Oct4 overexpression facilitates proliferation of porcine fibroblasts and development of cloned embryos.

Octamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency of embryonic stem cells. Recent reports have shown that Oct4 also controls cell-cycle progression and enhances the proliferation of various types of cells. As the high proliferation of donor fibroblasts is critical to the production of transgenic pigs, using the somatic cell nuclear transfer technique, we analysed the effect of Oct4 overexpression on the proliferation of porcine fibroblasts and embryos. Porcine endogenous Oct4 cDNA was cloned, sequenced and inserted into an expression vector. The vector was transfected into porcine fibroblasts, and a stable Oct4-overexpressed cell line was established by antibiotic selection. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells. Real-time RT-PCR results showed that p16, Bcl2 and Myc were upregulated in Oct4-overexpressed cells. Somatic cell nuclear transfer was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared with that of wild-type cloned embryos. The cleavage and blastocyst formation rates were improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as day 5 in culture, while blastocysts were observed from day 6 in wild-type cloned embryos. In conclusion, the overexpression of Oct4 enhanced the proliferation of both porcine fibroblasts and embryos.

Quantitative analysis of lncRNA in formalin-fixed paraffin-embedded tissues of oral squamous cell carcinoma.

The study evaluated expression profiles of few regulatory lncRNAs in oral squamous cell carcinoma and normal mucosa adjacent to oral cancer using paired fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues stored at a different duration of time (1-5 years) using real-time quantitative PCR. The quantity and quality of total RNA isolated from FFPE tissues was less compared with that of fresh frozen tissues, which resulted in a noncorrelation of quantification cycle values. Following normalization, the expression of lncRNAs in the paired tissues did not differ significantly. The differential expression of the lncRNAs in the study was consistent with The Cancer Genome Atlas head and neck squamous cell carcinoma database. The study findings demonstrate the possibility of performing accurate quantitative analysis of lncRNAs using short amplicons and standardized real-time quantitative PCR assays in oral squamous cell carcinoma FFPE samples.

Functional and long-lived melanocytes from human pluripotent stem cells with transient ectopic expression of JMJD3.

BACKGROUND: Melanocytes are an essential part of the epidermis, and their regeneration has received much attention because propagation of human adult melanocytes in vitro is too slow for clinical use. Differentiation from human pluripotent stem cells to melanocytes has been reported, but the protocols to produce them require multiple and complex differentiation steps. METHOD: We differentiated human embryonic stem cells (hESCs) that transiently express JMJD3 to pigmented cells. We investigated whether the pigmented cells have melanocytic characteristics and functions by qRT-PCR, immunocytochemical analysis and flow cytometry. We also investigated their biocompatibility by injecting the cells into immunodeficient mice for clinical use. RESULT: We successfully differentiated and established a pure culture of melanocytes. The melanocytes maintained their growth rate for a long time, approximately 200 days, and were functional. They exhibited melanogenesis and transfer of melanin to peripheral keratinocytes. Moreover, melanocytes simulated the developmental processes from melanoblasts to melanocytes. The melanocytes had high engraftability and biocompatibility in the immunodeficient mice. CONCLUSION: The robust generation of functional and long-lived melanocytes are key to developing clinical applications for the treatment of pigmentary skin disorders.

In Silico Comparative Analysis of the Functional, Structural, and Evolutionary Properties of SARS-CoV-2 Variant Spike Proteins.

Background: A recent global outbreak of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) created a pandemic and emerged as a potential threat to humanity. The analysis of virus genetic composition has revealed that the spike protein, one of the major structural proteins, facilitates the entry of the virus to host cells. Objective: The spike protein has become the main target for prophylactics and therapeutics studies. Here, we compared the spike proteins of SARS-CoV-2 variants using bioinformatics tools. Methods: The spike protein sequences of wild-type SARS-CoV-2 and its 6 variants-D614G, alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), gamma (P.1), and omicron (B.1.1.529)-were retrieved from the NCBI database. The ClustalX program was used to sequence multiple alignment and perform mutational analysis. Several online bioinformatics tools were used to predict the physiological, immunological, and structural features of the spike proteins of SARS-CoV-2 variants. A phylogenetic tree was constructed using CLC software. Statistical analysis of the data was done using jamovi 2 software. Results: Multiple sequence analysis revealed that the P681R mutation in the delta variant, which changed an amino acid from histidine (H) to arginine (R), made the protein more alkaline due to arginine's high pKa value (12.5) compared to histidine's (6.0). Physicochemical properties revealed the relatively higher isoelectric point (7.34) and aliphatic index (84.65) of the delta variant compared to other variants. Statistical analysis of the isoelectric point, antigenicity, and immunogenicity of all the variants revealed significant correlation, with P values ranging from <.007 to .04. The generation of a 2D gel map showed the separation of the delta spike protein from a grouping of the other variants. The phylogenetic tree of the spike proteins showed that the delta variant was close to and a mix of the Rousettus bat coronavirus and MERS-CoV. Conclusions: The comparative analysis of SARS-CoV-2 variants revealed that the delta variant is more aliphatic in nature, which provides more stability to it and subsequently influences virus behavior.

