Mitochondria-containing extracellular vesicles from mouse vs . human brain endothelial cells for ischemic stroke therapy.

Ischemic stroke-induced mitochondrial dysfunction in the blood-brain barrier-forming brain endothelial cells ( BECs ) results in long-term neurological dysfunction post-stroke. We previously reported that intravenous administration of human BEC ( hBEC )-derived mitochondria-containing extracellular vesicles ( EVs ) showed a potential efficacy signal in a mouse middle cerebral artery occlusion ( MCAo ) model of stroke. We hypothesized that EVs harvested from donor species homologous to the recipient species ( e.g., mouse) may improve therapeutic efficacy, and therefore, use of mouse BEC ( mBEC )-derived EVs may improve post-stroke outcomes in MCAo mice. We investigated if EVs derived from the same species as the recipient cell (mBEC-EVs and recipient mBECs or hBECs-EVs and recipient hBECs) show a greater EV mitochondria delivery efficiency than cross-species EVs and recipient cells (mBEC-EVs and recipient hBECs or vice versa ). Our results showed that mBEC-EVs outperformed hBEC-EVs in transferring EV mitochondria to the recipient ischemic mBECs, and improved mBEC mitochondrial function via increasing oxygen consumption rate. mBEC-EVs significantly reduced brain infarct volume and improved behavioral recovery compared to vehicle-injected MCAo mice. Our data suggests that mBEC-EVs show superior therapeutic efficacy in a mouse MCAo stroke model compared to hBEC-EVs-supporting the continued use of mBEC-EVs to optimize the therapeutic potential of mitochondria-containing EVs in preclinical studies.

Pigmented Microbial Extract (PMB) from Exiguobacterium Species MB2 Strain (PMB1) and Bacillus subtilis Strain MB1 (PMB2) Inhibited Breast Cancer Cells Growth In Vivo and In Vitro.

Breast cancer (BC) continues to be one of the major causes of cancer deaths in women. Progress has been made in targeting hormone and growth factor receptor-positive BCs with clinical efficacy and success. However, little progress has been made to develop a clinically viable treatment for the triple-negative BC cases (TNBCs). The current study aims to identify potent agents that can target TNBCs. Extracts from microbial sources have been reported to contain pharmacological agents that can selectively inhibit cancer cell growth. We have screened and identified pigmented microbial extracts (PMBs) that can inhibit BC cell proliferation by targeting legumain (LGMN). LGMN is an oncogenic protein expressed not only in malignant cells but also in tumor microenvironment cells, including tumor-associated macrophages. An LGMN inhibition assay was performed, and microbial extracts were evaluated for in vitro anticancer activity in BC cell lines, angiogenesis assay with chick chorioallantoic membrane (CAM), and tumor xenograft models in Swiss albino mice. We have identified that PMB from the Exiguobacterium (PMB1), inhibits BC growth more potently than PMB2, from the Bacillus subtilis strain. The analysis of PMB1 by GC-MS showed the presence of a variety of fatty acids and fatty-acid derivatives, small molecule phenolics, and aldehydes. PMB1 inhibited the activity of oncogenic legumain in BC cells and induced cell cycle arrest and apoptosis. PMB1 reduced the angiogenesis and inhibited BC cell migration. In mice, intraperitoneal administration of PMB1 retarded the growth of xenografted Ehrlich ascites mammary tumors and mitigated the proliferation of tumor cells in the peritoneal cavity in vivo. In summary, our findings demonstrate the high antitumor potential of PMB1.

Design, Synthesis, and Anti-Breast Cancer Potential of Imidazole-Pyridine Hybrid Molecules In Vitro and Ehrlich Ascites Carcinoma Growth Inhibitory Activity Assessment In Vivo.

