Unconventional role of Rab4 in the secretory pathway in Leishmania.

Leishmania donovani is an auxotroph for heme. Parasite acquires heme by clathrin-mediated endocytosis of hemoglobin by specific receptor. However, the regulation of receptor recycling pathway is not known in Leishmania. Here, we have cloned, expressed and characterized the Rab4 homologue from L. donovani. We have found that LdRab4 localizes in both early endosomes and Golgi in L. donovani. To understand the role of LdRab4 in L. donovani, we have generated transgenic parasites overexpressing GFP-LdRab4:WT, GFP-LdRab4:Q67L, and GFP-LdRab4:S22N. Our results have shown that overexpression of GFP-LdRab4:Q67L or GFP-LdRab4:S22N does not alter the cell surface localization of hemoglobin receptor in L. donovani. Surprisingly, we have found that overexpression of GFP-LdRab4:S22N significantly blocks the transport of Ldgp63 to the cell surface whereas the trafficking of Ldgp63 is induced to the cell surface in GFP-LdRab4:WT and GFP-LdRab4:Q67L overexpressing parasites. Consequently, we have found significant inhibition of gp63 secretion by GFP-LdRab4:S22N overexpressing parasites whereas secretion of Ldgp63 is enhanced in GFP-LdRab4:WT and GFP-LdRab4:Q67L overexpressing parasites in comparison to untransfected control parasites. Moreover, we have found that survival of transgenic parasites overexpressing GFP-LdRab4:S22N is severely compromised in macrophages in comparison to GFP-LdRab4:WT and GFP-LdRab4:Q67L expressing parasites. These results demonstrated that LdRab4 unconventionally regulates the secretory pathway in L. donovani.

Acanthosis nigricans independently predicts hepatic fibrosis in people with type 2 diabetes in North India.

BACKGROUND: Acanthosis nigricans (AN) is a skin condition characterized by hyperpigmentation and thickening, often found in individuals with insulin resistance. Despite this well-established association, the potential link between AN and hepatic fibrosis in people with type 2 diabetes (T2D) has yet to be thoroughly explored. METHODOLOGY: We recruited a total of 300 people with T2D, half of whom had AN (n, 150), and the other half without AN (n, 150). We evaluated body composition, biochemistry, and hepatic fat analysis (using the controlled attenuation parameter, CAP), as well as assessments of hepatic stiffness (using the kilopascal, kPa) using Fibroscan. We used multivariable regression analysis to find independent predictors of AN and their relationship to hepatic fibrosis. Furthermore, we developed a prediction equation and AUC for hepatic fibrosis. RESULTS: Upon comparison between AN vs. NAN group, following were significatly higher; weight, BMI, hepatic transaminases, liver span, CAP, and kPa. After adjusting for age, weight, body mass index, diabetes duration, and specific anti-hyperglycaemic drugs (gliclazide, DPP-4 inhibitors, pioglitazone, and Glucagon-like peptide-1 receptor agonists), adjusted OR for AN were, liver span, 1.78 (95% CI: 0.91-3.49, p = 0.09), CAP, 7.55 (95% CI: 0.93-61.1, p = 0.05), and kPa, 2.47 (95% CI: 1.50-4.06, p = 0.001). A ROC analysis of predictive score for hepatic fibrosis showed optimal sensitivity and specificity at a score cut-off of 25.2 (sensitivity 62%, specificity 63%), with an AUC of 0.6452 (95% CI: 0.61235-0.76420). CONCLUSION: Acanthosis nigricans has the potential to be used as an easy-to-identify clinical marker for risk of hepatic fat and fibrosis in Asian Indians with T2D, allowing for early detection and management strategies.

Machine learning based multipurpose medical image watermarking.

Digital data security has become an exigent area of research due to a huge amount of data availability at present time. Some of the fields like medical imaging and medical data sharing over communication platforms require high security against counterfeit access, manipulation and other processing operations. It is essential because the changed/manipulated data may lead to erroneous judgment by medical experts and can negatively influence the human's heath. This work offers a blind and robust medical image watermarking framework using deep neural network to provide effective security solutions for medical images. During watermarking, the region of interest (ROI) data of the original image is preserved by employing the LZW (Lampel-Ziv-Welch) compression algorithm. Subsequently the robust watermark is inserted into the original image using IWT (integer wavelet transform) based embedding approach. Next, the SHA-256 algorithm-based hash keys are generated for ROI and RONI (region of non-interest) regions. The fragile watermark is then prepared by ROI recovery data and the hash keys. Further, the LSB replacement-based insertion mechanism is utilized to embed the fragile watermark into RONI embedding region of robust watermarked image. A deep neural network-based framework is used to perform robust watermark extraction for efficient results with less computational time. Simulation results verify that the scheme has significant imperceptibility, efficient robust watermark extraction, correct authentication and completely reversible nature for ROI recovery. The relative investigation with existing schemes confirms the dominance of the proposed work over already existing work.

