Tissue proteome analysis for profiling proteins associated with lymph node metastasis in gallbladder cancer.

Lymph node (LN) metastasis is the earliest sign of metastatic spread and an established predictor of poor outcome in gallbladder cancer (GBC). Patients with LN positive GBC have a significantly worse survival (median survival- 7 months) than patients with LN negative disease (median survival- ~ 23 months) in spite of standard treatment which includes extended surgery followed by chemotherapy, radiotherapy and targeted therapy. This study aims at understanding the underlying molecular processes associated with LN metastasis in GBC. Here, we used iTRAQ-based quantitative proteomic analysis using tissue cohort comprising of primary tumor of LN negative GBC (n = 3), LN positive GBC (n = 4) and non-tumor controls (Gallstone disease, n = 4), to identify proteins associated with LN metastasis. A total of 58 differentially expressed proteins (DEPs) were found to be specifically associated with LN positive GBC based on the criteria of p value ≤ 0.05, fold change ≥ 2 and unique peptides ≥ 2. These include the cytoskeleton and associated proteins such as keratin, type II cytoskeletal 7 (KRT7), keratin type I cytoskeletal 19 (KRT19), vimentin (VIM), sorcin (SRI) and nuclear proteins such as nucleophosmin Isoform 1 (NPM1), heterogeneous nuclear ribonucleoproteins A2/B1 isoform X1 (HNRNPA2B1). Some of them are reported to be involved in promoting cell invasion and metastasis. Bioinformatic analysis of the deregulated proteins in LN positive GBC using STRING database identified 'neutrophil degranulation' and 'HIF1 activation' to be among the top deregulated pathways. Western blot and IHC analysis showed a significant overexpression of KRT7 and SRI in LN positive GBC in comparison to LN negative GBC. KRT7, SRI and other proteins may be further explored for their diagnostics and therapeutic applications in LN positive GBC.

Impact of the Covid-19 Pandemic on the Prevalence of MTP Cases and Their Clinicodemographic Profile in India: A Retrospective Multicentric Study.

Background: During the Covid-19 pandemic, reproductive health of women was disproportionately affected due to difficult access to safe abortion and contraceptive services. This study aims to assess the impact of the Covid-19 pandemic on the prevalence of MTP cases and to find out the clinicodemographic profiles of women undergoing MTP during three Covid-waves in different hospitals-Government and private sectors in India. Methods: This retrospective multicentric cohort study was conducted during three Covid-19 pandemic waves. The records were retrieved from the centers' medical record section and the MTP register from the Department of Obstetrics and Gynaecology. Results: On an average, 1.1 women/day underwent MTP during covid waves compared to 1.9 women/day during the pre-covid 2019. The first Covid wave's average MTP/day was very low (0.71) compared to the third (2.88) and second wave (1.12), respectively. These differences were statistically significant (p<0.0001). The most common indication for MTP was contraceptive failure 245(50.9%), followed by eugenic/congenital anomalies 88(18.9%). A total of 244 cases (50.6%) reported for MTP ≤ seven weeks and 114(23.6%) presented between 7 and 12 weeks. More than half (54%) of the women underwent surgical methods for abortion as the unavailability of medical abortion (MA) drugs. IUCD and sterilization were severely affected during the first and second Covid waves. Conclusion: Safe abortions are essential services for reproductive-age women. With the uncertainty of future Covid-like an emergency, we should strengthen our telemedicine network so that women can reach out early and MMA can be initiated to reduce the number of surgical abortions and unwanted pregnancies.

Proteomic profiling of cell line-derived extracellular vesicles to identify candidate circulatory markers for detection of gallbladder cancer.

