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Abstract The NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) is a critical intracellular inflammasome sensor and an important clinical target against inflammation-driven human diseases. Recent studies have elucidated its transition from a closed cage to an activated disk-like inflammasome, but the intermediate activation mechanism remains elusive. Here we report the cryo-electron microscopy structure of NLRP3, which forms an open octamer and undergoes a ~ 90° hinge rotation at the NACHT domain. Mutations on open octamer’s interfaces reduce IL-1β signaling, highlighting its essential role in NLRP3 activation/inflammasome assembly. The centrosomal NIMA-related kinase 7 (NEK7) disrupts large NLRP3 oligomers and forms NEK7/NLRP3 monomers/dimers which is a critical step preceding the assembly of the disk-like inflammasome. These data demonstrate an oligomeric cooperative activation of NLRP3 and provide insight into its inflammasome assembly mechanism.

Background: Afibrinogenemia is a rare disorder, with autosomal recessive inheritance, most often associated with consanguinity. To date, very few cases have been reported from India. The aim was to study the clinical heterogenicity of patients with afibrinogenemia. Case series: We present a retrospective study of five patients with afibrinogenemia diagnosed at our institute. All five patients had bleeding as a primary symptom. The median age for the first episode of bleeding was 150 days (range: 2 days - 18 months). Four were born of third degree consanguineous couple. The male to female ratio was 0.66. One patient had thrombosis along with bleeding. All patients had normal platelet count with deranged prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, and low to nil fibrinogen levels. All had at least one episode of deep bleeding. The sites of bleeding included: a) intracranial (n = 3), b) intramuscular (n = 1), and, c) hemarthrosis (n = 1). Conclusion: Afibrinogenemia should be considered as a differential diagnosis in patients with bleeding from any site with prolongation of PT and APTT. Paradoxical arterial and venous thrombosis may be a manifestation, but is rare. Thrombotic phenotype requires management with fibrinogen infusion along with anti-coagulants.

Abstract The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.

Objective: To analyze the clinical profile and available treatment modalities in pure red cell aplasia (PRCA). Methodology: Retrospective analysis of children diagnosed with PRCA from 2014 to 2019. Clinical profile, investigations, and treatment details were analyzed. Results: Eleven patients were diagnosed with PRCA; 5 congenital (mean age: 8.5 months) and 6 acquired (mean age: 13.5 years). In the congenital group, 5 had Diamond Blackfan Anemia (DBA); genetically proven in 3. Children with DBA recieved steroid trial for a mean duration of 9 months. Four patients required chronic transfusion, while one (with RPS 19 mutation) responded to steroids. Of the six in the acquired group, two each had transient erythroblastopenia of childhood, and parvovirus B19 infection. Two patients with PRCA post-allogenic HSCT (major ABO incompatibility) responded to multiple modalities of treatment with satisfactory results up to day +89 post-transplant. Conclusion: PRCA is a multi-faceted disease with many clinico-hematological presentations. We describe varied outcomes in children with various forms of PRCA receiving multiple treatment modalities.

Introduction: Patients with sickle cell disease (SCD) show a high prevalence of red blood cell (RBC) alloimmunization, however, few studies have focused on children. The present study was undertaken to determine the prevalence of alloimmunization and autoimmunity in children with SCD and to assess any correlation with age, number of transfusions received, and the number and frequency of crisis. Material & methods: Sixty children affected with homozygous SCD, aged between five to eighteen years and having received more than one packed red blood cell (PRBC) transfusion were enrolled in the study.The preliminary tests included antiglobulin test (indirect and direct), alloantibody screening by three panel, antibody typing using 11 panel and auto-control test to detect autoantibodies. Results: Direct antiglobulin test was negative in all the 60 patients, whereas indirect antiglobulin test was positive in 4/60 (6.6%) patients. Specific typing of alloantibodies revealed anti-D and anti-Kell antibodies in 2 (3.3%) each. Patients with alloantibody (4) had a higher mean age at onset of first symptom (7.05 ± 2.12 years) and age at first transfusion (7.5 ± 2.12 years) as compared to those without alloantibody, (4.48 ± 3.09 and 4.91 ± 3.12 years respectively). Patients with alloantibody received more PRBC transfusions, maintained low hemoglobin, required higher doses of hydroxyurea and also had a larger liver and spleen. Conclusion: The present study found a low prevalence of antibodies in patients with SCD. Patients presenting with symptoms of SCD at younger age received hydroxyurea at an earlier age. Starting hydroxyurea at an earlier age may decrease subsequent alloantibodies development. Larger studies to compare groups of patients related to administration of hydroxyurea are required to confirm this hypothesis.