INCIDENCE OF INFLAMMATORY BOWEL DISEASE: A SINGLE CENTRE RETROSPECTIVE STUDY.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder affecting the gastrointestinal tract. The etiology of this alarming condition is multifactorial. A Recently increasing trend in IBD is noted in our country. OBJECTIVE: The present study was designed with the main objective to assess the incidence and to identify the associated risk factors including demographic, geographical areas, and dietary patterns of IBD population of Northern of Karnataka viz. Hubli-Dharwad city. METHODS: A retrospective investigation was conducted on a cohort of 226 patients with a working diagnosis of IBD and those who were admitted between 2015 to 2019 the department of gastroenterology, SDMCMS&H. The diagnosis of IBD was made based on clinical, radiological, endoscopic, and histopathologic findings. The patients were categorized into IBD and those who have symptoms suggestive of IBD but did not fit into the diagnostic criteria into, non-IBD groups. The data about of on demography, diet patterns, and laboratory parameters were recorded. RESULTS: Among 226 patients enrolled in this study 2015-2019, IBD was confirmed in 54 Ulcerative colitis - 44 (19.46%), Crohn's disease - 10 (4.42%) patients with varying distribution of disease among different age groups and both genders, Ulcerative colitis (UC) [M: F: 28 (63.6%): 16 (36.4%)] and Crohn's disease (CD) [M: F: 07 (70.0%):03 (30.0%)]. Dietary pattern and other habitats had no significant contribution to illness and its symptoms. Urban (U) and Rural (R) divide was UC [U: R: 32 (72.7%): 12 (27.3%)], CD [U:R:07(70.0%):03(30.0%)] maintained. CONCLUSION: Incidence of IBD was high with UC as compared to CD. The incidence of IBD among patients presenting with symptoms suggestive of IBD is 19.46% with UC being major as compared to CD (4.42%). Male predominant patterns of IBD incidences were noted. Year by year increasing trend in disease burden was observed. The Dietary pattern has no direct correlation with IBD disease prevalence and incidences.

Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI).

BACKGROUND: Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI). METHODS: To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations. RESULTS: The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen. CONCLUSIONS: iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.

Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells.

Ammonia has a cytotoxic effect and can therefore be used as a selection agent for enrichment of zone I hepatocytes. However, it has not yet been determined whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed an ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human embryonic stem cells (ESCs) and from induced pluripotent stem cells (iPSCs). The resistance to cytotoxicity or cell death by ammonia is likely attributable to the metabolism of ammonia in the cells. In addition to ammonia metabolism-related genes, ammonia-selected hepatocytes showed increased expression of the cytochrome P450 genes. Additionally, the ammonia-selected cells achieved immortality or at least an equivalent life span to human pluripotent stem cells without affecting expression of the liver-associated genes. Ammonia treatment in combination with in vitro propagation is useful for obtaining large quantities of hepatocytes.

Incidência de doença inflamatória intestinal: estudo retrospectivo de um único centro / Incidence of inflammatory bowel disease: a single centre retrospective study