Gallbladder cancer (GBC) is the sixth most common gastrointestinal tract cancer with a very low overall survival and poor prognosis. Profiling of cancer-derived extracellular vesicles (EVs) is an emerging strategy for identification of candidate biomarkers for the detection and prognosis of the disease. The aim of the study was to analyse the protein content from GBC cell line- derived EVs with emphasis on proteins which could be used as candidate biomarkers for the detection of GBC. NOZ and OCUG-1 cell lines were cultured and EVs were isolated from conditioned media. LC-MS/MS analysis of total EV proteins led to the identification of a total of 268 proteins in both the cell lines. Of these, 110 proteins were identified with ≥2 unique peptides with ≥2 PSMs in at least two experimental and technical replicate runs. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database was used to perform bioinformatics analysis of 110 proteins which showed 'cell adhesion molecule binding', 'integrin binding', 'cadherin binding' among the top molecular functions and 'focal adhesion' to be among the top pathways associated with the EV proteins. A total of 42 proteins including haptoglobin (HP), pyruvate kinase (PKM), annexin A2 (ANXA2), thrombospondin 1 (THBS1), were reported to be differentially abundant in GBC tissue. Of these, 16 proteins were reported to be differentially abundant in plasma and plasma-derived EVs. We infer these proteins to be highly important to be considered as potential circulatory biomarkers for the detection of GBC. To check the validity of this hypothesis, one of the proteins, haptoglobin (HP) as a representative case, was analysed in plasma by quantitative Enzyme- linked immunosorbent assay (ELISA) and we observed its increased levels in GBC in comparison to controls (p value= 0.0063). Receiver operating characteristic (ROC) curve analysis for GBC vs controls showed an Area under the ROC Curve (AUC) of 0.8264 for HP with 22% sensitivity against 100% specificity. We propose that HP along with other candidate proteins may be further explored for their clinical application.

Epidemiology, Clinical Features and Management of Rhino Orbital Mucormycosis in Post COVID 19 Patients.

COVID 19 infections may be associated with a wide range of bacterial and fungal co-infections. Mucormycosis is a fungal infection primarily affecting immunocompromised individuals. We have observed sudden rise of mucormycosis cases in post COVID 19 patients. Here we have reported 100 cases of mucormycosis associated with COVID 19. To study epidemiology and clinical features of rhino orbital mucormycosis in post COVID 19 patients. To evaluate efficacy of medical as well as surgical treatment in such patients. This was an observational mixed (retrospective + prospective) study with a duration of 2 months. After noting demographic data, necessary radiological investigation was advised and representative tissue was sent for KOH and histopathological examination. Medical and surgical treatment was planned accordingly. Most patients (55%) presented with complaint of headache and facial pain. Hard palate involvement was observed in 45% patients. Unilateral presentation (68%) was more common. Only 25% patients who presented early had normal vision. We reported 22 patients with complete loss of vision. Eye movements were restricted in 58% patients. Diabetes mellitus is most common predisposing factor (65%). 9 patients required orbital exentration. Only 18% patients required Amphotericin for more than 14 days. Immune dysregulation caused by COVID 19 infection in addition to widespread use of steroids and broad-spectrum antibiotics may lead to the development mucormycosis. Diabetes Mellitus type II is another important risk factor and the presence of both have additional effect in causing mucormycosis. Headache and facial pain should be considered highly suspicious of mucormycosis. Early diagnosis with efficient treatment can improve prognosis.

Quantitative tissue proteome profile reveals neutrophil degranulation and remodeling of extracellular matrix proteins in early stage gallbladder cancer.