Breast cancer remains a challenging medical issue and is a high priority for biomedical research despite significant advancements in cancer research and therapy. The current study aims to determine the anticancer activity of a group of imidazole-pyridine-based scaffolds against a variety of breast cancer cell lines differing in their receptor expression (estrogen receptor (ER), progesterone receptor (PR), and HER-2). A series of 10 molecules (coded 5a-5j) were synthesized through multicomponent and alkylation reactions. FTIR, MS, 1H, and 13C NMR spectral analyses confirmed the structures and purity of the synthesized molecules. Subsequently, these molecules were tested for their ability to inhibit the viability of cell lines representing carcinoma of the breast, viz., MDA-MB-468 (ER-, PR-, and HER-), BT-474 (ER+, PR+, and HER+), T-47D (ER+, PR+, and HER-), and MCF-7 (ER+, PR+, and HER-) in vitro. Among these 10 molecules, 5a, 5c, 5d, and 5e exhibited better potency, as evidenced by IC50 < 50 µM at 24 h of treatment against BT-474 and MDA-MB-468 cell lines. However, except for 5d, the IC50 value is much higher than 50 µM when tested against T47D and MCF-7 cell lines at 24h. Extended treatment for 48 h reduced the effect of these molecules, as an increase in IC50 was observed. In mice, intraperitoneal administration of 5e retarded the Ehrlich ascites carcinoma (EAC) growth without causing any organ toxicity at the doses tested. In summary, we report the synthesis scheme and key structural requirements for a new series of imidazole-pyridine molecules for in vitro inhibition of the feasibility of breast cancer cells and in vivo inhibition of EAC tumors.

Volatile profiling as a potential biochemical marker for validation of gamma irradiation derived putative mutants in polyembryonic genotypes of mango (Mangifera indica L.).

Introduction: Putative mutants were generated through gamma irradiation in the polyembryonic mango genotype Nekkare. The putative mutant progenies along with control seedlings and mother plants were evaluated by comparing the compositions and relative proportions of their major volatile compounds. Methods: Volatile profiling was done using headspace-solid phase micro-extraction (HS SPME) method coupled with gas chromatography-mass spectrometry (GC-MS MS). Furthermore, characterisation of putative mutants and control seedlings was carried out using simple sequence repeat (SSR) markers to ascertain the genetic diversity present in the samples under study. Results: Monoterpenes were the most abundant volatile compound in all the studied samples (ranging from 34.76% to 91.41%) out of which I-Phellandrene and cis-Ocimene formed the major fraction in mother plants (20.45%-21.86% and 16.17%-21.27%, respectively) and control seedlings (23.32%-24.95% and 18.95%-20.81%, respectively), while beta-Phellandrene was dominant in the selected putative mutant samples (2.34%-29.53%). Among sesquiterpenes, trans-Caryophyllene was detected only in the putative mutant samples (0.10%-30.18%). Grouping together of mother plants and control seedlings was seen in the cluster analysis, while the putative mutants grouped apart from them suggesting genetic diversity. Genetic distance between the mother plants and control seedlings ranged from 0.97 to 2.73, while between putative mutants, control seedlings, and mother plants, it ranged from 6.54 to 9.82. SSR-based characterisation of putative mutant seedlings showed that mutation caused variability in the treated population. This was evident from the high allelic richness ranging from 4 to 12 with a mean of 7 and a higher mean Shannon's Information Index (1.50) of the putative mutant population. Discussion: The study demonstrates that volatile profiling and molecular characterisation using SSR markers could be used as a tool to detect variation in a mutated population. In addition, volatile profiling can be used to validate putative mutants in polyembryonic mango genotypes where the seedlings of nucellar origin are similar to mother plants.

Human papillomavirus-driven repression of NRF2 signalling confers chemo-radio sensitivity and predicts prognosis in head and neck squamous cell carcinoma.

PURPOSE: To investigate the role of NRF2 signalling in conferring superior prognosis in patients with HPV positive (HPV+ve) head & neck squamous cell carcinomas (HNSCC) compared to HPV negative (HPV-ve) HNSCC and develop molecular markers for selection of HPV+ve HNSCC patients for treatment de-escalation trials. METHODS: NRF2 activity (NRF2, KEAP1, and NRF2-transcriptional targets), p16, and p53 levels between HPV+ve HNSCC and HPV-ve HNSCC in prospective and retrospective tumor samples as well as from TCGA database were compared. Cancer cells were transfected with HPV-E6/E7 plasmid to elucidate if HPV infection represses NRF2 activity and sensitizes to chemo-radiotherapy. RESULTS: Prospective analysis revealed a marked reduction in expression of NRF2, and its downstream genes in HPV+ve tumors compared to HPV-ve tumors. A retrospective analysis by IHC revealed significantly lower NQO1 in p16high tumors compared to p16low tumors and the NQO1 expression correlated negatively with p16 and positively with p53. Analysis of the TCGA database confirmed low constitutive NRF2 activity in HPV+ve HNSCC compared to HPV-ve HNSCC and revealed that HPV+ve HNSCC patients with 'low NQO1' expression showed better overall survival compared to HPV+ve HNSCC patients with 'high NQO1' expression. Ectopic expression of HPV-E6/E7 plasmid in various cancer cells repressed constitutive NRF2 activity, reduced total GSH, increased ROS levels, and sensitized the cancer cells to cisplatin and ionizing radiation. CONCLUSION: Low constitutive NRF2 activity contributes to better prognosis of HPV+ve HNSCC patients. Co-expression of p16high, NQO1low, and p53low could serve as a predictive biomarker for the selection of HPV + ve HNSCC patients for de-escalation trials.

