Residues at the interface between zinc binding and winged helix domains of human RECQ1 play a significant role in DNA strand annealing activity

RECQ1 is the shortest among the five human RecQ helicases comprising of two RecA like domains, a zinc-binding domain and a RecQ C-terminal domain containing the winged-helix (WH). Mutations or deletions on the tip of a β-hairpin located in the WH domain are known to abolish the unwinding activity. Interestingly, the same mutations on the β-hairpin of annealing incompetent RECQ1 mutant (RECQ1^T1) have been reported to restore its annealing activity. In an attempt to unravel the strand annealing mechanism, we have crystallized a fragment of RECQ1 encompassing D2–Zn–WH domains harbouring mutations on the β-hairpin. From our crystal structure data and interface analysis, we have demonstrated that an α-helix located in zinc-binding domain potentially interacts with residues of WH domain, which plays a significant role in strand annealing activity. We have shown that deletion of the α-helix or mutation of specific residues on it restores strand annealing activity of annealing deficient constructs of RECQ1. Our results also demonstrate that mutations on the α-helix induce conformational changes and affects DNA stimulated ATP hydrolysis and unwinding activity of RECQ1. Our study, for the first time, provides insight into the conformational requirements of the WH domain for efficient strand annealing by human RECQ1.     

Breast Cancer Stem Cell Therapeutics,Multiple Strategies Versus Using Engineered Mesenchymal Stem Cells With Notch Inhibitory Properties: Possibilities and Perspectives

Relapse cases of cancers are more vigorous and difficult to control due to the preponderance of cancer stem cells (CSCs). Such CSCs that had been otherwise dormant during the first incidence of cancer gradually appear as radiochemoresistant cancer cells. Hence, cancer therapeutics aimed at CSCs would be an effective strategy for mitigating the cancers during relapse. Alternatively, CSC therapy can also be proposed as an adjuvant therapy, along‐with the conventional therapies. As regenerative stem cells (RSCs) are known for their trophic effects, anti‐tumorogenicity, and better migration toward an injury site, this review aims to address the use of adult stem cells such as dental pulp derived; cord blood derived pure populations of regenerative stem cells for targeting CSCs. Indeed, pro‐tumorogenicity of RSCs is of concern and hence has also been dealt with in relation to breast CSC therapeutics. Furthermore, as notch signaling pathways are upregulated in breast cancers, and anti‐notch antibody based and sh‐RNA based therapies are already in the market, this review focuses the possibilities of engineering RSCs to express notch inhibitory proteins for breast CSC therapeutics. Also, we have drawn a comparison among various possibilities of breast CSC therapeutics, about, notch1 inhibition. J. Cell. Biochem. 119: 141–149, 2018. © 2017 Wiley Periodicals, Inc.     

Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet‐like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five‐step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti‐hyperglycaemic hormone‐Glp1 (glucagon‐like peptide 1) analogue Liraglutide with prolonged t_½ and Exendin 4. The differentiated islet‐like 3D clusters expressed bonafide mature and functional β‐cell markers‐PDX1 (pancreatic and duodenal homoeobox‐1), C‐peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C‐peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non‐obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long‐term in vivo functionally viable islets from hESC.     

In vitro studies on erythrosine-based photodynamic therapy of malignant and pre-malignant oral epithelial cells

Photodynamic Therapy (PDT) involves the administration of a tumor localizing photosensitizing agent, which upon activation with light of an appropriate wavelength leads to the destruction of the tumor cells. The aim of the present study was to determine the efficacy of erythrosine as a photosensitizer for the PDT of oral malignancies. The drug uptake kinetics of erythrosine in malignant (H357) and pre-malignant (DOK) oral epithelial cells and their susceptibility to erythrosine-based PDT was studied along with the determination of the subcellular localization of erythrosine. This was followed by initial investigations into the mechanism of cell killing induced following PDT involving both high and low concentrations of erythrosine. The results showed that at 37 °C the uptake of erythrosine by both DOK and H357 cells increased in an erythrosine dose dependent manner. However, the percentage of cell killing observed following PDT differed between the 2 cell lines; a maximum of ~80% of DOK cell killing was achieved as compared to ~60% killing for H357 cells. Both the DOK and H357 cell types exhibited predominantly mitochondrial accumulation of erythrosine, but the mitochondrial trans-membrane potential (ΔΨ(m)) studies showed that the H357 cells were far more resistant to the changes in ΔΨ(m) when compared to the DOK cells and this might be a factor in the apparent relative resistance of the H357 cells to PDT. Finally, cell death morphology and caspase activity analysis studies demonstrated the occurrence of extensive necrosis with high dose PDT in DOK cells, whereas apoptosis was observed at lower doses of PDT for both cell lines. For H357 cells, high dose PDT produced both apoptotic as well as necrotic responses. This is the first instance of erythrosine-based PDT's usage for cancer cell killing.     

Genome Sequencing and Phylogenetic Analysis of 39 Human Parainfluenza Virus Type 1 Strains Isolated from 1997–2010

Thirty-nine human parainfluenza type 1 (HPIV-1) genomes were sequenced from samples collected in Milwaukee, Wisconsin from 1997–2010. Following sequencing, phylogenetic analyses of these sequences plus any publicly available HPIV-1 sequences (from GenBank) were performed. Phylogenetic analysis of the whole genomes, as well as individual genes, revealed that the current HPIV-1 viruses group into three different clades. Previous evolutionary studies of HPIV-1 in Milwaukee revealed that there were two genotypes of HPIV-1 co-circulating in 1991 (previously described as HPIV-1 genotypes C and D). The current study reveals that there are still two different HPIV-1 viruses co-circulating in Milwaukee; however, both groups of HPIV-1 viruses are derived from genotype C indicating that genotype D may no longer be in circulation in Milwaukee. Analyses of genetic diversity indicate that while most of the genome is under purifying selection some regions of the genome are more tolerant of mutation. In the 40 HPIV-1 genomes sequenced in this study, the nucleotide sequence of the L gene is the most conserved while the sequence of the P gene is the most variable. Over the entire protein coding region of the genome, 81 variable amino acid residues were observed and as with nucleotide diversity, the P protein seemed to be the most tolerant of mutation (and contains the greatest proportion of non-synonymous to synonymous substitutions) while the M protein appears to be the least tolerant of amino acid substitution.     

7-Phenyl-pyrido[2,3-d]pyrimidine-2,4-diamines: Novel and highly selective protein tyrosine phosphatase 1B inhibitors

High throughput screening of the Roche compound collection led to the identification of diaminopyrroloquinazoline series as a novel class of PTP1B inhibitors. Structural modification of diaminopyrroloquinazoline series resulted in pyrido[2,3-d]pyrimidine-2,4-diamine series which was further optimized to give compounds 5 and 24 as potent, selective (except T-cell phosphatase) PTP1B inhibitors with good mouse PK properties.     

Eco-physiological Responses of Artificial Night Light Pollution in Plants

Russian Journal of Plant Physiology - Early in the 20th century, disparate human developmental processes culminate excess artificial light during night time and distort the phenological,...