Background: Osteoporosis in Thalassemia Major (TM) represents a prominent cause of morbidity. Calcium is important for bone health throughout the life. If not well transfused, the medullary as well as extramedullary erythropoiesis is stimulated. This leads to thinning of the cortices of long bones and marked dilatation of the medullary cavities. Aims and Objectives: To evaluate the efficacy of bisphosphonates in thalassemia patients with osteopenia/osteoporosis and to study the safety profile of bisphosphonates. Material and Methods: The prospective interventional study was conducted from January 2019-March 2020 at thalassemia day care centre of a tertiary care hospital after approval from Institutional Ethics Committee. Of the 150 thalassemia patients, 40 diagnosed with B-thalassemia major with more than 7 years of age, who were on regular transfusion and with Dual X-Ray Absorptiometry (DEXA) scan showing osteopenic/osteoporotic changes were exclusively included in the study. Age appropriate consent, history, clinical examination and biochemical investigations were done. All patients whose Bone Mineral Density (BMD) was showing osteoporosis were started on Alendronate 25 mg/week orally in the morning on empty stomach. The patients were followed up monthly for one year, after which they were evaluated with repeat biochemical tests and DEXA scan. Results: Majority of children were in the age group of 10-15 years (50%) with male predominance (55%). At pre-treatment, 24 (60%) and 20 (50%) patients had backache and bone pain respectively which significantly reduced to 4 (10%) each at post-treatment. Mean serum calcium improved from 8.27 g% before treatment to 9.26 g% post-treatment. Of the 40 patients, 8 (20%) had osteopenia and 32 (80%) had osteoporosis before starting the therapy. Post-treatment, there was significant increase in bone mass, 10 (25%) patients had normal Z score, 14 (35%) patients had osteopenia and only 16 (40%) had osteoporosis with improved BMD. Only 2 (5%) children had developed twitching secondary to hypocalcemia. Conclusion: DEXA scan must be done routinely in all thalassemia patients. Bisphosphonates along with adequate calcium can effectively reduce bone related morbidity in thalassemics with good safety profile.

Introduction: Delta-beta thalassemia and hereditary persistence of fetal hemoglobin (HPFH) results from a deletion in both the delta and beta genes on chromosome 11. Heterozygous/Homozygous delta-beta thalassemia and HPFH have a varied clinical features including age of presentation, transfusion requirement and mild to severe anemia. Aim: To study the clinical profile of patients with compound heterozygous delta beta thalassemia/HPFH. Methods: Retrospective analysis of 5 patients diagnosed as compound heterozygous delta-beta thalassemia/HPFH and followed up in the tertiary care hospital. The diagnosis was done by High Performance Liquid Chromatography (HPLC) of the patients and their parents. The data was collected from medical records of the hospital. Results: Five patients diagnosed as compound heterozygous delta-beta thalassemia/HPFH on HPLC were studied, of which 3 were males and 2 females. The mean age was 7.6 years (range 2–15). All patients had pallor and spleno-hepatomegaly at presentation. The mean hemoglobin at presentation was 6.14 gm% (range 4.2–9.2) with peripheral smear suggestive of thalassemia like picture. Mutation studies were available for 4 patients. Three patients were transfusion independent; 2 were on transfusion regimen. Conclusion: Compound heterozygous delta-beta thalassemia/HPFH has varied clinical features with severity ranging from non-transfusion dependent to transfusion dependent. As there is limited literature on compound heterozygous delta-beta thalassemia/HPFH, such cases need to be identified and reported.

Introduction: Sleep is essential for physical and mental well being. Cancer and its therapy affect sleep which adds on to the burden of healthy living. Various studies have shown that disturbed sleep patterns have a bearing on the quality of life. Objective: To determine the incidence of abnormal sleep patterns and study its associations with treatment for cancer in children. Materials and methods: This cross-sectional study was conducted at a tertiary health-care centre of a metropolitan city in Western India from January to March 2017. Sleep was assessed in children between age group 3–15 years undergoing therapy for cancer using BEARS screening tool and the Children’s Sleep Habits Questionnaire (CSHQ). Questions were administered to parents/guardians accompanying the child. Results: A total of 63 children were included. The prevalence of sleep disturbance as assessed by CSHQ score was 69.8%. On BEARS screening 38(60%), reported a problem with sleep. Presence of parents had a positive impact on sleep. The mean CSHQ scores of the 25 children who had no parent reported was 46.2 and 20 of them had scores more than 41. The sleep domains significantly affected in these children were Bedtime Resistance, Sleep duration, Sleep anxiety, Night Awakenings, Parasomnia and Fatigue. Conclusion: Evaluation for the cause of sleep problems and interventions to address these issues is necessary for better care of these children. Further studies on impact of sleep disturbances on outcomes of cancer therapy and quality of life are the need of the hour.

The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.