Bacterial flagellin is a dominant, stable innate immune activator in the gastrointestinal contents of mice and rats.

Intestinal contents comprise the largest repository of immunogenic ligands of microbial origin. We undertook this study to assess the predominant microbe-associated molecular patterns (MAMPs) present therein and the receptors) that mediate the innate immune responses to them. Here, we demonstrated that intestinal contents from conventional, but not germ-free, mice and rats triggered robust innate immune responses in vitro and in vivo. Such immune responses were abrogated in the absence of either myeloid differentiation factor 88 (MyD88) or Toll-like receptor (TLR) 5, but not TLR4, suggesting that the stimuli was flagellin (i.e., protein subunit of flagella that drives bacterial motility). Accordingly, pre-treating intestinal extracts with proteinase, thereby degrading flagellin, was sufficient to block their ability to activate innate immune responses. Taken together, this work serves to underscore flagellin as a major, heat-stable and bioactive MAMP in the intestinal content that confers this milieu strong potential to trigger innate immune responses.

Providing Diabetes Education through Phone Calls Assisted in the Better Control of Hyperglycemia and Improved the Knowledge of Patients on Diabetes Management.

Purpose: A recent single-arm pilot study from our group showed a significant decrease in HbA1C in Type-2 diabetes individuals provided with SMS and phone call-based education on glycemic control. Considering the preference of participants to phone call-based education, a randomized control trial (RCT) with parallel design was conducted to determine the impact of phone call-based diabetes educational intervention on the control of hyperglycemia and improvement in the knowledge about diabetes management. Objectives: To determine the impact of phone call-based educational intervention on the control of hyperglycemia and improvement in the knowledge about diabetes management. Methodology: The study was conducted for a period of 12 months on a total of 273 Type-2 diabetic patients (interventional group (n = 135); non-interventional group (n = 138)) who had provided consent to participate. Subjects in the case group received weekly phone calls on diabetes education; whereas the control group received no education. HbA1C investigations were carried out at baseline and at every fourth month until the completion of the study period for the subjects in both the groups. The impact of phone call-based education was measured by comparing HbA1C values as well as by measuring the questionnaire-based knowledge scores on diabetes management. Results: At the end of the study period, there was a significant reduction in HbA1C in 58.8% participants (n = 65) and a manifold (2-5-fold) increase in knowledge on diabetes management among participants in the case group (n = 110). However, no significant difference in HbA1C and knowledge score was observed in participants from the control group (n = 115). Conclusion: Phone call-based diabetes education is a viable option to empower patients for better management of Type-2 diabetes.

Mitochondria-containing extracellular vesicles (EV) reduce mouse brain infarct sizes and EV/HSP27 protect ischemic brain endothelial cultures.

Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of BEC-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous 27 kDa heat shock protein (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, that subsequently colocalized with the mitochondrial network of the recipient primary human BECs. Recipient BECs treated with m/lEVs showed increased relative ATP levels and mitochondrial function. To determine if the m/lEV-meditated increase in recipient BEC ATP levels was associated with m/lEV mitochondria, we isolated m/lEVs from donor BECs pre-treated with oligomycin A (OGM, mitochondria electron transport complex V inhibitor), referred to as OGM-m/lEVs. BECs treated with naïve m/lEVs showed a significant increase in ATP levels compared to untreated OGD cells, OGM-m/lEVs treated BECs showed a loss of ATP levels suggesting that the m/lEV-mediated increase in ATP levels is likely a function of their innate mitochondrial load. In contrast, sEV-mediated ATP increases were not affected by inhibition of mitochondrial function in the donor BECs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EVs with a cationic polymer, poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity may be a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.