Gallbladder cancer (GBC) is an aggressive malignancy of the gastrointestinal tract with a poor prognosis. It is important to understand the molecular processes associated with the pathogenesis of early stage GBC and identify proteins useful for diagnostic and therapeutic strategies. Here, we have carried out an iTRAQ-based quantitative proteomic analysis of tumor tissues from early stage GBC cases (stage I, n=7 and stage II, n=5) and non-tumor controls (n=6) from gallstone disease (GSD). We identified 357 differentially expressed proteins (DEPs) based on ≥ 2 unique peptides and ≥ 2 fold change with p value < 0.05. Pathway analysis using the STRING database showed, 'neutrophil degranulation' to be the major upregulated pathway that includes proteins such as MPO, PRTN3, S100A8, MMP9, DEFA1, AZU, and 'ECM organization' to be the major downregulated pathway that includes proteins such as COL14A1, COL1A2, COL6A1, COL6A2, COL6A3, BGN, DCN. Western blot and/or IHC analysis confirmed the elevated expression of MPO, PRTN3 and S100A8 in early stage of the disease. Based on the above results, we hypothesize that there is an increased neutrophil infiltration in tumor tissue and neutrophil degranulation leading to degradation of extracellular matrix (ECM) proteins promoting cancer cell invasion in the early stage GBC. Some of the proteins (MPO, MMP9, DEFA1) associated with 'neutrophil degranulation' showed the presence of 'signal sequence' suggesting their potential as circulatory markers for early detection of GBC. Overall, the study presents a protein dataset associated with early stage GBC.

Plasma-derived candidate biomarkers for detection of gallbladder carcinoma.

Gallbladder carcinoma (GBC) is a major cancer of the gastrointestinal tract with poor prognosis. Reliable and affordable biomarker-based assays with high sensitivity and specificity for the detection of this cancer are a clinical need. With the aim of studying the potential of the plasma-derived extracellular vesicles (EVs), we carried out quantitative proteomic analysis of the EV proteins, using three types of controls and various stages of the disease, which led to the identification of 86 proteins with altered abundance. These include 29 proteins unique to early stage, 44 unique to the advanced stage and 13 proteins being common to both the stages. Many proteins are functionally relevant to the tumor condition or have been also known to be differentially expressed in GBC tissues. Several of them are also present in the plasma in free state. Clinical verification of three tumor-associated proteins with elevated levels in comparison to all the three control types-5'-nucleotidase isoform 2 (NT5E), aminopeptidase N (ANPEP) and neprilysin (MME) was carried out using individual plasma samples from early or advanced stage GBC. Sensitivity and specificity assessment based on receiver operating characteristic (ROC) analysis indicated a significant association of NT5E and ANPEP with advanced stage GBC and MME with early stage GBC. These and other proteins identified in the study may be potentially useful for developing new diagnostics for GBC.

Modifying gap junction communication in cancer therapy.

AIM: Drug delivery is crucial for therapeutic efficacy and gap junction communication channels (GJIC) facilitate movement within the tumour. Pro-drug activation, a modality of cancer therapy leads to Ganciclovir triphosphate (GCV-TP) incorporation into newly synthesized DNA resulting in cell death. The objective was to enhance, with Histone deacetylase inhibitors (HDACi) and All Trans Retinoic Acid (ATRA), GJIC, crucial for drug delivery, and with combination, abrogate the observed detrimental effect of Dexamethasone (DXM). METHODS: Cell lines (NT8E, and HeLa) were pre-treated with Valproic Acid (VPA) (1 mM), 4 Phenyl Butyrate (4PB) (2 mM), ATRA (10 µM) and Dexamethasone (1 µM). Protein quantitated with the Bicinchoninic (BCA) assay for cell lysates, membrane and soluble fractions was assessed with Western blotting for Connexins (43, 26 and 32) and E-Cadherin. A qRT-PCR was done for CX 43-GJA1, CX 26-GJB2, CX 32-GJB1 and E-Cadherin, and normalized with Glyceraldehyde Phosphate dehydrogenase (GAPDH). Further, localization of Connexins (CX) and E-Cadherin, GJIC competence, pre-clinical in-vitro studies and the mechanism of cell death were evaluated. RESULTS: There was no toxicity or change in growth patterns observed with the drugs. In both the cell lines CX 43 localized to the membrane whereas CX 32 and CX 26 were present but not membrane bound. E-Cadherin was present on the membrane in NT8E and completely absent in HeLa cells. Effects of HDACi, DXM and ATRA were seen on the expression of Connexins and E-Cadherin in both the cell lines. NT8E and HeLa cell lines showed enhanced GJIC with 4PB [30 %], VPA [36 %] and ATRA [54 %] with a 60 % increase in cytotoxicity and an abrogation of Dexamethasone inhibition on combination with VPA or ATRA. CONCLUSION: An enhancement of GJIC function by HDACi and ATRA increased cytotoxicity and could be effective in the presence of Dexamethasone, when combined with ATRA or VPA.

