An Insight into Vital Genes Responsible for ß-cell Formation.

The regulation of glucose homeostasis and insulin secretion by pancreatic ß-cells, when disturbed, will result in diabetes mellitus. Replacement of dysfunctional or lost ß-cells with fully functional ones can tackle the problem of ß-cell generation in diabetes mellitus. Various pancreatic-specific genes are expressed during different stages of development, which have essential roles in pancreatogenesis and ß-cell formation. These factors play a critical role in cellular-based studies like transdifferentiation or de-differentiation of somatic cells to multipotent or pluripotent stem cells and their differentiation into functional ß-cells. This work gives an overview of crucial transcription factors expressed during various stages of pancreas development and their role in ß-cell specification. In addition, it also provides a perspective on the underlying molecular mechanisms.

Soluble expression, purification, and secondary structure determination of human PDX1 transcription factor.

The transcription factor PDX1 is a master regulator essential for proper development of the pancreas, duodenum and antrum. Furthermore, it is an indispensable reprogramming factor for the derivation of human ß-cells, and recently, it has been identified as a tumor suppressor protein in gastric cancer. Here, we report the soluble expression and purification of the full-length human PDX1 protein from a heterologous system. To achieve this, the 849 bp coding sequence of the PDX1 gene was first codon-optimized for expression in Escherichia coli (E. coli). This codon-optimized gene sequence was fused to a protein transduction domain, a nuclear localization sequence, and a His-tag, and this insert was cloned into the protein expression vector for expression in E. coli strain BL21(DE3). Next, screening and identification of the suitable gene construct and optimal expression conditions to obtain this recombinant fusion protein in a soluble form was performed. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Importantly, the secondary structure of the protein was retained after purification. Further, this recombinant PDX1 fusion protein was applied to human cells and showed the ability to enter the cells as well as translocate to the nucleus. This recombinant tool can be used as a safe tool and can potentially replace its genetic and viral forms in the reprogramming process to induce a ß-cell-specific transcriptional profile in an integration-free manner. Additionally, it can also be used to elucidate its role in cellular processes and for structural and biochemical studies.

Recent Advances in the Generation of ß-Cells from Induced Pluripotent Stem Cells as a Potential Cure for Diabetes Mellitus.

Diabetes mellitus (DM) is a group of metabolic disorders characterized by high blood glucose levels due to insufficient insulin secretion, insulin action, or both. The present-day solution to diabetes mellitus includes regular administration of insulin, which brings about many medical complications in diabetic patients. Although islet transplantation from cadaveric subjects was proposed to be a permanent cure, the increased risk of infections, the need for immunosuppressive drugs, and their unavailability had restricted its use. To overcome this, the generation of renewable and transplantable ß-cells derived from autologous induced pluripotent stem cells (iPSCs) has gained enormous interest as a potential therapeutic strategy to treat diabetes mellitus permanently. To date, extensive research has been undertaken to derive transplantable insulin-producing ß-cells (iß-cells) from iPSCs in vitro by recapitulating the in vivo developmental process of the pancreas. This in vivo developmental process relies on transcription factors, signaling molecules, growth factors, and culture microenvironment. This review highlights the various factors facilitating the generation of mature ß-cells from iPSCs. Moreover, this review also describes the generation of pancreatic progenitors and ß-cells from diabetic patient-specific iPSCs, exploring the potential of the diabetes disease model and drug discovery. In addition, the applications of genome editing strategies have also been discussed to achieve patient-specific diabetes cell therapy. Last, we have discussed the current challenges and prospects of iPSC-derived ß-cells to improve the relative efficacy of the available treatment of diabetes mellitus.

Severe postural hypotension and sinus bradycardia with thalidomide in patients with erythema nodosum leprosum.

Two men in their 60s and 40s were diagnosed with erythema nodosum leprosum based on the development of recurrent painful ulcers and nodules, respectively, for the previous 6 months. Thalidomide 100 mg four times a day, along with MB-MDT, was started in both patients. Both patients experienced severe dizziness on rising from a seated posture soon after initiation of thalidomide and a decrease in blood pressure and heart rate. Cardiovascular/neurology examination and routine blood investigations were normal. An autonomic nervous system assessment indicated bradycardia, postural hypotension and decreased cardiac autonomic function. The dosage of thalidomide was then gradually reduced over 4-5 days to 100 mg/day following a suspicion that thalidomide was the cause of postural hypotension. The dizziness subsided, and blood pressure and heart rate returned to normal.We concluded that thalidomide was the culprit behind bradycardia and dose- dependent postural hypotension.