The global spread of the SARS-CoV-2 virus has resulted in emergence of lineages which impact the effectiveness of immunotherapies and vaccines that are based on the early Wuhan isolate. All currently approved vaccines employ the spike protein S, as it is the target for neutralizing antibodies. Here we describe two SARS-CoV-2 isolates with unusually large deletions in the N-terminal domain (NTD) of the spike. Cryo-EM structural analysis shows that the deletions result in complete reshaping of the NTD supersite, an antigenically important region of the NTD. For both spike variants the remodeling of the NTD negatively affects binding of all tested NTD-specific antibodies in and outside of the NTD supersite. For one of the variants, we observed a P9L mediated shift of the signal peptide cleavage site resulting in the loss of a disulfide-bridge; a unique escape mechanism with high antigenic impact. Although the observed deletions and disulfide mutations are rare, similar modifications have become independently established in several other lineages, indicating a possibility to become more dominant in the future. The observed plasticity of the NTD foreshadows its broad potential for immune escape with the continued spread of SARS-CoV-2.

Numerous studies conducted in the last few years have produced figures showing that the incidence of musculoskeletal issues is continuously rising and that a variety of treatment options are available. Musculoskeletal illnesses (MSDs), including fractures, arthritis, and osteoporosis, are increasingly treated with electromagnetic fields (EMFs). As a non-invasive, secure, and efficient treatment tool with no apparent side effects, Pulsed Electromagnetic field (PEMF) are well recognized. The present study aims to evaluate the literature by reviewing the data already available on PEMF's effectiveness in the treatment of different musculoskeletal disorders. For locating the literature articles published in English language on various musculoskeletal diseases treated by PEMFs were included. Information was looked for in the databases of PubMed, Google Scholar, Cochrane, and SCOPUS. The result of the study shows that due to its great efficacy and few risk considerations, PEMF has a lot of potentials to become a separate or complementary treatment strategy for treating numerous musculoskeletal disorders. The present study concludes that numerous issues are still unresolved. Further research from well-designed, high-quality studies are required to standardise therapy parameters and identify the most effective process for healthcare decision-making prior to widespread clinical application. In this study, we aim to provide up-to-date details on the therapeutic applications, mechanism of action, and ethical issues surrounding PEMFs in musculoskeletal disorders.

Lactoferrin (LF) is an iron-binding glycoprotein released from mucous secreting cells and neutrophils. LF can be used in a broad range of eye diseases related to the retina, cornea, and optic nerve. The retina is particularly affected by oxidative stress inside the photoreceptor being constantly exposed to light which induces accumulation of reactive oxygen species (ROS) in the retinal pigmented epithelium (RPE) causing damage to photoreceptor recycling. Retinitis pigmentosa (RP) and macular degeneration are inherited retinopathies that consist of different disease-causing genes, that cause mutations with highly varied clinical consequences. Age-related macular degeneration is a chronic disease of the retina and one of the major causes of sight loss. This review provides an application of lactoferrin and LF-based nano-formulations or nanoparticles in the field of retinal diseases or corneal diseases such as retinitis pigmentosa, retinoblastoma, age-related macular degeneration (AMD), keratoconus and uveitis. Several studies have found that lactoferrin's antibacterial activity is not limited to its iron sequestration, but also its ability as a nanoparticle that acts as a carrier to deliver drugs by crossing the blood–retina barrier (BRB) and its involvement in cell cycle control, which is not possible by many transferrin proteins.

Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to determine the structures of ME3 in various ligand-binding states at near-atomic resolutions. ME3 is captured in the open form existing as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 Domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.

Various herpesvirus therapeutics target the viral DNA polymerase. Here, the authors present the crystal structure of herpesvirus polymerase in the elongating state with bound primer-template DNA and the broad-spectrum non-nucleoside inhibitor PNU-183792, which is of interest for further drug design.

Background: Metabolic syndrome is defined as a state of metabolic dysregulation characterized by insulin resistance, a predisposition to Type 2 diabetes mellitus and atherosclerotic vascular disease. The changing hormonal milieu with altered estrogen and testosterone ratio is the cause of metabolic syndrome at menopausal transition. Aims and Objectives: This study was designed to determine the incidence of metabolic syndrome and its various components in premenopausal and postmenopausal women from Punjab. Materials and Methods: This study was conducted on 200 females in the age group of 45–60 years attending outpatient department over a period of 2 years in the Department of Obstetrics and Gynaecology, Bebe Nanki Mother and Child Care Centre, Government Medical College, Amritsar. A detailed history and physical examination were done and recorded on a pro forma. Biochemical assessment comprising fasting blood glucose, triglycerides, and cholesterol was done. Metabolic syndrome was assessed as per the modified NCEP ATP III criteria. Results: Twenty-nine percent women were found to have newly onset metabolic syndrome, the incidence in premenopausal group was 16%, and in postmenopausal group was 42%. Among the components, the incidence of hypertension (58%) was the highest followed by waist circumference (42%). Maximum correlation of metabolic syndrome was found with high-density lipoprotein (odds ratio - 7.250) followed by waist circumference (odds ratio - 7.111). Conclusion: The incidence of metabolic syndrome was found to be higher in postmenopausal women than in premenopausal women. Currently, the need of the hour is lifestyle modification to reduce the emergence of metabolic syndrome and cardiovascular diseases.