Immunoproteomics approach revealed elevated autoantibody levels against ANXA1 in early stage gallbladder carcinoma.

BACKGROUND: Early diagnosis is important for the timely treatment of gallbladder carcinoma (GBC) patients and may lead to increased survival outcomes. Here, we have applied serological proteome analysis (SERPA), an immunoproteomics approach, for the detection of 'tumor-associated antigens (TAAs) that elicit humoral response' in early stage GBC patients. METHODS: Total protein from pooled tumor tissue of GBC patients (n = 7) was resolved by two-dimensional gel electrophoresis (2-DE) followed by immunoblotting using pooled blood plasma from healthy volunteers (n = 11) or gallstone disease (GSD) cases (n = 11) or early stage GBC (Stage I and II) (n = 5) or GBC stage IIIA (n = 9). 2-D gel and immunoblot images were acquired and analyzed using PDQuest software to identify immunoreactive spots in GBC cases in comparison to controls. Proteins from immunoreactive spots were identified by liquid chromatography- tandem mass spectrometric analysis (LC-MS/MS). Autoantibody levels for two of the functionally relevant proteins were investigated in individual plasma samples (52 cases and 89 controls) by dot blot assay using recombinant proteins. RESULTS: Image analysis using PDQuest software identified 25 protein spots with significantly high or specific immunoreactivity in GBC cases. Mass spectrometric analysis of 8 corresponding protein spots showing intense immunoreactivity (based on densitometric analysis) in early stage GBC or GBC stage IIIA cases led to the identification of 27 proteins. Some of the identified proteins include ANXA1, HSPD1, CA1, CA2, ALDOA and CTSD. Among the two proteins, namely ANXA1 and HSPD1 verified using a cohort of samples, significantly elevated autoantibody levels against ANXA1 were observed in early stage GBC cases in comparison to healthy volunteers or GSD cases (unpaired t-test, p < 0.05). Receiver operating characteristic (ROC) curve analysis for ANXA1 showed an Area under the Curve (AUC) of 0.69, with 41.7% sensitivity against a specificity of 89.9% for early stage GBC. IHC analysis for ANXA1 protein showed 'high' expression levels in 72% of GBC cases whereas all the controls showed 'low' expression levels. CONCLUSIONS: The study suggests that the ANXA1 autoantibody levels against ANXA1 may be potentially employed for early stage detection of GBC patients. Other proteins could also be explored and verified in a large cohort of clinical samples.

Ambient PM2.5 exposure and premature mortality burden in the holy city Varanasi, India.

More than 3 million population residing in the holy city Varanasi and sub-urban areas is exposed to very high level of fine particulate matter (PM2.5) from various sources. Continuous monitoring by Central Pollution Control Board started only in 2015; therefore what was the pollution level in the past and how it has changed over the years are not known. We use MODIS aerosol products to infer PM2.5 and examine 15-year climatology. Data shows a rapid (1.5-3% per year) increase in PM2.5 in the last 15 years and high (87% days in a year) persistence of PM2.5 above the national air quality standard. It translates to a burden of 5700 (2800-7500) annual premature deaths (0.16% of the population), of which 29%, 18%, 33%, 19% and remaining 1% are attributed to ischemic heart disease, stroke, chronic obstructive pulmonary disease, acute lower respiratory infection and lung cancer respectively. If the region achieves the Indian (WHO) air quality standard, 1900 (3800) premature deaths can be avoided every year.

Small group learning: effect on item analysis and accuracy of self-assessment of medical students.