ABSTRACT Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder affecting the gastrointestinal tract. The etiology of this alarming condition is multifactorial. A Recently increasing trend in IBD is noted in our country. Objective: The present study was designed with the main objective to assess the incidence and to identify the associated risk factors including demographic, geographical areas, and dietary patterns of IBD population of Northern of Karnataka viz. Hubli-Dharwad city. Methods: A retrospective investigation was conducted on a cohort of 226 patients with a working diagnosis of IBD and those who were admitted between 2015 to 2019 the department of gastroenterology, SDMCMS&H. The diagnosis of IBD was made based on clinical, radiological, endoscopic, and histopathologic findings. The patients were categorized into IBD and those who have symptoms suggestive of IBD but did not fit into the diagnostic criteria into, non-IBD groups. The data about of on demography, diet patterns, and laboratory parameters were recorded. Results: Among 226 patients enrolled in this study 2015-2019, IBD was confirmed in 54 Ulcerative colitis - 44 (19.46%), Crohn's disease - 10 (4.42%) patients with varying distribution of disease among different age groups and both genders, Ulcerative colitis (UC) [M: F: 28 (63.6%): 16 (36.4%)] and Crohn's disease (CD) [M: F: 07 (70.0%):03 (30.0%)]. Dietary pattern and other habitats had no significant contribution to illness and its symptoms. Urban (U) and Rural (R) divide was UC [U: R: 32 (72.7%): 12 (27.3%)], CD [U:R:07(70.0%):03(30.0%)] maintained. Conclusion Incidence of IBD was high with UC as compared to CD. The incidence of IBD among patients presenting with symptoms suggestive of IBD is 19.46% with UC being major as compared to CD (4.42%). Male predominant patterns of IBD incidences were noted. Year by year increasing trend in disease burden was observed. The Dietary pattern has no direct correlation with IBD disease prevalence and incidences.

Resumo Contexto: A Doença inflamatória intestinal (DII) é uma doença inflamatória crônica que afeta o trato gastrointestinal. A etiologia desta condição alarmante é multifatorial. Uma tendência recentemente crescente na DII é notada em nosso país. Objetivo: O presente estudo foi desenhado com o objetivo principal de avaliar a incidência e identificar os fatores de risco associados, incluindo demográficos, áreas geográficas e padrões alimentares da população com DII do Norte de Karnataka viz. Cidade de Hubli-Dharwad. Metodos: Uma investigação retrospectiva foi realizada em uma coorte de 226 pacientes com diagnóstico de DII e que foram admitidos entre 2015 e 2019 no departamento de gastroenterologia, SDMCMS&H. O diagnóstico de DII foi feito com base em achados clínicos, radiológicos, endoscópicos e histopatológicos. Os pacientes foram categorizados em DII e aqueles que apresentam sintomas sugestivos de DII, mas não se enquadraram nos critérios diagnósticos em grupos sem DII. Os dados sobre a demografia, padrões de dieta e parâmetros laboratoriais foram registrados. Resultados: Entre os 226 pacientes inscritos neste estudo entre 2015-2019, DII foi confirmada em 54 [RCUI - 44 (19,46%), DC - 10 (4,42%)] com distribuição variada da doença entre diferentes faixas etárias e ambos os sexos, colite ulcerativa (RCUI) [M: F: 28 (63,6%):16 (36,4%)] e doença de Crohn (DC) [M: F: 07 (70,0%): 03 (30,0%)]. O padrão alimentar e outros hábitos não tiveram contribuição significativa para a doença e seus sintomas. Urbanos (U) e rurais (R) dividiram-se em RCUI [U: R: 32 (72,7%):12 (27,3%)], DC [07 (70,0%): 03 (30,0%)]. Conclusão A incidência de DII foi elevada para RCUI em relação a DC. A incidência de DII entre os pacientes com sintomas sugestivos de DIB é de 19,46% com a RCUI sendo maior em relação a DC (4,42%). Foram observados padrões predominantes masculinos de incidência de DII. Ano a ano foi observada tendência crescente de carga da doença. O padrão dietético não tem correlação direta com a prevalência e incidências da DII.

Autologous bone marrow mesenchymal cell induced chondrogenesis for the treatment of osteoarthritis of knee.

Healthy and high quality of life has become the main issue with increasing human life span. Many biological treatments for osteoarthritis of the knee have been tried with limited success. We compared data from 7 patients who underwent total knee arthroplasty and 46 patients who underwent autologous bone-marrow mesenchymal cell induced chondrogenesis (MCIC) for osteoarthritis of grade IV of the Kellgren-Lawrence classification and grade IV of modified Outerbridge classification from 50 to 65 years of age. Clinical evaluation of the 2 groups showed significant improvement in the mean telephone Knee Society Scoring system (tKSS)-A (pain) and tKSS-B (function) scores throughout the postoperative follow-up period. There was no difference in the patients' satisfaction between the 2 groups. MCIC is a treatment option at least for delaying disease progression of osteoarthritis of the knee. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s13770-016-9125-y and is accessible for authorized users.