Socio-Demographic and Clinical Profile of Drug Resistant Tuberculosis Patients in a Tertiary Care Centre of Kolkata

Background: Drug resistant tuberculosis is a major public health threat that hinders progress in tuberculosis control worldwide. In 2019, India contributed highest cases of TB (27%) Worldwide. In 2019, 3.3% of new TB cases and 18% of previously treated cases had MDR/RRTB worldwide and India (27%) had the largest share of the global burden. This study was done to find out the sociodemographic and clinical profile of drug resistant TB patients. Methodology: It is an observational descriptive study with cross-sectional design, conducted at the drug resistant tuberculosis centre of R. G. Kar Medical College of Kolkata, West Bengal. Study Population consisted of the patients who were registered for the DR-TB regimen. Study duration was July 2016 –March 2017. Pre-tested, semi-structured schedule was designed to capture their sociodemographic profile, treatment history, clinical findings and available medical records. Data were compiled in MS Excel and analyzed in IBM SPSS 23.0. Results: Out of 159 cases, 27% patients were between 21-30 years. Males were predominant (68.6%). Most patients (56.6%) were underweight (BMI<18.5kg/m2). HIV seropositivity was found in 4 (2.5%) cases. Among all cases, 81.1% had history of taking ATD and 34.4% was cured in previous episode of treatment. The commonest associated comorbidity was DM (15.7%). Rifampicin resistance (93.1%) was most common followed by Isoniazid (8.2%). Pallor was found among 94.3% patients. Most patients had bilateral (62.3%) and moderately extensive (57.2%) lesions in chest x-ray. Conclusion: DR-TB control should focus adequately on younger age group as numbers of resistance is increasing among them. Relapse in previously cured cases was found to be major contributor of DR-TB suspect cases. Though, Rifampicin resistance was so common but prevalence of resistance to 2nd line drugs is still low. Focus should be given on early detection of drug resistance in all TB cases and improvement of nutritional status of the TB patient

Genomic Exploration of Distinct Molecular Phenotypes Steering Temozolomide Resistance Development in Patient-Derived Glioblastoma Cells

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells’ molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as “adaptive” (ADA) or “non-adaptive” (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor’s ability to survive. Depending on the tumor’s adaptability potential, subpopulations with acquired resistance mechanisms may arise.</p

Ex vivo drug sensitivity screening predicts response to temozolomide in glioblastoma patients and identifies candidate biomarkers

Background: Patient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ). Methods: DNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data. Results: Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets. Conclusion:GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.</p

A lethal Cardiotoxic–Cytotoxic Protein from the Indian Monocellate Cobra (Naja kaouthia) Venom

A lethal cardiotoxic–cytotoxic protein (mol. wt. 6.76 kDa) has been purified from the Indian monocellate cobra (Naja kaouthia) venom by ion-exchange chromatography and HPLC. CD spectra indicated the presence of 23% a helix, 19% b sheets and 35% coil. Complete amino acid sequence was determined by MALDI, which showed similar homology with cardiotoxins/cytotoxins isolated from venom of other Naja species. Intraperitoneal LD50 was 2.5 mg kg�1 in BalbC male mice. In vitro cardiotoxicity studies on isolated guinea pig auricle showed that the molecule produced auricular blockade that was abolished after trypsin treatment. Cytotoxicity studies on human leukemic U937 and K562 cells showed that it significantly inhibited cell proliferation in a dose and time dependent manner, as observed by trypan blue exclusion method and tetrazolium bromide reduction assay. IC50 on U937 and K562 cells were 3.5 mg/ml and 1.1 mg/ml respectively. Morphometry and cell sorting studies indicated apoptosis induction in toxin treated leukemic cells. Apoptosis was caspase 3 and 9 dependent and the treated leukemic cells were arrested in sub-G1 stage. There was an increase in Bax–Bcl2 ratio, decrease in HSP (Heat shock protein) 70 and HSP90 and induction of PARP cleavage after NK-CT1 treatment. The toxin showed low cytotoxic effect on normal human leukocytes as compared with imatinib mesylate. Further detailed cytotoxic and cardiotoxic effects at the molecular level are in progress

Cytotoxic and Antioxidant Property of a Purified Fraction (NN-32) of Indian Naja Naja Venom on Ehrlich Ascites Carcinoma in BALB/c Mice