BACKGROUND: Small group sessions are regarded as a more active and student-centered approach to learning. Item analysis provides objective evidence of whether such sessions improve comprehension and make the topic easier for students, in addition to assessing the relative benefit of the sessions to good versus poor performers. Self-assessment makes students aware of their deficiencies. Small group sessions can also help students develop the ability to self-assess. This study was carried out to assess the effect of small group sessions on item analysis and students' self-assessment. METHODS: A total of 21 female and 29 male first year medical students participated in a small group session on topics covered by didactic lectures two weeks earlier. It was preceded and followed by two multiple choice question (MCQ) tests, in which students were asked to self-assess their likely score. The MCQs used were item analyzed in a previous group and were chosen of matching difficulty and discriminatory indices for the pre- and post-tests. RESULTS: The small group session improved the marks of both genders equally, but female performance was better. The session made the items easier; increasing the difficulty index significantly but there was no significant alteration in the discriminatory index. There was overestimation in the self-assessment of both genders, but male overestimation was greater. The session improved the self-assessment of students in terms of expected marks and expectation of passing. DISCUSSION: Small group session improved the ability of students to self-assess their knowledge and increased the difficulty index of items reflecting students' better performance.

Asymptomatic bacteriuria & obstetric outcome following treatment in early versus late pregnancy in north Indian women.

BACKGROUND & OBJECTIVES: Asymptomatic bacteriuria during pregnancy if left untreated, may lead to acute pyelonephritis, preterm labour, low birth weight foetus, etc. Adequate and early treatment reduces the incidence of these obstetric complications. The present study was done to determine presence of asymptomatic bacteriuria (ASB) and obstetric outcome following treatment in early versus late pregnancy. METHODS: A prospective cohort study was conducted at a tertiary care teaching hospital of north India. Pregnant women till 20 wk (n=371) and between 32 to 34 wk gestation (n=274) having no urinary complaints were included. Their mid stream urine sample was sent for culture and sensitivity. Women having > 10 [5] colony forming units/ml of single organism were diagnosed positive for ASB and treated. They were followed till delivery for obstetric outcome. Relative risk with 95% confidence interval was used to describe association between ASB and outcome of interest. RESULTS: ASB was found in 17 per cent pregnant women till 20 wk and in 16 per cent between 32 to 34 wk gestation. Increased incidence of preeclamptic toxaemia (PET) [RR 3.79, 95% CI 1.80-7.97], preterm premature rupture of membrane (PPROM)[RR 3.63, 45% CI 1.63-8.07], preterm labour (PTL) [RR 3.27, 95% CI 1.38-7.72], intrauterine growth restriction (IUGR)[RR 3.79, 95% CI 1.80-79], low birth weight (LBW) [RR1.37, 95% CI 0.71-2.61] was seen in late detected women (32-34 wk) as compared to ASB negative women, whereas no significant difference was seen in early detected women (till 20 wk) as compared to ASB negative women. INTERPRETATION & CONCLUSIONS: Early detection and treatment of ASB during pregnancy prevents complications like PET, IUGR, PTL, PPROM and LBW. Therefore, screening and treatment of ASB may be incorporated as routine antenatal care for safe motherhood and healthy newborn.

Anatomical variations in the branching pattern of human aortic arch: a cadaveric study from central India.

Variations of the branches of aortic arch are due to alteration in the development of certain branchial arch arteries during embryonic period. Knowledge of these variations is important during aortic instrumentation, thoracic, and neck surgeries. In the present study we observed these variations in fifty-two cadavers from Indian populations. In thirty-three (63.5%) cadavers, the aortic arch showed classical branching pattern which includes brachiocephalic trunk, left common carotid artery, and left subclavian artery. In nineteen (36.5%) cadavers it showed variations in the branching pattern, which include the two branches, namely, left subclavian artery and a common trunk in 19.2% cases, four branches, namely, brachiocephalic trunk, left common carotid artery, left vertebral artery, and left subclavian artery in 15.3% cases, and the three branches, namely, common trunk, left vertebral artery, and left subclavian artery in 1.9% cases.

Use of formative assessment as an educational tool.