Bilateral multifocal choroiditis with disc edema in a 15-year-old girl following COVID-19 vaccination.

A few cases of posterior uveitis following COVID-19 vaccination have been reported but none in the pediatric age group. A 15-year-old girl presented with history of headache and bilateral blurred vision of five days duration. The symptoms developed five days after vaccination with the first dose of Covaxin (inactivated SARS-CoV-2 vaccine). Her anterior segment was normal in both eyes (BE), whereas the posterior segment showed mild vitritis with disc edema and multiple yellowish lesions at the level of choroid clustered at the macula and associated with multiple serous detachments. BE uveitis resolved, and the vision was completely recovered three weeks after treatment with steroids. Hence, ophthalmologists should be aware of uveitis following vaccination-a condition that is usually benign, transient, and results in excellent outcomes with timely diagnosis and early treatment with steroids.

Enterohepatic Shunt-Driven Cholemia Predisposes to Liver Cancer.

BACKGROUND & AIMS: Pathogenesis of hepatocellular carcinoma (HCC), which kills millions annually, is poorly understood. Identification of risk factors and modifiable determinants and mechanistic understanding of how they impact HCC are urgently needed. METHODS: We sought early prognostic indicators of HCC in C57BL/6 mice, which we found were prone to developing this disease when fed a fermentable fiber-enriched diet. Such markers were used to phenotype and interrogate stages of HCC development. Their human relevance was tested using serum collected prospectively from an HCC/case-control cohort. RESULTS: HCC proneness in mice was dictated by the presence of congenitally present portosystemic shunt (PSS), which resulted in markedly elevated serum bile acids (BAs). Approximately 10% of mice from various sources exhibited PSS/cholemia, but lacked an overt phenotype when fed standard chow. However, PSS/cholemic mice fed compositionally defined diets, developed BA- and cyclooxygenase-dependent liver injury, which was exacerbated and uniformly progressed to HCC when diets were enriched with the fermentable fiber inulin. Such progression to cholestatic HCC associated with exacerbated cholemia and an immunosuppressive milieu, both of which were required in that HCC was prevented by impeding BA biosynthesis or neutralizing interleukin-10 or programmed death protein 1. Analysis of human sera revealed that elevated BA was associated with future development of HCC. CONCLUSIONS: PSS is relatively common in C57BL/6 mice and causes silent cholemia, which predisposes to liver injury and HCC, particularly when fed a fermentable fiber-enriched diet. Incidence of silent PSS/cholemia in humans awaits investigation. Regardless, measuring serum BA may aid HCC risk assessment, potentially alerting select individuals to consider dietary or BA interventions.

Selective IgA Deficiency in Spontaneously Hypertensive Rats With Gut Dysbiosis.

BACKGROUND: The spontaneously hypertensive rat (SHR) is extensively used to study hypertension. Gut microbiota dysbiosis is a notable feature in SHR for reasons unknown. Immunoglobulin A (IgA) is a major host factor required for gut microbiota homeostasis. We hypothesized that inadequate IgA contributes to gut microbiota dysbiosis in SHR. METHODS: IgA was measured in feces, cecum, serum, liver, gut-associated lymphoid tissue, and milk from SHR and Wistar Kyoto rats. IgA regulatory factors like IgM, IgG, and pIgR (polymeric immunoglobulin receptor) were analyzed. IgA and IgG antibodies and blood pressure (BP) were measured before and after administrating a bacterial antigen (ie, flagellin). RESULTS: Compared with Wistar Kyoto rats, SHR displayed remarkably near-deficient IgA levels accompanied by compensatory increases in serum IgM and IgG and gut-liver pIgR expression. Inadequate milk IgA in SHR emphasized this immune defect stemmed from the neonatal stage. Reduced IgA+ B cells in circulation and Peyer patches indicated a possible reason for the lower IgA in SHR. Noteworthy, a genetic insufficiency was unlikely because administering flagellin to SHR induced anti-flagellin IgA antibodies. This immune response surprisingly accelerated hypertension development in SHR, suggesting IgA quiescence may help maintain lower BP. CONCLUSIONS: This study is the first to reveal IgA deficiency in SHR as one host factor associated with gut microbiota dysbiosis and invigorates future research to determine the pathophysiological role of IgA in hypertension.