Abstract Introduction Both inflammation and oxidative stress (OS) stimulate each other and act together in providing defense against recurrent vulvovaginal infections (RVVI). Both processes are in such a tight link, that defect in one may lead to defect in other; therefore, recognition and treatment of primary anomaly are of great clinical importance. Objective To investigate the relationship between PRRs, i.e., mannose-binding lectin (MBL) and dendritic cell-associated C-type lectin-1 (Dectin-1) and oxidative stress parameters, i.e., total antioxidant status (TAC), total oxidant status (TOS), and oxidative stress index (OSI), along with their dependency on system factors in modulating susceptibility to RVVI. Study design This case-control study included 258 RVVI cases and 203 age-matched controls. TAC, TOS, and OSI were determined according to modified standard protocols. MBL and Dectin-1 levels were assessed by commercially available kits. Results RVVI and bacterial vaginosis showed significantly low TAC than controls. Significantly low TOS was observed in cases than controls. RVVI and vulvovaginal candidiasis showed significantly low OSI than controls. Significantly low MBL and high Dectin-1 levels were observed in cases than controls. Significant, but unexpected weak relationship, was observed between OS parameters and PRRs. Serum biomarkers were found to be dependent on confounding variables. Conclusion Low MBL levels were found to be the primary source of defect in the present study that leads to the dysregulated immune system with no observed protective oxidative stress, increasing susceptibility to RVVI. Therefore, substituting the required MBL in representative Indian population may restore host homeostasis and provide recovery from RVVI.

Abstract Background Human mannose binding lectin (MBL) and dendritic cell-associated C-type lectin-1 (Dectin-1) are the two prototypical PRRs of innate immunity, whose direct role in recurrent vulvovaginal infections (RVVI) defense has been defined. Previously, MBL insufficiency was proposed as a possible risk factor for the rapid progression of RVVI while, Dectin-1 was found to be playing an active role in the defense. However, the complete genetic bases for the observed low MBL levels are still lacking as our previous studies in harmony with others demonstrated the un-expected genotype–phenotype patterns. This suggested the presence of unidentified regulatory variants that may modulate sMBL levels and risk of RVVI. Therefore, the present study was designed for more inclusive locus-wide MBL2 analysis and for the possible non-linear interaction analysis of two PRRs that may impact RVVI susceptibility. Methods The present study has extended the previous findings by investigating (1) the role of chosen additional SNPs falling in the 5′ near region relating to sMBL levels and RVVI susceptibility, using polymerase chain reaction-restriction fragment length polymorphism, (2) interactions among SNPs within gene by comprehensive locus-wide haplotype analyses of two MBL2 blocks, (3) gene–gene interaction analyses between two PRRs, using multifactor dimensionality reduction. Results rs11003124_G, rs7084554_C, rs36014597_G, and rs11003123_A were observed as the minor alleles in the representative North Indian cohort. RVVI cases and its types showed an appreciably high frequency of C allele, its homozygosity and heterozygosity, explaining the observed dominant mode of inheritance of rs7084554 polymorphism in contributing 1.81 fold risk of RVVI. The rs36014597 polymorphism showed the overdominant mode of inheritance, which further depicts that the carrier of a heterozygous genotype of this polymorphism had more extreme phenotype than either of its homozygous carriers in developing 4.07 fold risk of RVVI. sMBL levels significantly varied for rs11003124, rs36014597 and rs11003123 polymorphisms in bacterial vaginosis, while for rs7084554 polymorphism in mixed infection. Independent analysis of 5′ and 3′ haplotype blocks suggested the risk-modifying effect of all the 5′ additional variants, Y/X secretor polymorphism and 3′-UTR SNP i.e. rs10824792. Combined 5′/3′ haplotype analyses depicted the importance of rs36014597; an additional 5′ variant, Y/X and rs10824792 polymorphisms from both the blocks in regulating sMBL levels and RVVI risk. Three gene–gene interaction models involving uni-variant, bi-variant and tri-variant appeared as significant predictors of RVVI risk with cross-validation consistency of 10/10, 9/10 and 5/10, respectively. Conclusions The study presented a low-cost reproducible screening design for additional 5′ variants i.e. rs11003124, rs7084554, rs36014597 and rs11003123 of MBL2 that can act as markers of susceptibility for RVVI or any other diseases. Two additional 5′ variants of MBL2 i.e. rs7084554 and rs36014597 were suggested as novel molecular markers that may contribute to RVVI risk by varying sMBL levels. Variants of two blocks were found to have more of a combined effect than the independent effect in modulating RVVI susceptibility and sMBL levels. The study presented weak synergistic interaction between MBL2 and CLEC7A in association with RVVI risk. The preliminary data will establish the foundation for the investigation of within gene and between genes interaction analyses towards RVVI susceptibility.