Gamma delta T cells in acute myeloid leukemia: biology and emerging therapeutic strategies.

γδ T cells play an important role in disease control in acute myeloid leukemia (AML) and have become an emerging area of therapeutic interest. These cells represent a minor population of T lymphocytes with intrinsic abilities to recognize antigens in a major histocompatibility complex-independent manner and functionally straddle the innate and adaptive immunity interface. AML shows high expression of phosphoantigens and UL-16 binding proteins that activate the Vδ2 and Vδ1 subtypes of γδ T cells, respectively, leading to γδ T cell-mediated cytotoxicity. Insights from murine models and clinical data in humans show improved overall survival, leukemia-free survival, reduced risk of relapse, enhanced graft-versus-leukemia effect, and decreased graft-versus-host disease in patients with AML who have higher reconstitution of γδ T cells following allogeneic hematopoietic stem cell transplantation. Clinical trials leveraging γδ T cell biology have used unmodified and modified allogeneic cells as well as bispecific engagers and monoclonal antibodies. In this review, we discuss γδ T cells' biology, roles in cancer and AML, and mechanisms of immune escape and antileukemia effect; we also discuss recent clinical advances related to γδ T cells in the field of AML therapeutics.

Evaluation of oral health education programs among patients with schizophrenia in India: An interventional study.

BACKGROUND: Schizophrenia is a mental disease that can drastically affect oral health. Hence, this remains a significant factor that affects oral health-related quality of life (OHRQoL). Assessing the OHRQoLin schizophrenic patients is one of the primary needs. The oral health impact profile is an impressive range of instruments that assesses the impact of oral conditions on well-being and quality of life. MATERIALS AND METHODS: A 100 schizophrenic individuals were randomly selected from the Psychiatry Department of Government Hospital, Patna, Bihar, India, and grouped into two groups: (a) Group I with.group-based sessions and (b) Group II with one-on-one interactions. Inclusion criteria: (a) Individuals who received the clinical confirmatory diagnosis of schizophrenia, (b) Patients who regularly reported to the psychologist for periodic evaluation, and (c) Patients who could follow instructions. Exclusion criteria: (a) Patients or their caregivers who were unwilling to participate in the study, (b) Patients with any other mental health disability other than schizophrenia, (c) Patients with muscular or nervous system disorders, and (d) Patients who could not follow instructions. Patients were educated on the modified Bass technique, and mean ± standard deviation plaque scores were compared at the baseline and after 12 weeks of intervention. RESULTS: Statistical analysis was performed using the Chi-square analytical test and paired "t-test." Statistically significant differences were observed in plaque scores in both groups (P < 0.001). CONCLUSION: Educational training tools help in improving oral health in schizophrenic patients.

Effect of oral doxycycline, azithromycin and isotretinoin on haematological inflammatory markers and interleukin-17A levels in acne vulgaris: a single blinded randomised interventional study.

Amidst the existing literature on the effect of isotretinoin on serum interleukin-17 levels in acne patients, the effects of oral antibiotics azithromycin and doxycycline on serum interleukin-17 is scarce. We conducted an investigator blinded randomized interventional study to compare the effect of doxycycline, azithromycin and isotretinoin on inflammatory markers and Interleukin-17A (IL-17A) levels in acne. Patients were randomized and received the treatment according to treatment arm till 12 weeks. At baseline and 12 weeks/treatment completion, clinical improvement and Red-cell-distribution width (RDW),Neutrophil-lymphocyte ratio(NLR),Platelet-lymphocyte ratio(PLR), Mean-Platelet volume(MPV), Platelet-distribution width(PDW) and Interleukin-17A levels were analysed. P-value < 0.05 was considered statistically significant. Out of 120 patients, 110 patients completed the study. Baseline Global acne grading scale (GAGS) in doxycycline, azithromycin or isotretinoin group was 24.32 ± 3.119, 24.12 ± 2.804 and 25.10 ± 3.985 respectively and post-treatment was 5.216 ± 1.88, 7.265 ± 2.17 and 2.769 ± 1.08. All the drugs caused a statistically significant decrease in RDW and IL-17 A levels. Baseline levels of IL-17 A were significantly higher in patients with higher GAGS and post-acne scarring. One of the limitations of our study was that we excluded severe nodulocystic acne patients thereby these results have to be carefully extrapolated.