Sodium-glucose cotransporter-2 inhibitors in patients with type 2 diabetes mellitus and moderate to severe hepatic fibrosis: A retrospective study.

BACKGROUND: Sodium-glucose cotransporter-2 inhibitors (SGLT2i) have been shown to decrease hepatic transaminases, steatosis, and in some studies, hepatic fibrosis. However, the safety and efficacy of SGLT2i has not been tested in patients who have moderate to severe hepatic fibrosis. METHODS: In a retrospective study of sixty patients with moderate to severe hepatic fibrosis (kPa estimated by Fibroscan > 10), SGLT2i were prescribed on top of other oral anti-hyperglycemic medications. The safety and efficacy of SGLT2i were evaluated. Using the Fibroscan, CAP scores (decibel/meter), and liver stiffness measurement (LSM) (kPa, kilopascals) were examined before and after treatment. RESULTS: The mean age of the T2DM patients was 54.7 ± 10.3 years, and the mean duration of T2DM was 8.3 ± 7.1 years. SGLT2i were given from 3 to 36 months. After treatment, a decrease in glycated hemoglobin (HbA1c), and hepatic transaminases (SGOT and SGPT) was recorded. Upon follow up, CAP and kPa scores decreased significantly. Importantly, no adverse drug reaction, such as balanoposthitis, vulvovaginitis, urosepsis, and postural drop in blood pressure, were reported in any patient. CONCLUSION: In this retrospective cohort study, patient with T2DM and moderate to severe hepatic fibrosis, use of SGLT2i is safe with respect to common adverse effects & may have contributed to improved hepatic profile.

Hemoglobin Endocytosis and Intracellular Trafficking: A Novel Way of Heme Acquisition by Leishmania.

Leishmania species are causative agents of human leishmaniasis, affecting 12 million people annually. Drugs available for leishmaniasis are toxic, and no vaccine is available. Thus, the major thrust is to identify new therapeutic targets. Leishmania is an auxotroph for heme and must acquire heme from the host for its survival. Thus, the major focus has been to understand the heme acquisition process by the parasites in the last few decades. It is conceivable that the parasite is possibly obtaining heme from host hemoprotein, as free heme is not available in the host. Current understanding indicates that Leishmania internalizes hemoglobin (Hb) through a specific receptor by a clathrin-mediated endocytic process and targets it to the parasite lysosomes via the Rab5 and Rab7 regulated endocytic pathway, where it is degraded to generate intracellular heme that is used by the parasite. Subsequently, intra-lysosomal heme is initially transported to the cytosol and is finally delivered to the mitochondria via different heme transporters. Studies using different null mutant parasites showed that these receptors and transporters are essential for the survival of the parasite. Thus, the heme acquisition process in Leishmania may be exploited for the development of novel therapeutics.

Multipurpose medical image watermarking for effective security solutions.

Digital medical images contain important information regarding patient's health and very useful for diagnosis. Even a small change in medical images (especially in the region of interest (ROI)) can mislead the doctors/practitioners for deciding further treatment. Therefore, the protection of the images against intentional/unintentional tampering, forgery, filtering, compression and other common signal processing attacks are mandatory. This manuscript presents a multipurpose medical image watermarking scheme to offer copyright/ownership protection, tamper detection/localization (for ROI (region of interest) and different segments of RONI (region of non-interest)), and self-recovery of the ROI with 100% reversibility. Initially, the recovery information of the host image's ROI is compressed using LZW (Lempel-Ziv-Welch) algorithm. Afterwards, the robust watermark is embedded into the host image using a transform domain based embedding mechanism. Further, the 256-bit hash keys are generated using SHA-256 algorithm for the ROI and eight RONI regions (i.e. RONI-1 to RONI-8) of the robust watermarked image. The compressed recovery data and hash keys are combined and then embedded into the segmented RONI region of the robust watermarked image using an LSB replacement based fragile watermarking approach. Experimental results show high imperceptibility, high robustness, perfect tamper detection, significant tamper localization, and perfect recovery of the ROI (100% reversibility). The scheme doesn't need original host or watermark information for the extraction process due to the blind nature. The relative analysis demonstrates the superiority of the proposed scheme over existing schemes.

Dapagliflozin Improves Body Fat Patterning, and Hepatic and Pancreatic Fat in Patients With Type 2 Diabetes in North India.

