles of the conserved motifs in SIM site-directed Arabidopsis mutants using several functional assays. We identified a potential cyclin-dependent kinase (CDK)-binding site, which bears no resemblance to other known CDK interaction motifs. We also identified a potential site of phosphorylation and two redundant nuclear localization sequences. Surprisingly, the only motif with similarity to the other family of plant CDK inhibitors, the INHIBITOR/INTERACTOR OF CDC2 KINASE/KIP-RELATED PROTEIN proteins, is not required for SIM function in vivo. Because even highly divergent members of the SMR family are able to replace SIM function in Arabidopsis trichomes, it is likely that the results obtained here for SIM will apply to other members of this plant-specific family of CDK inhibitors.

Purification, characterization and antibacterial spectrum of a compound produced by Bacillus cereus MTCC 10072.

Awareness of the consumer has increased the demand of safe and chemical-free foods, and consequently it has increased the demand of antibacterial bioactive compounds. In the present study, antibacterial compound produced by a local bacterial isolate NSD MTCC 10072, showing antagonistic activity against six human pathogens, was isolated, partially purified and characterized. Maximum production of antibacterial compound was observed between 51 and 60 h after seeding. The antibacterial activity of the compound was found to be thermostable up to 80 °C for 60 min and its efficacy was very good between pH 4 and 12. Minimum inhibitory concentration (25.84 µg/µl) of the antibacterial compound was observed against Streptococcus aureus NICM 2901. GC-MS analysis of the bacterium secreted chemical compound (C11H18N2O2) was used to identify the antimicrobial compound as Pyrrolo(1,2-a) pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl). In Silico studies showed that the antimicrobial compound is non-toxic, non-irritating and followed Lipinski-type properties which suggested that the compound could be used as potential drug against different human pathogens.

Genetic basis of unexplained erythrocytosis in Indian patients.

OBJECTIVE: To evaluate the spectrum of genetic defects in Indian patients with unexplained erythrocytosis. METHODS: Fifteen families (18 patients) with unexplained erythrocytosis were enrolled after excluding polycythemia vera and secondary erythrocytosis. Focused Sanger sequencing from genomic DNA was performed for EPOR (exon 8), VHL (exons 2-3), EGLN1 (exons 2-5), EPAS1 (exon 12), and all exons of HBB, HBA1, and HBA2 genes. RESULTS: Eleven of the 18 patients (including two pairs of brothers) had Chuvash polycythemia, that is, homozygosity for VHL:c.598C > T (p.Arg200Trp). Three patients (two of whom were brothers) had HBB mutations associated with increased oxygen-affinity hemoglobin-one had a heterozygous Hb McKees Rocks HBB:c.438T > A (p.Tyr146*), and two brothers showed heterozygous Hb Rainier HBB:c.437A > G (p.Tyr146Cys). No pathogenic variants were found in the remaining four cases. CONCLUSION: A gene-by-gene Sanger sequencing approach could determine a genetic basis for erythrocytosis in 11 of the 15 (73%) Indian families, with homozygous VHL:c.598C > T (p.Arg200Trp) being the commonest pathogenic variant. This first study from the Indian subcontinent provides a rationale for analyzing this variant in patients with suspected congenital erythrocytosis from this region. Rare first occurrences of Hb McKees Rocks and Hb Rainier in Indians are also being reported.