A cytotoxic and antioxidant protein (NN-32) from the Indian spectacled cobra Naja naja venom was identified and its probable mode of action on murine Ehrlich ascites carcinoma (EAC) was established. The venom purified through ion exchange chromatography produced several peaks, among which fraction 32 produced cytotoxic-cardiotoxic properties. This fraction (NN-32) showed a single peak (retention time 38.3 min) by HPLC using C4 column. The molecular mass determined by MALDI-MS, found to be 6.7 kDa and the first ten N-terminal sequence was determined (LKCNKLVPLF) by Edmann degradation method using applied Biosystem procise sequencer. It was observed that the sequence shared 100% homology with other cytotoxin cardiotoxin identified from the venom of Naja species. NN-32 showed cytotoxicity on EAC cells, increased survival time of inoculated EAC mice, reduced solid tumor volume and weight. NN-32 increased proapoptotic protein caspase 3 and 9 activity and Bax-Bcl2 ratio. It also increased the antioxidant markers glutathione, glutathione peroxidase, glutathione transferase, superoxide dismutase and catalase activity. NN-32 increased serum IL-10 level and decreased murine keratinocytederived chemokine level. The cardiotoxicity of NN-32 was established on isolated guinea pig auricle, where 100% irreversible blockade of auricular contraction was observed. Thus, it may be concluded that, NN-32 induced anticancer activity in EAC mice was partly mediated through its apoptogenic – antioxidant property

Occurrence of non-protein low molecular weight cardiotoxin in Indian King Cobra (<i style="">Ophiophagus hannah</i>) Cantor 1836, venom

279-285Pathophysiology due to snakebite is a combined effect of various actions of the complex venom constituents. Importance of protein toxins in snake envenomation is well known. The present investigation reports the existence of non-protein/nonpetide low molecular weight toxin in Indian King Cobra venom, which plays an important role in envenomation consequences in experimental animal models. A group of non-peptidic toxins (OH-NPT1) was isolated from Indian King Cobra Ophiophagus hannah by thin layer chromatography and silica gel column chromatography. UV, IR, NMR and (ESI) TOF-MS studies characterized the OH-NPT1 as a mixture of aliphatic acids having molecular weights 256, 326 and 340Da. The minimum lethal dose of OH-NPT1 was found to be 2.5 g/20g (iv) and 4g/20g (ip) in male albino mice. The cardiotoxic property of OH-NPT1 was established through studies on isolated guinea pig heart and auricle preparations, ECG studies in albino rat and estimation of LDH1/LDH and CPK-MB/CPK ratio in Swiss albino mice. Commercial antiserum failed to neutralize the lethality and cardiotoxicity of the toxin. However, calcium and magnesium effectively neutralized the lethal action

A Heat Stable Protein Toxin (drCT-I) from the Indian Viper (Daboia russelli russelli) Venom having Antiproliferative, Cytotoxic and Apoptotic Activities

A heat stable 7.2 kDa protein toxin (drCT-I) has been purified and crystallized from Indian Daboia russelli russelli venom (Roy Choudhury et al., 2006. Acta Cryst. F Struct Biol Cryst Commun, 62(Pt. 3), 292). The N-terminal (first 20) amino acid sequence of drCT-I was LKCNKLVPLFYKTCPAGKNL, which showed sequence homology to cytotoxins isolated from Naja venom. drCT-I has been evaluated for anticancer activity against EAC cells in vivo and human leukemic cells (U937, K562) in vitro. drCT-I (125 mg/kg, i.p/day for 10 days) significantly decreased EAC cell count, cell viability (po0:001) and significantly increased the survival time of tumour bearing mice (T/C% 178.64, po0:01) in comparison to untreated tumour bearing control. drCT-I, produced dose and time-dependent inhibition of U937 and K562 cell growth and had an IC50 of 8.9 and 6.7 mg/ml respectively after 24 h treatment. The reduced MTT values after drCT-I treatment indicated its cytotoxic nature, which supported its antiproliferative action. Scanning electron microscopy and confocal microscopy in U937 and K562 cells after drCT-I treatment indicated certain features of apoptosis such as membrane blebbing, perforations, nuclear fragmentation. The induction of apoptosis was further confirmed by phosphatidylserine externalization observed using annexinV-FITC/PI staining and flow cytometric analysis. drCT-I brought about apoptosis by G1 phase arrest of the cell cycle. The effect of drCT-I on normal human peripheral blood mononuclear cell (PBMNC) viability and cytotoxicity was studied in culture and was found to be lower than that on U937 and K562 cells. Thus both in vivo and in vitro experimental results suggested that drCT-I possessed anticancer potential

Comparative analysis of deeply phenotyped GBM cohorts of 'short-term' and 'long-term' survivors

Background: Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). Methods: Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age Results: Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. Conclusion: Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM.</p

Genomic exploration of distinct molecular phenotypes steering temozolomide resistance development in patient-derived glioblastoma cells

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells' molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as "adaptive" (ADA) or "non-adaptive" (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor's ability to survive. Depending on the tumor's adaptability potential, subpopulations with acquired resistance mechanisms may arise. </p

Comparative analysis of deeply phenotyped GBM cohorts of 'short-term' and 'long-term' survivors.

peer reviewedBACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). METHODS: Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age < 70 years old; Stupp protocol as first line treatment, IDH wild type), and a multi-omic analysis of LTS and STS GBM samples was performed. RESULTS: Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. CONCLUSION: Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM