Probiotic potential of ß­galactosidase­producing lactic acid bacteria from fermented milk and their molecular characterization.

Probiotics have attained significant interest in recent years as a result of their gut microbiome modulation and gastrointestinal health benefits. Numerous fermented foods contain lactic acid bacteria (LAB) which are considered as GRAS and probiotic bacteria. The present study aimed to investigate indigenous LAB from homemade fermented milk samples collected in remote areas of Karnataka (India), in order to isolate the most potent and well-adapted to local environmental conditions bacteria, which were then evaluated using a step-by-step approach focused on the evaluation of probiotic traits and ß-galactosidase-producing ability. LAB were screened using 5-bromo-4-chloro-3-indole-D-galactopyranoside (X-Gal) and O-nitrophenyl-ß-D-galactopyranoside (ONPG) as substrate, and exhibited ß-galactosidase activity ranging from 728.25 to 1,203.32 Miller units. The most promising isolates were selected for 16S rRNA gene sequence analysis and identified as Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lactiplantibacillus pentosus and Lactiplantibacillus sp. Furthermore, these isolates were evaluated by in vitro, viz., survival in gastrointestinal tract, antibiotic susceptibility, antimicrobial activity, cell surface characteristics, and haemolytic activity. All eight isolates demonstrated strong adherence and prevented pathogen penetration into HT-29 cells, indicating potential of the bacteria to scale up industrial level production of milk products for lactose intolerants.

Generation of a recombinant version of a biologically active cell-permeant human HAND2 transcription factor from E. coli.

Transcription factor HAND2 has a significant role in vascularization, angiogenesis, and cardiac neural crest development. It is one of the key cardiac factors crucial for the enhanced derivation of functional and mature myocytes from non-myocyte cells. Here, we report the generation of the recombinant human HAND2 fusion protein from the heterologous system. First, we cloned the full-length human HAND2 gene (only protein-coding sequence) after codon optimization along with the fusion tags (for cell penetration, nuclear translocation, and affinity purification) into the expression vector. We then transformed and expressed it in Escherichia coli strain, BL21(DE3). Next, the effect (in terms of expression) of tagging fusion tags with this recombinant protein at two different terminals was also investigated. Using affinity chromatography, we established the one-step homogeneous purification of recombinant human HAND2 fusion protein; and through circular dichroism spectroscopy, we established that this purified protein had retained its secondary structure. We then showed that this purified human protein could transduce the human cells and translocate to its nucleus. The generated recombinant HAND2 fusion protein showed angiogenic potential in the ex vivo chicken embryo model. Following transduction in MEF2C overexpressing cardiomyoblast cells, this purified recombinant protein synergistically activated the α-MHC promoter and induced GFP expression in the α-MHC-eGFP reporter assay. Prospectively, the purified bioactive recombinant HAND2 protein can potentially be a safe and effective molecular tool in the direct cardiac reprogramming process and other biological applications.

Tissue-Restricted Stem Cells as Starting Cell Source for Efficient Generation of Pluripotent Stem Cells: An Overview.

Induced pluripotent stem cells (iPSCs) have vast biomedical potential concerning disease modeling, drug screening and discovery, cell therapy, tissue engineering, and understanding organismal development. In the year 2006, a groundbreaking study reported the generation of iPSCs from mouse embryonic fibroblasts by viral transduction of four transcription factors, namely, Oct4, Sox2, Klf4, and c-Myc. Subsequently, human iPSCs were generated by reprogramming fibroblasts as a starting cell source using two reprogramming factor cocktails [(i) OCT4, SOX2, KLF4, and c-MYC, and (ii) OCT4, SOX2, NANOG, and LIN28]. The wide range of applications of these human iPSCs in research, therapeutics, and personalized medicine has driven the scientific community to optimize and understand this reprogramming process to achieve quality iPSCs with higher efficiency and faster kinetics. One of the essential criteria to address this is by identifying an ideal cell source in which pluripotency can be induced efficiently to give rise to high-quality iPSCs. Therefore, various cell types have been studied for their ability to generate iPSCs efficiently. Cell sources that can be easily reverted to a pluripotent state are tissue-restricted stem cells present in the fetus and adult tissues. Tissue-restricted stem cells can be isolated from fetal, cord blood, bone marrow, and other adult tissues or can be obtained by differentiation of embryonic stem cells or trans-differentiation of other tissue-restricted stem cells. Since these cells are undifferentiated cells with self-renewal potential, they are much easier to reprogram due to the inherent characteristic of having an endogenous expression of few pluripotency-inducing factors. This review presents an overview of promising tissue-restricted stem cells that can be isolated from different sources, namely, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, limbal epithelial stem cells, and spermatogonial stem cells, and their reprogramming efficacy. This insight will pave the way for developing safe and efficient reprogramming strategies and generating patient-specific iPSCs from tissue-restricted stem cells derived from various fetal and adult tissues.

Ubiquitin Mediated Degradation of EGFR by 17 ß-estradiol in Triple Negative MDA-MB-231 (TNBC) Breast Cancer Cells Line.

BACKGROUND: Triple Negative Breast Cancer (TNBC) commonly displays Epidermal growth factor receptor (EGFR). Effective EGFR degradation results in the suppression of tumor in various models. Studies have addressed the relevance of this strategy in the treatment of TNBC. In the present study, we examined the effect of 17 ß- estradiol on EGFR expression in MDA-MB-231 (TNBC) cell line and assessed whether 17 ß-estradiol degrades EGFR by ubiquitination pathway. OBJECTIVES: The objective of this study is to treat MDA-MB-231 cell lines with Cycloheximide with or without 17ß-estrdiol to observe whether 17ß-estradiol leads to EGFR degradation and to treat with MG-132 to assess whether degradation occurs through ubiquitination pathway. METHODS: MDA-MB-231 cells were treated with 17ß-estradiol (E2) and EGFR expression was studied by western blotting at different intervals by using Cycloheximide chase. To assess ubiquitination pathway of degradation of EGFR in MDA-MB-231 cell line, MG-132 was used. RESULTS: EGFR expression was reduced with ß-estradiol treatment in MDA-MB-231 cell line with Cycloheximide chase. Upon Treatment with MG-132 and E2, EGFR expression did not reduce, suggesting that Estrogen degrades EGFR by ubiquitination pathway. CONCLUSION: Estrogen degrades EGFR in MDA-MB-231 cells and this degradation occurs by ubiquitination.

An Overview on Promising Somatic Cell Sources Utilized for the Efficient Generation of Induced Pluripotent Stem Cells.

Human induced Pluripotent Stem Cells (iPSCs) have enormous potential in understanding developmental biology, disease modeling, drug discovery, and regenerative medicine. The initial human iPSC studies used fibroblasts as a starting cell source to reprogram them; however, it has been identified to be a less appealing somatic cell source by numerous studies due to various reasons. One of the important criteria to achieve efficient reprogramming is determining an appropriate starting somatic cell type to induce pluripotency since the cellular source has a major influence on the reprogramming efficiency, kinetics, and quality of iPSCs. Therefore, numerous groups have explored various somatic cell sources to identify the promising sources for reprogramming into iPSCs with different reprogramming factor combinations. This review provides an overview of promising easily accessible somatic cell sources isolated in non-invasive or minimally invasive manner such as keratinocytes, urine cells, and peripheral blood mononuclear cells used for the generation of human iPSCs derived from healthy and diseased subjects. Notably, iPSCs generated from one of these cell types derived from the patient will offer ethical and clinical advantages. In addition, these promising somatic cell sources have the potential to efficiently generate bona fide iPSCs with improved reprogramming efficiency and faster kinetics. This knowledge will help in establishing strategies for safe and efficient reprogramming and the generation of patient-specific iPSCs from these cell types.

Culture and characterization of human dental pulp­derived stem cells as limbal stem cells for corneal damage repair.

Limbal stem cell deficiency (LSCD) is one of the leading causes of corneal damage. Injury or inflammation in the cornea causes LSCD, which may be unilateral or bilateral depending upon the cause. Limbal epithelial cell implants successfully improve vision in patients with chemical injury­induced LSCD. Transplantation of cultured epithelial stem cells has become a treatment of choice for numerous patients with LSCD. Bilateral LSCD is frequently observed in the general population, where no residual stem cells are available for ex vivo culture. Allografts are associated with a high risk of rejection, neoplasia, and disease transmission. In this respect, allogenic cell populations from other regions in the patient may substitute for allogenic material. In the present study, dental pulp stem cells were cultured in limbal stem cell media and these cells were characterized against limbal stem cells, revealing the significance of using dental pulp stem cell treatment in bilateral LSCD. The morphology and culture pattern of both limbal and dental pulp stem cells grown in limbal stem­specific media were similar. Polymerase chain reaction analysis revealed that stem cell markers were highly expressed in limbal stem cells compared to in dental pulp stem cells, regardless of the medium and scaffold in which they were grown. Although dental pulp stem cell molecular expression is quite low at the transcript level, the functional protein level according to immunocytochemistry and western blot analyses demonstrated that stem cells and corneal differentiation molecule levels were quite high, indicating their potential as limbal stem cells in the respective microenvironment.