Varietal identification and fingerprinting of Pearl Millet (Pennisetum glaucum L.) varieties and hybrid using morphological descriptors and SSR markers

Pearl Millet (Pennisetum glaucum) is the sixth most important cereal crop in the world. The genomic resources available in Pearl millet can be utilized for fingerprinting and screening of hybrids using SSR markers and will be helpful for the assessment of seed purity. Hence, the present study was focused on fingerprint popular pearl millet varieties and hybrids of Tamil Nadu for varietal identification and hybrid purity test. The varieties used for DNA fingerprinting were CO (Cu) 9, CO 10, Pearl Millet hybrid CO 9 along with the parents, A' line ICMA 93111A and R' line PT 6029-30. The morphological features were recorded to screen the cultivars. The Pearl millet hybrid CO 9 scored the highest value for more than four quantitative characters via., Number of productive tillers (4-6), Leaf blade length (60-68cm), Leaf blade width (4.0-4.5cm), number of nodes (8-10), and 1000 seed weight (13-14g) which is at par and comparable with the composite CO 10  and higher than that of the variety CO (Cu) 9. PCR was performed using 36 SSR primers to find out polymorphism among the varieties. The SSR markers ICMP3021 and PSMP2089 were able to selectively identify CO (Cu) 9 from the other varieties. Whereas, the SSR markers ICMP3018, PSMP2219, and PSMP2220 were used to distinguish CO 10 from the other varieties. Further, the CO10 variety produced additional alleles for all the markers due to its composite nature. Among the thirty-six SSR primers screened, none of them were found suitable to distinguish the TNAU hybrid CO 9 from its parents. The unique DNA fingerprints developed in the present study can be utilized for seed purity testing and varietal identification

Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

Background & Aims Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. Methods We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. Results Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. Conclusions In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer

Challenges and opportunities in mixed method data collection on mental health issues of health care workers during COVID-19 pandemic in India

Background: The present paper describes the key challenges and opportunities of mixed method telephonic data collection for mental health research using field notes and the experiences of the investigators in a multicenter study in ten sites of India. The study was conducted in public and private hospitals to understand the mental health status, social stigma and coping strategies of different healthcare personnel during the COVID-19 pandemic in India.Methods: Qualitative and quantitative interviews were conducted telephonically. The experiences of data collection were noted as a field notes/diary by the data collectors and principal investigators.Results: The interviewers reported challenges such as network issues, lack of transfer of visual cues and sensitive content of data. Although the telephonic interviews present various challenges in mixed method data collection, it can be used as an alternative to face-to-face data collection using available technology.Conclusions: It is important that the investigators are well trained keeping these challenges in mind so that their capacity is built to deal with these challenges and good quality data is obtained

Factors associated with stigma and manifestations experienced by Indian health care workers involved in COVID-19 management in India: A qualitative study

Healthcare personnel who deal with COVID-19 experience stigma. There is a lack of national-level representative qualitative data to study COVID-19-related stigma among healthcare workers in India. The present study explores factors associated with stigma and manifestations experienced by Indian healthcare workers involved in COVID-19 management. We conducted in-depth interviews across 10 centres in India, which were analysed using NVivo software version 12. Thematic and sentiment analysis was performed to gain deep insights into the complex phenomenon by categorising the qualitative data into meaningful and related categories. Healthcare workers (HCW) usually addressed the stigma they encountered when doing their COVID duties under the superordinate theme of stigma. Among them, 77.42% said they had been stigmatised in some way. Analyses revealed seven interrelated themes surrounding stigma among healthcare workers. It can be seen that the majority of the stigma and coping sentiments fall into the mixed category, followed by the negative sentiment category. This study contributes to our understanding of stigma and discrimination in low- and middle-income settings. Our data show that the emergence of fear of the virus has quickly turned into a stigma against healthcare workers

The interplay of Carbonic Anhydrase IX and the glutamine transporter SLC1A5 in the hypoxic tumor microenvironment