Phytochemical-rich Fractions from Foxtail Millet (Setaria italica (L.) P. Beauv) Seeds Exhibited Antioxidant Activity and Reduced the Viability of Breast Cancer Cells In Vitro by Inducing DNA Fragmentation and Promoting Cell Cycle Arrest.

BACKGROUND: The demand for millet-based diets has increased significantly in recent years due to their beneficial effects on human health. Foxtail Millet (Setaria italica (L.) P. Beauv, previously known as Panicum italicum L., referred as FTM in this manuscript) seeds have been not only used as astringent and diuretic agents, but they are also used to treat dyspepsia and rheumatism. Recent studies have shown that solvent extracts from FTM seeds exhibited antioxidant and antiinflammatory activities. However, the nature and antiproliferative potential of phytochemical constituents of solvent extracts are not much explored. OBJECTIVES: Major objectives of this study are to generate and characterize the phytochemical-rich fractions from Foxtail millet seeds, test the antioxidant activity, and antiproliferative potential against cell lines representing carcinomas of the breast, and determine the mechanisms of cell growth inhibition. METHOD: Phytochemical-rich fractions were generated by extracting the seeds using 70% ethanol (FTM-FP) and 10% alkali (FTM-BP). Antioxidant potential was determined by ferric reducing antioxidant power (FRAP) assay and DPPH radical scavenging activity assays. The antiproliferative potential was determined using sulforhodamine-B assay. The impact on cell cycle and DNA fragmentation was analyzed by staining the cells with DAPI followed by analyzing the stained cells using NC-3000. RESULTS: Analysis of the results showed the presence of phenolics and flavonoids in the FTM-FP and FTM-BP fractions. Both fractions exhibited antiproliferative potential against breast cancer cell lines. Mechanistically, both fractions induced G2/M cell cycle arrest and increased the fragmentation of DNA, which lead to the accumulation of cells in the Sub-G1 phase. CONCLUSION: In summary, results of this study demonstrated the potential of foxtail millet phytochemical fractions for retarding the proliferative potential of breast cancer cells.

Melanogenesis Markers Expression in Premature Graying of Hair: A Cross-Sectional Study.

BACKGROUND: Studies on mice and aging human hair follicles provide compelling evidence that graying of hair results from premature differentiation of melanocyte stem cells in the niche/bulge. OBJECTIVE: The aim of this study was to analyze whether differentiation of melanocyte stem cells is responsible for premature graying of hair (PGH). METHODS: Twenty-five patients with PGH (n = 25) attending the dermatology department were recruited. Five unpigmented and 5 pigmented hairs were obtained per patient by separating individual follicles after 1 mm punch biopsies. The hairs were dissected at a distance of 2 mm from the bulb to separate the stem cells (upper segment - US) from the melanocytes (lower segment - LS). RNA was extracted from hair follicle US and LS, and expression of GP100, tyrosinase (TYR), and tyrosinase-related protein-1 (TYRP1) genes was quantified using Qiagen one-step RT-PCR kit. RESULTS: We found melanogenesis gene expression in both temporary (US) and permanent (LS) segments of unpigmented and pigmented hair follicles. When compared between the US and LS of white hair, the expression of TYR and GP100 was much higher in US than LS, suggestive of melanogenesis in the bulge. Similarly, when compared between white and black US, the expression of all 3 genes was higher in white US than black US, although not statistically significant. LIMITATIONS: Low samples size and lack of data pertaining to the expression of genes at protein level are the limitations of current study. CONCLUSION: Even though this pilot study data yielded key information about the expression of GP100, TYR, and TYRP-1 at the mRNA level, further studies quantifying the expression of these genes at protein level are needed to provide additional clues to further address the results in detail.

Involvement of husband in maternal and child health care in rural field practice area of a tertiary medical college in South India-A mixed method study.

BACKGROUND: To commemorate the 25th anniversary of International Conference on Population and Development (ICPD) in the pursuit of Sexual and Reproductive Health Rights views of duty bearers (men) who are mostly not involved in antenatal care in a patriarchal society like India needs to be explored. DESIGN: It is a mixed method study (Triangulation). SETTING AND POPULATION: It was conducted in a rural field practice area of a private medical college in South India covering a population of 19,200. OBJECTIVES: 1) To determine the involvement of husband in maternal and child care. 2) To find out the perceptions of the husbands of antenatal pregnant women in maternal and child health (MCH) care. METHODS: (Quan) A semi-structured questionnaire to find out the areas where husband is involved maximum during antenatal care (Qual). In-depth interviews was conducted to find out the factors associated with their involvement. RESULTS: About 72.5% came for antenatal visits while it decreased to 27.5% during labor and further decreased to 20.3% during immunization. The reasons for decreased participation were (1) Professional Commitments, (2) Views of a Patriarchal society like India, (3) Financial Difficulties, and (4) Health Facility Related Challenges. CONCLUSION: There is a need to educate the husband regarding the importance of husband's involvement during delivery and immunization. Programs should also include men as the stakeholders for accountability and better MCH care for women.