Introduction: Anemia has been widely reported to predict a poorer prognosis for HIV-infected patients, both in terms of progression to AIDS and in survival. This study aimed to determine the etiology of anemia and its immunological correlation in HIV-infected children. Materials and Methods: Four hundred and eighty-nine HIV-infected children were screened, of which 86 HIV-infected children with anemia were enrolled. Standard WHO definitions were used for anemia, HIV staging, and growth parameters. Chi-square test, t-tests, and univariate and multivariate logistic regression analyses were used to analyze the data. Results: Anemia was present in 17.58% (86/489) of HIV-infected children, including 84.6% with moderate anemia, 11.5% with severe anemia, and 2.32% with mild anemia. The mean hemoglobin (Hb) among patients with CD4 count 350 cell/mm3 (P = 0.02). Children with severe immunological stage had a significantly lower mean Hb (adjusted estimate: −1.61, 95% confidence interval: −2.65, −0.56) compared to those who had normal immune status. No statistically significant differences in mean Hb at baseline when compared to various demographic and clinical characteristics were observed in unadjusted and adjusted regression models. Conclusion: Hb is an easy and inexpensive tool to measure and can be used for monitoring disease progression in a resource-limited setting.

The problem of antibiotic resistance has prompted researchers around the globe to search for new antimicrobial agents. Antimicrobial proteins and peptides are naturally secreted by almost all the living organisms to fight infections and can be safer alternatives to chemical antibiotics. Lactoferrin (LF) is a known antimicrobial protein present in all body secretions. In this study, LF was digested by trypsin, and the resulting hydrolysates were studied with respect to their antimicrobial properties. Among the hydrolysates, a 21-kDa basic fragment of LF (termed lactosmart) showed promise as a new potent antimicrobial agent. The antimicrobial studies were performed on various microorganisms including Shigella flexneri, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli as well as fungal pathogens such as Candida albicans, Candida tropicalis, and Candida glabrata. In addition, the lipopolysaccharide (LPS)-binding properties of lactosmart were studied using surface plasmon resonance technique in vitro, along with docking of LPS and molecular dynamics (MD) simulation studies. The results showed that lactosmart had better inhibitory effects against pathogenic microorganisms compared to LF. The results of docking and MD simulation studies further validated the tighter binding of LPS to lactosmart compared to LF. The two LPS-binding sites have been characterized structurally in detail. Through these studies, it has been demonstrated that in native LF, only one LPS-binding site remains exposed due to its location being on the surface of the molecule. However, due to the generation of the lactosmart molecule, the second LPS-binding site gets exposed too. Since LPS is an essential and conserved part of the bacterial cell wall, the pro-inflammatory response in the human body caused by LPS can be targeted using the newly identified lactosmart. These findings highlight the immense potential of lactosmart in comparison to native LF in antimicrobial defense. We propose that lactosmart can be further developed as an antibacterial, antifungal, and antibiofilm agent.

Introduction: Endometrial Hyperplasia (EH) is a nonneoplastic proliferation of endometrial glands, resulting from the action of unopposed estrogen, which if unchecked for long, can cause certain genetic alterations that eventually lead to the development of endometrial adenocarcinoma. Paired Box 2 (PAX2) is a gene coding for a transcription factor required during embryonic development, which has been found to mutate early during endometrial carcinogenesis. Aim: Our study aimed at evaluating loss of PAX2 gene expression in different types of EHs and analyzing its utility in detecting Endometrial Intra-epithelial Neoplasia (EIN). Materials and Methods: It was a cross-sectional study conducted in the duration of 1.5 years, from January 2014 to June 2015. Total 50 cases diagnosed as EH were categorised as one of the four WHO sub-types. Further, these were catergorised as per EIN classification. Thereafter, PAX2 immunohistochemical staining was applied and percentage loss of PAX2 staining was evaluated and the results obtained were analysed statistically. Mainly Chi-square test and ANOVA test were applied. Results: Simple hyperplasia without atypia was found to be the most common sub type where as simple hyperplasia with atypia the least common. 29/33 (87.89%) cases of simple hyperplasia without atypia showed 50%. 13/14 (92.86%) of the EIN cases showed PAX2 loss >50%, thus showing a more consistent loss of PAX2. Conclusion: Hence, it is concluded that >50% loss of PAX2 staining indicates early endometrial carcinogenesis, and is suggested as an aid in the diagnosis of EIN.