Mortality and Outcomes in Cerebrovascular Disease Patients With Emphasis on COVID-19: A Cross-Sectional Analysis of the National Inpatient Sample 2020.

Background COVID-19-related pulmonary complications have been explored extensively in the recent past. There is also a significant amount of literature on the neurological manifestations of COVID-19. However, there exists an unmet need to assess the impact of COVID-19 on patients with cerebrovascular diseases and its role in affecting mortality in such patients. Methods In this cross-sectional study, we analyzed 401,318 hospitalized patients with cerebrovascular diseases using the discharge data from the National Inpatient Sample 2020 to assess the association of COVID-19 with multiple clinical conditions, along with additional factors, such as length of stay in the hospital, total charges incurred, region and type of hospital, and primary insurance/payer in the United States of America. We used a multivariable logistic regression model to predict factors relating to mortality in such patients. Results The mortality during hospitalization in patients with cerebrovascular disease who were also diagnosed with COVID-19 was significantly higher than the patients without COVID-19 (22.50% vs 5.44%, p-value <0.0001). COVID-19 independently increased the odds of death significantly in patients with cerebrovascular diseases (adjusted OR = 4.81, p-value <0.0001). Other statistically and clinically significant factors that contributed to increased odds of mortality in such patients were comorbidities such as moderate/severe liver disease, myocardial infarction, congestive heart failure, and complications such as the development of a saddle pulmonary embolus. Conclusion COVID-19 was associated with higher mortality in patients with cerebrovascular diseases. It also significantly increased the duration of hospital stay and odds of mortality in such patients.

Production of Bioactive Human PAX4 Protein from E. coli.

Paired box 4 (PAX4) is a pivotal transcription factor involved in pancreatogenesis during embryogenesis, and in adults, it is key for ß-cell proliferation and survival. Additionally, PAX4 also functions as a tumor suppressor protein in human melanomas. The present study demonstrates the production of bioactive recombinant human PAX4 transcription factor. At first, the inserts (PAX4 protein-coding sequence having tags at either ends) were cloned in an expression vector to give rise to pET28a(+)-HTN-PAX4 and pET28a(+)-PAX4-NTH genetic constructs, and these were then transformed into Escherichia coli (E. coli) for their expression. The HTN-PAX4 and PAX4-NTH fusion proteins produced were purified with a yield of ~ 3.15 mg and ~ 0.83 mg, respectively, from 1.2 L E. coli culture. Further, the secondary structure retention of the PAX4 fusion proteins and their potential to internalize the mammalian cell and its nucleus was demonstrated. The bioactivity of these fusion proteins was investigated using various assays (cell migration, cell proliferation and cell cycle assays), demonstrating it to function as a tumor suppressor protein. Thus, this macromolecule can prospectively help understand the function of human PAX4 in cellular processes, disease-specific investigations and direct cellular reprogramming.

Protein Production and Purification of a Codon-Optimized Human NGN3 Transcription Factor from E. coli.

Neurogenin 3 (NGN3) transcription factor is vital for the development of endocrine cells of the intestine and pancreas. NGN3 is also critical for the neural precursor cell determination in the neuroectoderm. Additionally, it is one of the vital transcription factors for deriving human ß-cells from specialized somatic cells. In the current study, the production and purification of the human NGN3 protein from Escherichia coli (E. coli) is reported. First, the 642 bp protein-coding nucleotide sequence of the NGN3 gene was codon-optimized to enable enhanced protein expression in E. coli strain BL21(DE3). The codon-optimized NGN3 sequence was fused in-frame to three different fusion tags to enable cell penetration, nuclear translocation, and affinity purification. The gene insert with the fusion tags was subsequently cloned into an expression vector (pET28a( +)) for heterologous expression in BL21(DE3) cells. A suitable genetic construct and the ideal expression conditions were subsequently identified that produced a soluble form of the recombinant NGN3 fusion protein. This NGN3 fusion protein was purified to homogeneity (purity > 90%) under native conditions, and its secondary structure was retained post-purification. This purified protein, when applied to human cells, did not induce cytotoxicity. Further, the cellular uptake and nuclear translocation of the NGN3 fusion protein was demonstrated followed by its biological activity in PANC-1 cells. Prospectively, this recombinant protein can be utilized for various biological applications to investigate its functionality in cell reprogramming, biological processes, and diseases.