BACKGROUND: Though formative assessments are popular in medical education, but data to establish their educational benefits are lacking. This study was conducted to determine whether participation and performance of MBBS students in regular formative assessments are associated with positive outcomes and has measurable effects on learning. METHODS: One hundred and fifty MBBS students of semester II attending Biochemistry classes were studied by dividing into two groups till the completion of a topic. End-of-topic summative assessment marks were analysed with respect to the effect of participation and performance in formative assessments. RESULTS: Participation in formative assessments had a statistically significant positive relationship with summative assessment marks. Mean difference in formative and summative assessment marks for group that participated in formative assessments is 1.6 (95% CI = 0.9-2.4, p < 0.001). The mean difference in summative assessment marks for two groups is 3.4 (95% CI = 2.3-4.6, p < 0.001). The mean difference in marks obtained by solving case studies given in Summative Assessment for two groups is 1.2 (95% CI = 0.7-1.6, p < 0.001). CONCLUSIONS: Formative assessment not only assesses students' achievements but it also enables students to recognise the areas in which they are having difficulty and to concentrate their future efforts on those areas. Adequate frequency of formative assessment with immediate feedback is beneficial as it stimulates meaningful and multifaceted learning. The results of this study encourage the use of formative assessment as an educational tool in all MBBS subjects for they have significant positive effects on learning.

Vitamin E, Its Beneficial Role in Diabetes Mellitus (DM) and Its Complications.

INTRODUCTION: Studies have shown that diabetes is accompanied by an increased oxidative damage to all the bimolecular. Enhanced oxidative stress contributes to the development of the diabetic complications. The key lipid soluble chain breaking antioxidant,-tocopherol, is known to be deficient in diabetes. Human intervention studies have indicated the role of vitamin E in improving the endothelial function, the retinal blood flow and the renal dysfunction. The aim of the study was to find the role of vitamin E in preventing the development and the progression of the diabetic complications. METHODOLOGY: Both type I and II DM patients with and without complications were included in this study. They were divided separately into the test (which received insulin/oral hypoglycemic and vitamin E) and the control groups (which received only insulin/oral hypoglycemic drugs). The Fasting Blood Sugar(FBS), Post-prandial Blood Sugar(PPBS) and the Total Cholesterol(TC) were estimated and the Blood Pressure (BP) was noted at 0(beginning),12,18 and 24 months. Cardiovascular disease, retinopathy, nephropathy and foot ulcer development and progression were monitored. The data was analyzed by the Z test for the means and for the proportions. RESULTS: It was evident from the analysis of the data that the PPBS, TC and the Diastolic Blood Pressure (DBP) declined gradually and significantly in the test groups. This was a beneficial development for the diabetic patients. The patients who were on the vitamin E supplementation had a delayed development and a slow progression of the complications. CONCLUSION: Vitamin E supplementation has an important role in delaying the onset of the diabetic complications as well as for slowing down the progression of the complications.

Transvaginal repair of complex and complicated vesicovaginal fistulae.

OBJECTIVE: To document the experience at a tertiary referral hospital in India with managing complex and complicated vesicovaginal fistulae (VVF) by the transvaginal route, and to document the complications and the long-term outcome of the patients. METHODS: The medical records of 102 patients with complex or complicated VVF who underwent transvaginal surgical repair during 2000-2009 were reviewed retrospectively. RESULTS: The mean age of the patients was 24 years and the mean duration of urinary incontinence was 3 years (range 6 months to 12 years). The patients were followed up for a median of 48 months. In total, 78 patients had obstetric fistulae, 20 patients had surgical (gynecologic) fistulae, and 4 patients had postradiation fistulae. The overall success rate for the transvaginal approach was 86.3%. Fourteen patients remained incontinent despite the surgical repair. Early failure of the repair was observed in 11.8% of the patients and delayed failure in 2.0%. Postoperative complications included stress urinary incontinence (9.8%), urge urinary incontinence (7.8%), dyspareunia (5.9%), and chronic labial pain (2.0%). CONCLUSION: The transvaginal approach to the repair of complex and complicated VVF gives good long-term results with low complication rates.