Targeted Inhibition of Anti-Inflammatory Regulator Nrf2 Results in Breast Cancer Retardation In Vitro and In Vivo.

Nuclear factor erythroid-2 related factor-2 (Nrf2) is an oxidative stress-response transcriptional activator that promotes carcinogenesis through metabolic reprogramming, tumor promoting inflammation, and therapeutic resistance. However, the extension of Nrf2 expression and its involvement in regulation of breast cancer (BC) responses to chemotherapy remain largely unclear. This study determined the expression of Nrf2 in BC tissues (n = 46) and cell lines (MDA-MB-453, MCF-7, MDA-MB-231, MDA-MB-468) with diverse phenotypes. Immunohistochemical (IHC)analysis indicated lower Nrf2 expression in normal breast tissues, compared to BC samples, although the difference was not found to be significant. However, pharmacological inhibition and siRNA-induced downregulation of Nrf2 were marked by decreased activity of NADPH quinone oxidoreductase 1 (NQO1), a direct target of Nrf2. Silenced or inhibited Nrf2 signaling resulted in reduced BC proliferation and migration, cell cycle arrest, activation of apoptosis, and sensitization of BC cells to cisplatin in vitro. Ehrlich Ascites Carcinoma (EAC) cells demonstrated elevated levels of Nrf2 and were further tested in experimental mouse models in vivo. Intraperitoneal administration of pharmacological Nrf2 inhibitor brusatol slowed tumor cell growth. Brusatol increased lymphocyte trafficking towards engrafted tumor tissue in vivo, suggesting activation of anti-cancer effects in tumor microenvironment. Further large-scale BC testing is needed to confirm Nrf2 marker and therapeutic capacities for chemo sensitization in drug resistant and advanced tumors.

Sex-Specific Differences in Autophagic Responses to Experimental Ischemic Stroke.

Ischemic stroke triggers a series of complex pathophysiological processes including autophagy. Differential activation of autophagy occurs in neurons derived from males versus females after stressors such as nutrient deprivation. Whether autophagy displays sexual dimorphism after ischemic stroke is unknown. We used a cerebral ischemia mouse model (middle cerebral artery occlusion, MCAO) to evaluate the effects of inhibiting autophagy in ischemic brain pathology. We observed that inhibiting autophagy reduced infarct volume in males and ovariectomized females. However, autophagy inhibition enhanced infarct size in females and in ovariectomized females supplemented with estrogen compared to control mice. We also observed that males had increased levels of Beclin1 and LC3 and decreased levels of pULK1 and p62 at 24 h, while females had decreased levels of Beclin1 and increased levels of ATG7. Furthermore, the levels of autophagy markers were increased under basal conditions and after oxygen and glucose deprivation in male neurons compared with female neurons in vitro. E2 supplementation significantly inhibited autophagy only in male neurons, and was beneficial for cell survival only in female neurons. This study shows that autophagy in the ischemic brain differs between the sexes, and that autophagy regulators have different effects in a sex-dependent manner in neurons.

Design, Synthesis, Characterization, and Crystal Structure Studies of Nrf2 Modulators for Inhibiting Cancer Cell Growth In Vitro and In Vivo.