Recurrent Vulvovaginal infections (RVVI) are the commonly reported microbiological syndrome affecting millions of women globally. Various molecules of innate immune system are instrumental in clearance of these microbial pathogens, thus suggested as one of the most important contributing factor in determining the disease outcome. Dendritic cell-associated C-type lectin-1 (Dectin-1) is an important molecule of innate immunity that is primarily known for its role in antifungal defenses. However, role of dectin-1 in recognition of other pathogens is also documented. The intracellular expression of dectin-1 was shown to be up-regulated by Mannose Binding Lectin (MBL)-mediated opsonophagocytosis of pathogens. Dectin-1 is encoded by CLEC7A, postulated to be a candidate gene in modulating risk of developing RVVI. In this study, we identified CLEC7A causal variants using in silico analysis. To assess their impact on susceptibility to RVVI, these causal variants along with serum dectin-1 levels (sDectin-1) were investigated using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) and Enzyme Linked Immnosorbent Assay (ELISA) respectively, under a case-control design. Furthermore, effect of these polymorphisms was also assessed on sMBL levels. In silico analysis revealed 9 putative functional conserved SNPs of CLEC7A. Association analysis revealed a significantly lower risk of developing RVVI and its types in carriers of CLEC7A rs3901533 G allele and its homozygous genotypes (p < 0.05). The heterozygous genotype was associated with significant protection against RVVI (p = 0.004). Haplotypes GGG and GTA showed significant protection against RVVI (p < 0.0001; p = 0.0003), Bacterial Vaginosis (p = 0.03; p = 0.002), Vulvovaginal Candidiasis (p = 0.03; p = 0.01) and Mixed Infections (p = 0.007; p = 0.04). Mean sDectin-1 levels were significantly high in RVVI and its types compared to controls (p < 0.05). Further, genotype-phenotype stratification showed significant differences within/between cases groups and controls. The CLEC7A rs3901533 polymorphism was also found to be associated with sMBL levels. The present study contributed novel insights into the role of dectin-1 in RVVI. CLEC7A rs3901533 polymorphism and high sDectin-1 levels along with low sMBL levels were found to be associated with RVVI susceptibility. Thus, screening of women with RVVI for these novel associations may lead to better diagnosis and treatment. Also genotyping method used in this study constitutes a simple and reliable assay, which can be confidently, used as a cheaper alternative for genotyping these variants in clinical settings. Finally, new restorative markers for other infectious diseases might be found by exploring nine functionally identified CLEC7A SNPs.

Women with endometriosis (EMS) appear to be at a higher risk of developing other autoimmune diseases predominantly multiple sclerosis (MS). Though EMS and MS are evidently diverse in their phenotype, they are linked by a common autoimmune condition or immunodeficiency which could play a role in the expansion of endometriosis and possibly increase the risk of developing MS in women with EMS. However, the common molecular links connecting EMS with MS are still unclear. We conducted a meta-analysis of microarray experiments focused on EMS and MS with their respective controls. The GEO2R web application discovered a total of 711 and 1516 genes that are differentially expressed across the experimental conditions in EMS and MS, respectively with 129 shared DEGs between them. The functional enrichment analysis of DEGs predicts the shared gene expression signatures as well as the overlapping biological processes likely to infer the co-occurrence of EMS with MS. Network based meta-analysis unveiled six interaction networks/crosstalks through overlapping edges between commonly dysregulated pathways of EMS and MS. The PTPN1, ERBB3, and CDH1 were observed to be the highly ranked hub genes connected with disease-related genes of both EMS and MS. Androgen receptor (AR) and nuclear factor-kB p65 (RelA) were observed to be the most enriched transcription factor in the upstream of shared down-regulated and up-regulated genes, respectively. The two disease sample sets compared through crosstalk interactions between shared pathways revealed commonly up- and down-regulated expressions of 10 immunomodulatory proteins as probable linkers between EMS and MS. This study pinpoints the number of shared genes, pathways, protein kinases, and upstream regulators that may help in the development of biomarkers for diagnosis of MS and endometriosis at the same time through improved understanding of shared molecular signatures and crosstalk.

Birds were surveyed in the Yamuna Nagar District, Haryana, India and the threat categories of the species observed were determined with the IUCN Red List. A total of 185 species belonging to 46 families were observed. Families Accipitridae, Passeridae, Corvidae and Scolopacidae dominated the list. There were 13 species with various IUCN Red List categories. One Critically Endangered species, the Red-headed Vulture Sarcogyps calvus, and two Endangered species Black-bellied Tern Sterna acuticauda and Egyptian Vulture Neophron percnopterus were observed. A prerequisite for all the measures focussed on management and conservation of species is that the status and population trends of the target species must be known, and future work should be in this direction.