CONTEXT: Excess hepatic and pancreatic fat may contribute to hyperglycemia. OBJECTIVE: The objective of this study was to examine the effect of dapagliflozin (an SGLT2 inhibitor) on anthropometric profile, liver, and pancreatic fat in patients with type 2 diabetes mellitus (T2DM). METHODS: This is an observational interventional paired study design without a control group. Patients (n = 30) were given dapagliflozin 10 mg/day (on top of stable dose of metformin and/or sulfonylureas) for 120 days. Changes in anthropometry (circumferences and skinfold thickness), surrogate markers of insulin resistance, body composition, liver, and pancreatic fat (as measured by magnetic resonance imaging (MRI)-derived proton density fat fraction [FF]) were evaluated. RESULTS: After 120 days of treatment with dapagliflozin, a statistically significant reduction in weight, body mass index (BMI), body fat, circumferences, and all skinfold thickness was seen. A statistically significant reduction in blood glucose, glycated hemoglobin A1c, hepatic transaminases, fasting insulin, homeostatic model assessment of insulin resistance (HOMA-IR), and postprandial C-peptide was noted, while HOMA-ß, postprandial insulin sensitivity, and fasting adiponectin were statistically significantly increased. There was no change in lean body mass. Compared to baseline there was a statistically significant decrease in mean liver FF (from 15.2% to 10.1%, P < .0001) and mean pancreatic FF (from 7.5% to 5.99%, P < .0083). Reduction in liver fat was statistically significant after adjustment for change in body weight. CONCLUSION: Dapagliflozin, after 120 days of use, reduced pancreatic and liver fat and increased insulin sensitivity in Asian Indian patients with T2DM.

High prevalence of hepatic steatosis and hepatic fibrosis in patients with type 2 diabetes mellitus.

AIM: To determine the prevalence of hepatic steatosis and fibrosis in patients with T2DM from North India. RESEARCH DESIGN AND METHODS: In this cross-sectional study, Asian Indian patients with T2DM (n,250) underwent liver ultrasonography (USG), Fibroscan for assessment of hepatic steatosis (Controlled Attenuation Parameter, CAP) and hepatic fibrosis (Kilopascals, kPa) respectively. Pearson's correlation analysis & logistic regression analysis for significant predictors of hepatic steatosis and fibrosis were done. The cut-off value of liver span was calculated by ROC-AUC analysis. RESULTS: Grade 3 hepatic steatosis was seen in 213 T2DM patients (85.2%). It was higher in males than females and in those with high BMI values. Any degree of fibrosis and severe fibrosis were seen in 205 (62%) and 46 (18.4%) patients, respectively; these were higher in males, specifically in those with BMI >30 kg/m2, and diabetes of a duration more than 5 years. BMI and SGPT were the significant predictors of hepatic steatosis. An increase of 1 unit of BMI above 23 kg/m2 led to 19.6 times increased risk of hepatic steatosis in T2DM patients aged 50 years and above. SGOT and GGTP were significant predictors of any degree of hepatic fibrosis. On ROC-AUC analysis, liver span cut-off values of ≥16.4 cms and ≥16.8 cm in males and females respectively, were predictive of hepatic fibrosis. CONCLUSION: High prevalence of grade 3 hepatic steatosis and hepatic fibrosis needs increased vigilance and corrective lifestyle and pharmacological measures. Asian Indian patients with T2DM and BMI >30 kg/m2, with duration of diabetes above 5 years & an ultrasound derived liver span ≥16.4 cms, should be further evaluated for hepatic fibrosis.

High prevalence of post COVID-19 fatigue in patients with type 2 diabetes: A case-control study.

BACKGROUND: Post COVID-19 syndrome (PCS) has emerged as a major roadblock in the recovery of patients infected with SARS-CoV-2. Amongst many symptoms like myalgia, headache, cough, breathlessness; fatigue is is most prevalent and makes the patient severely debilitated. Research on PCS, in particular fatigue, in patients with diabetes has not been done. METHODOLOGY: In this prospective study, we included patients with type 2 diabetes (T2D) who had COVID-19 (mild to moderate severity), and matched T2D patients who did not suffer from COVID-19. Demography, anthropometry, glycemic measures, treatment, and details of COVID-19 were recorded. Symptoms were scored using Chalder Fatigue Scale (reported as fatigue score, FS) and handgrip strength (in kg) was recorded by Jamar Hydraulic Hand Dynamometer. RESULTS: A total of 108 patients were included (cases, 52, controls, 56). Both groups were matched for age, duration of diabetes, BMI, TSH, serum albumin and vitamin D levels. T2D patients who had COVID-19 showed significantly more fatigue when compared with patients who did not have COVID-19 but both groups had comparable handgrip strength. Furthermore, patients with T2D with previous COVID-19 infection and who had FS > 4 have had significant higher inflammation markers during acute illness, and post COVID-19, had increased post prandial blood glucose levels, lost more weight, had reduced physical activity and showed significantly lower handgrip strength as compared to those with FS < 4. CONCLUSION: Patients with T2D who had COVID-19 infection as compared to those without had significantly more fatigue after the acute illness, and those with higher FS had reduced handgrip strength indicating sarcopenia, even after careful matching for common contributory factors to fatigue at baseline. Rehabilitation of those with FS>4 after acute infection would require careful attention to nutrition, glycemic control and graduated physical activity protocol.