Carbonic Anhydrase IX (CAIX) is a membrane-bound enzyme that plays a vital role in the pH regulation of hypoxic tumor cells. CAIX is highly expressed in several solid tumors and is an indicator of poor prognosis and response to therapy. Although the importance of CAIX’s role in mediating tumor progression is well-known, the underlying mechanisms remain unclear. To identify the interactors of CAIX in the hypoxic tumor microenvironment, a proteomic analysis was performed using proximity-dependent biotin identification (BioID) and identified metabolic transporters involved in amino acid transport and pH regulation as high confidence interactors. In my thesis, I show that CAIX interacts with the amino acid transporters, SLC1A5; SLC3A2; SLC7A5, and the bicarbonate transporter, SLC4A7. These findings lay a premise for possible dynamic associations of these metabolic transporters with CAIX in the tumor microenvironment to mediate various functions and support tumor progression. In my thesis, I have focused on investigating the interaction of CAIX with SLC1A5, a glutamine (Gln) transporter crucial for supporting tumor growth. Through in-vitro studies, I show that CAIX associates with SLC1A5 in hypoxic cancer cells and regulates Gln uptake in a SLC1A5-depndent manner. Loss of CAIX expression or CAIX activity results in increased Gln transport. I found that the loss of CAIX activity increases cellular ROS, and cells respond by increasing their Gln utilization to synthesize the antioxidant, glutathione (GSH). This helps the cells to maintain redox homeostasis, preventing the lethal effects of oxidative stress and protecting them from cell death. To identify strategies for overcoming resistance to CAIX inhibition, I found that inhibiting the CAIX activity in combination with blocking Gln metabolism or GSH synthesis induces an iron-dependent, oxidative cell death called ferroptosis. Together, these data reveal an important mechanism by which CAIX cooperatively works with SLC1A5 to protect hypoxic cancer cells from oxidative stress and promotes survival. Furthermore, I have demonstrated four different co-targeting strategies that effectively induce ferroptosis in hypoxic cancer cells. In summary, my work has revealed the co-targeting of CAIX activity and Gln metabolism as a potential strategy to effectively treat aggressive, solid tumors.Medicine, Faculty ofGraduat

Psychological distress and burnout among healthcare worker during COVID-19 pandemic in India-A cross-sectional study.

BackgroundCOVID-19 has inundated the entire world disrupting the lives of millions of people. The pandemic has stressed the healthcare system of India impacting the psychological status and functioning of health care workers. The aim of this study is to determine the burnout levels and factors associated with the risk of psychological distress among healthcare workers (HCW) engaged in the management of COVID 19 in India.MethodsA cross-sectional study was conducted from 1 September 2020 to 30 November 2020 by telephonic interviews using a web-based Google form. Health facilities and community centres from 12 cities located in 10 states were selected for data collection. Data on socio-demographic and occupation-related variables like age, sex, type of family, income, type of occupation, hours of work and income were obtained was obtained from 967 participants, including doctors, nurses, ambulance drivers, emergency response teams, lab personnel, and others directly involved in COVID 19 patient care. Levels of psychological distress was assessed by the General health Questionnaire -GHQ-5 and levels of burnout was assessed using the ICMR-NIOH Burnout questionnaire. Multivariable logistic regression analysis was performed to identify factors associated with the risk of psychological distress. The third quartile values of the three subscales of burnout viz EE, DP and PA were used to identify burnout profiles of the healthcare workers.ResultsOverall, 52.9% of the participants had the risk of psychological distress that needed further evaluation. Risk of psychological distress was significantly associated with longer hours of work (≥ 8 hours a day) (AOR = 2.38, 95% CI(1.66-3.41), income≥20000(AOR = 1.74, 95% CI, (1.16-2.6); screening of COVID-19 patients (AOR = 1.63 95% CI (1.09-2.46), contact tracing (AOR = 2.05, 95% CI (1.1-3.81), High Emotional exhaustion score (EE ≥16) (AOR = 4.41 95% CI (3.14-6.28) and High Depersonalisation score (DP≥7) (AOR = 1.79, 95% CI (1.28-2.51)). About 4.7% of the HCWs were overextended (EE>18); 6.5% were disengaged (DP>8) and 9.7% HCWs were showing signs of burnout (high on all three dimensions).ConclusionThe study has identified key factors that could have been likely triggers for psychological distress among healthcare workers who were engaged in management of COVID cases in India. The study also demonstrates the use of GHQ-5 and ICMR-NIOH Burnout questionnaire as important tools to identify persons at risk of psychological distress and occurrence of burnout symptoms respectively. The findings provide useful guide to planning interventions to mitigate mental health problems among HCW in future epidemic/pandemic scenarios in the country