Background: Neuraxial labor analgesia using new local anesthetics such as levobupivacaine has become very popular by virtue of the safety and lesser motor blockade caused by these agents. Combined spinal-epidural analgesia (CSEA) has become the preferred method for labor analgesia as it combines benefits of both spinal analgesia and flexibility of the epidural catheter. Adding opioids to local anesthetic drugs provide rapid onset and prolonged analgesia but may be associated with several maternal and fetal adverse effects. The purpose of this study is to compare fentanyl and tramadol used in CSEA in terms of duration of analgesia and frequency of the adverse fetomaternal outcome. Materials and Methods: A total of 60 primiparas with a singleton pregnancy in active labor were given CSEA after randomly allocating them in two groups of 30 each. Group I received intrathecal 2.5 mg levobupivacaine + 25 μg fentanyl followed by epidural top ups of 20 ml 0.125% solution of the same combination. Group II received 25 mg tramadol instead of fentanyl. Epidural top ups were given when parturient complained of two painful contractions (visual analogue scale ≥ 4). Data collected were demographic profile of the patients, analgesic qualities, side- effects and the fetomaternal outcome. Results: Patients in Group II had significantly prolonged analgesia (145 ± 9 minutes) than in Group I (95 ± 7 minutes). Patients receiving fentanyl showed rapid onset of analgesia, but there were more incidence of side-effects like shivering, pruritus, transient fetal bradycardia, hypotension, nausea and vomiting. Only side-effect in the tramadol group was nausea and vomiting. During labor, maternal satisfaction was excellent. Conclusions: Adding tramadol to local anesthetic provides prolonged analgesia with minimal side effects. Fentanyl, when used as adjuvant to local anesthetic, has a rapid onset of analgesia but has certain fetomaternal side-effects.

Peptidyl-tRNA hydrolase (Pth) catalyses the release of tRNA and peptide components from peptidyl-tRNA molecules. Pth from a Gram-positive bacterium Streptococcus pyogenes (SpPth) was cloned, expressed, purified and crystallised. Three-dimensional structure of SpPth was determined by X-ray crystallography at 2.19 Å resolution. Structure determination showed that the asymmetric unit of the unit cell contained two crystallographically independent molecules, designated A and B. The superimposition of Cα traces of molecules A and B showed an r.m.s. shift of 0.4 Å, indicating that the structures of two crystallographically independent molecules were identical. The polypeptide chain of SpPth adopted an overall α/β conformation. The substrate-binding cleft in SpPth is formed with three loops: the gate loop, Ile91–Leu102; the base loop, Gly108–Gly115; and the lid loop, Gly136–Gly150. Unlike in the structures of Pth from Gram-negative bacteria, the entry to the cleft in the structure of SpPth appeared to be virtually closed. However, the conformations of the active site residues were found to be similar.

Objective: Magnesium has been used as an adjuvant by various routes, including intravenous, intrathecal, and epidural in different dosage regimens. The effect of single bolus dose of magnesium as an adjuvant to fentanyl for postoperative analgesia has not been studied. This prospective randomized controlled trial was done to evaluate the efficacy of single bolus administration of magnesium epidurally as an adjuvant to epidural fentanyl for postoperative analgesia in patients undergoing total hip replacement under combined spinal epidural anesthesia. Methods : Sixty patients received combined spinal-epidural anesthesia with 2 mL of 0.5% hyperbaric bupivacaine intrathecally. After the surgery, patients were randomized into Group F [epidural fentanyl (1 μg/kg) in 10 mL saline] and Group FM [epidural magnesium (75 mg) along with fentanyl (1 μg/kg) in 10 mL saline]. Supplementary analgesia was provided by 50 mg intravenous tramadol if Verbal Rating Score (VRS) >4. Patient′s first analgesic requirement and duration of analgesia were recorded. Results : The duration of analgesia was significantly longer for Group FM, 340±28.8 min, compared with Group F, 164±17.1 min (P=0.001). The frequency of rescue analgesics required in 24-h postoperative period in Group FM (2.3±0.5) was significantly less than that in Group F (4.3±0.5) (P=0.001). VRS was significantly lower in Group FM up to 4 h in the postoperative period (P=0.001). Bromage scale was statistically insignificant at all points of time. Conclusions : The administration of magnesium (75 mg) as an adjuvant to epidural fentanyl (1 μg/ kg) for postoperative analgesia results in significantly lower VRS with prolonged duration of analgesia as compared with epidural fentanyl (1 μg/kg) alone. Concomitant administration of magnesium also reduces the requirement of breakthrough analgesics with no increased incidence of side effects.

Freedom from pain has almost developed to be a fundamental human right. Providing pain relief via epidural catheters in thoracic and upper abdominal surgeries is widely accepted. Pain relief through this technique not only provides continuous analgesia but also reduces post-operative pulmonary complications and also hastens recovery. But being a blind procedure it is accompanied by certain complications. Hypotension, dura puncture, high epidural, total spinal, epidural haematoma, spinal cord injury and infection are some of the documented side effects of epidural block. There are case reports eliciting neurological complications, catheter site infections, paresthesias, radicular symptoms and worsening of previous neurological conditions. Few technical problems related to breakage of epidural catheter are also mentioned in the literature. The patient had no sequelae on long term follow up even when a portion of catheter was retained. We present a case report where epidural catheter punctured pleura in a patient undergoing thoracotomy for carcinoma oesophagus.