Nrf2 is one of the important therapeutic targets studied extensively in several cancers including the carcinomas of the colon and rectum. However, to date, not many Nrf2 inhibitors showed promising results for retarding the growth of colorectal cancers (CRCs). Therefore, in this study, first, we have demonstrated the therapeutic effect of siRNA-mediated downmodulation of Nrf2 on the proliferation rate of CRC cell lines. Next, we have designed, synthesized, characterized, and determined the crystal structures for a series of tetrahydrocarbazoles (THCs) and assessed their potential to modulate the activity of Nrf2 target gene NAD(P)H:quinone oxidoreductase (NQO1) activity by treating colorectal carcinoma cell line HCT-116. Later, the cytotoxic potential of compounds was assessed against cell lines expressing varying amounts of Nrf2, viz., breast cancer cell lines MDA-MB-231 and T47D (low functionally active Nrf2), HCT-116 (moderately active Nrf2), and lung cancer cell line A549 (highly active Nrf2), and the lead compound 5b was tested for its effect on cell cycle progression in vitro and for retarding the growth of Ehrlich ascites carcinomas (EACs) in mice. Data from our study demonstrated that among various compounds 5b exhibited better therapeutic index and retarded the growth of EAC cells in mice. Therefore, compound 5b is recommended for further development to target cancers.

Long Noncoding RNA Fos Downstream Transcript Is Developmentally Dispensable but Vital for Shaping the Poststroke Functional Outcome.

Background and Purpose: Stroke induces the expression of several long noncoding RNAs in the brain. However, their functional significance in poststroke outcome is poorly understood. We recently observed that a brain-specific long noncoding RNA called Fos downstream transcript (FosDT) is induced rapidly in the rodent brain following focal ischemia. Using FosDT knockout rats, we presently evaluated the role of FosDT in poststroke brain damage. Methods: FosDT knockout rats were generated using CRISPR-Cas9 genome editing on a Sprague-Dawley background. Male and female FosDT−/− and FosDT+/+ cohorts were subjected to transient middle cerebral artery occlusion. Postischemic sensorimotor deficits were evaluated between days 1 and 7 and lesion volume on day 7 of reperfusion. The developmental expression profile of FosDT was determined with real-time polymerase chain reaction and mechanistic implications of FosDT in the ischemic brain were conducted with RNA-sequencing analysis and immunostaining of pathological markers. Results: FosDT expression is developmentally regulated, with the adult cerebral cortex showing significantly higher FosDT expression than neonates. FosDT−/− rats did not show any anomalies in growth and development, fertility, brain cytoarchitecture, and cerebral vasculature. However, when subjected to transient focal ischemia, FosDT−/− rats of both sexes showed enhanced sensorimotor recovery and reduced brain damage. RNA-sequencing analysis showed that improved poststroke functional outcome in FosDT−/− rats is partially associated with curtailed induction of inflammatory genes, reduced apoptosis, mitochondrial dysfunction, and oxidative stress. Conclusions: Our study shows that FosDT is developmentally dispensable, mechanistically important, and a functionally promising target to reduce ischemic brain damage and facilitate neurological recovery.

Optimal diagnostic tool for surveillance of oesophageal varices during COVID-19 pandemic.

AIM: To study the diagnostic accuracy and utility of triphasic abdominal computed tomography (CT) in the diagnosis and grading of oesophageal varices (OVs) as an alternative to endoscopy during the COVID-19 pandemic. MATERIALS AND METHODS: A prospective analysis was undertaken of retrospective data from cirrhotic patients who underwent oesophago-gastro-duodenoscopy (OGD) and a triphasic abdominal CT from January to December 2019. Endoscopists and radiologists provided their respective independent assessment of OV grading after being blinded to the clinical details. Performance of CT grading of OVs was compared with the reference standard endoscopic grading using weighted kappa (k). Non-invasive scores such, as aspartate transaminase (AST)-to-platelet ratio index (APRI), Fibrosis-4 (FIB-4) Index, platelet: spleen (PS) ratio were correlated between the two techniques. RESULTS: OV grading between endoscopists and radiologists showed 81.73% agreement (85 out of 104 patients) in the comparative analysis of 104 cirrhotic patients, of which no varices (57.1%, n=4), small (85.1%, n=23), medium (72.2%%, n=26), and large varices (94.1%, n=32) with a weighted k score of 0.88 (95% confidence interval 0.82-0.94). Overall, the sensitivity of CT in the diagnosis of no, small, medium, and large OVs was 66.6%, 79.3%, 89.6%, and 94.1%, respectively, with an area under the receiver operating curve (AUROC) score of 0.775, 0.887, 0.839, and 0.914. Performance of APRI, FIB-4, and PS ratio correlated well with the severity of OVs with no difference between OGD and CT grading. CONCLUSION: Triphasic abdominal CT can be an invaluable tool in the diagnosis and grading of OVs during the COVID-19 pandemic.