Rab5b function is essential to acquire heme from hemoglobin endocytosis for survival of Leishmania.

Previously, we showed that Rab5a and Rab5b differentially regulate fluid-phase and receptor-mediated endocytosis in Leishmania, respectively. To unequivocally demonstrate the role of Rab5b in hemoglobin endocytosis in Leishmania, we generated null-mutants of Rab5b parasites by sequentially replacing both copies of LdRab5b with the hygromycin and neomycin resistance gene cassettes. LdRab5b-/- null-mutant parasite was confirmed by qPCR analysis of genomic DNA using LdRab5b specific primers. LdRab5b-/- cells showed severe growth defect indicating essential function of LdRab5b in parasite. To characterize the role of Rab5b in Hb endocytosis in parasites, LdRab5b-/- cells were rescued by exogenous addition of hemin in growth medium. Our results showed that LdRab5b-/- cells are relatively smaller in size. Ultrastructural analysis revealed the presence of relatively enlarged flagellar pocket and bigger intracellular vesicles in these cells in comparison to control cells. Both promastigotes and amastigotes of Rab5b null-mutant parasites were unable to internalize Hb but fluid phase endocytosis of different markers was not affected. However, complementation of LdRab5b:WT in LdRab5b-/- cells (LdRab5b-/-:pRab5b:WT) rescued Hb internalization in these cells. Interestingly, LdRab5b-/- cells showed significantly less Hb-receptor on cell surface in comparison to control cells indicating a block in HbR trafficking. Finally, we showed that LdRab5b-/- parasites can infect the macrophages but are unable to survive after 96 h of infection in comparison to control cells. However, supplementation of hemin in the growth medium significantly rescued LdRab5b-/-Leishmania survival in macrophage indicating that LdRab5b function is essential for the acquisition of heme from internalized Hb for the survival of Leishmania.

Identification and characterization of the hemoglobin-binding domain of hemoglobin receptor in Leishmania.

Leishmania internalize hemoglobin (Hb) via a specific receptor (HbR) for their survival. To identify the Hb-binding domain of HbR, we cloned and expressed several truncated proteins of HbR and determined their ability to bind Hb. Our findings reveal that 90% of Hb-binding activity is retained in HbR41-80 in comparison with HbR1-471 . We synthesized a 40 amino acid peptide (SSEKMKQLTMYMIHEMVEGLEGRPSTVRMLPSFVYTSDPA) corresponding to HbR41-80 and found that it specifically binds Hb. Subsequently, we found that the HbR41-80 peptide completely blocks Hb uptake in both promastigote and amastigote forms of Leishmania and, thereby, inhibits the growth of the parasite. These results demonstrate that HbR41-80 is the Hb-binding domain of HbR, which might be used as a potential therapeutic agent to inhibit the growth of Leishmania.

Breakthrough COVID19 infections after vaccinations in healthcare and other workers in a chronic care medical facility in New Delhi, India.

BACKGROUND AND AIMS: Vaccinations for COVID19 are now open to all adults in India. However, spread of COVID19 infection continues unabated. We aimed to ascertain number of breakthrough COVID19 infections after vaccinations in a chronic care, diabetes-centric healthcare facility. METHODS: We reviewed rigorously maintained data of vaccinations, health status, symptoms of COVID19 & RT-PCR testing of all staff (doctors, nurses, paramedical workers, and other staff) in our health care facility from January 16, 2021 till date. RESULTS: Out of 123 employees, 113 were vaccinated (Covaxin, 28, Covishield, 85). Second dose was completed in 107 (94.7%) and first dose in 6 persons (5.3%). Symptomatic COVD19 infections occurred in 19 persons (16.9%) post any dose of vaccine. Symptomatic breakthrough infections > 14 days after second dose occurred in 15 persons (13.3%). Except one (required hospitalization), all 14 had mild COVID19 disease. CONCLUSIONS: We report mild symptomatic breakthrough infections as seen in our health care facility. Research in breakthrough infections in India should be extended to other institutions and community to obtain larger data.