Enteric fever is a major cause of morbidity in several parts of the Indian subcontinent. The treatment for typhoid fever majorly includes the fluoroquinolone group of antibiotics. Excessive and indiscriminate use of these antibiotics has led to development of acquired resistance in the causative organism Salmonella Typhi. The resistance towards fluoroquinolones is associated with mutations in the target gene of DNA Gyrase. We have estimated the Minimum Inhibitory Concentration (MIC) of commonly used fluoroquinolone representatives from three generations, ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin, for 100 clinical isolates of Salmonella Typhi from patients in the Indian subcontinent. The MICs have been found to be in the range of 0.032 to 8 μg/ml. The gene encoding DNA Gyrase was subsequently sequenced and point mutations were observed in DNA Gyrase in the quinolone resistance determining region comprising Ser83Phe/Tyr and Asp87Tyr/Gly. The binding ability of these four fluoroquinolones in the quinolone binding pocket of wild type as well as mutant DNA Gyrase was computationally analyzed by molecular docking to assess their differential binding behaviour. This study has revealed that mutations in DNA Gyrase alter the characteristics of the binding pocket resulting in the loss of crucial molecular interactions and consequently decrease the binding affinity of fluoroquinolones with the target protein. The present study assists in understanding the underlying molecular and structural mechanism for decreased fluoroquinolone susceptibility in clinical isolates as a consequence of mutations in DNA Gyrase.

Lactoferrin is an 80 kDa bilobal, iron binding glycoprotein which is primarily antimicrobial in nature. The hydrolysis of lactoferrin by various proteases in the gut produces several functional fragments of lactoferrin which have varying molecular sizes and properties. Here, bovine lactoferrin has been hydrolyzed by trypsin, the major enzyme present in the gut, to produce three functional molecules of sizes approximately 21 kDa, 38 kDa and 45 kDa. The molecules have been purified using ion exchange and gel filtration chromatography and identified using N-terminal sequencing, which reveals that while the 21 kDa molecule corresponds to the N2 domain (21LF), the 38 kDa represents the whole C-lobe (38LF) and the 45 kDa is a portion of N1 domain of N-lobe attached to the C-lobe (45LF). The iron binding and release properties of 21LF, 38LF and 45LF have been studied and compared. The sequence and structure analysis of the portions of the excision sites of LF from various species have been done. The antibacterial properties of these three molecules against bacterial strains, Streptococcus pyogenes, Escherichia coli, Yersinia enterocolitica and Listeria monocytogenes were investigated. The antifungal action of the molecules was also evaluated against Candida albicans. This is the first report on the antimicrobial actions of the trypsin cleaved functional molecules of lactoferrin from any species.

Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens.

Lactoferrin is an iron-binding diferric glycoprotein present in most of the exocrine secretions. The major role of lactoferrin, which is found abundantly in colostrum, is antimicrobial action for the defense of mammary gland and the neonates. Lactoferrin consists of two equal halves, designated as N-lobe and C-lobe, each of which contains one iron-binding site. While the N-lobe of lactoferrin has been extensively studied and is known for its enhanced antimicrobial effect, the C-lobe of lactoferrin mediates various therapeutic functions which are still being discovered. The potential of the C-lobe in the treatment of gastropathy, diabetes, and corneal wounds and injuries has been indicated. This review provides the details of the proteolytic preparation of C-lobe, and interspecies comparisons of its sequence and structure, as well as the scope of its therapeutic applications.

The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.

Peptidoglycan recognition proteins (PGRPs) are part of the innate immune system. The 19 kDa Short PGRP (PGRP-S) is one of the four mammalian PGRPs. The concentration of PGRP-S in camel (CPGRP-S) has been shown to increase considerably during mastitis. The structure of CPGRP-S consists of four protein molecules designated as A, B, C and D forming stable intermolecular contacts, A-B and C-D. The A-B and C-D interfaces are located on the opposite sides of the same monomer leading to the the formation of a linear chain with alternating A-B and C-D contacts. Two ligand binding sites, one at C-D contact and another at A-B contact have been observed. CPGRP-S binds to the components of bacterial cell wall molecules such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) from both gram-positive and gram-negative bacteria. It also binds to fatty acids including mycolic acid of the Mycobacterium tuberculosis (Mtb). Previous structural studies of binary complexes of CPGRP-S with LPS and stearic acid (SA) have shown that LPS binds to CPGRP-S at C-D contact (Site-1) while SA binds to it at the A-B contact (Site-2). The binding studies using surface plasmon resonance showed that LPS and SA bound to CPGRP-S in the presence of each other. The structure determination of the ternary complex showed that LPS and SA bound to CPGRP-S at Site-1 and Site-2 respectively. LPS formed 13 hydrogen bonds and 159 van der Waals contacts (distances ≤4.2 Å) while SA formed 56 van der Waals contacts. The ELISA test showed that increased levels of productions of pro-inflammatory cytokines TNF-α and IFN-γ due to LPS and SA decreased considerably upon the addition of CPGRP-S.