Tethering-facilitated DNA ‘opening’ and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively ‘opening’ these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a ‘kinetic gating’ mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how ‘opening’ is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed ‘open’ undamaged DNA in solution and that such ‘opening’ can largely occur without one or the other of the β-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound ‘open’ DNA adopts multiple conformations in solution notwithstanding the DNA’s original structure or the β-hairpins. Molecular dynamics simulations reveal compensatory roles of the β-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA ‘opening’ of undamaged sites and the dynamic nature of ‘open’ DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.     

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.     

Population ecological parameters and stock assessment of Russian sturgeon Acipenser gueldenstaedti Brandt & Ratzeburg, 1833 in the Southern Caspian Sea

Russian sturgeon (Acipenser gueldenstaedti Brandt & Ratzeburg, 1833) is one of the major commercial sturgeon species, but there is no adequate information and previous‐published about population dynamics and stock assessment of this species in the southern Caspian Sea. This paper examines the age structure, growth parameters, maturity, age at first capture, optimum length, natural and fishing mortality and amount of biomass in the southern Caspian Sea (Iranian waters), during a two decades time series period from 1990–1991 to 2008–2009. For a pooled data, the growth parameters were estimated as L_∞ = 214.0 cm, K = 0.054, t₀ = ?4.5 year. Size at fifty percent sexual maturity was at FL = 118 cm for females and 113 cm for males. The age at first capture (t_c) estimated to be 12.1 years. In the catch composition, bulk of the catch (75%) belonged to 13–17 years old. The instantaneous coefficient of natural mortality (M) was estimated as 0.120 year^?1 and the instantaneous coefficient of fishing mortality (F) varied during the 19‐year period between 0.130 to 0.505 year^?1. The biomass showed a descending trend from 1,941.2 mt in 1990–1991 collapsed to about 55 mt in 2004–2005, and then decreased to the lowest level and represented 18.5 mt in 2008–2009. The result revealed that, the stock of Russian sturgeon is being over‐fished. We concluded that to manage the sturgeons stocks, a coordinated regional and international effort are needed to provide immediate implementation of stock enhancement and management in the Caspian Sea.     

Applying the Taguchi Design for Optimized Formulation of Sustained Release Gliclazide Chitosan Beads: An In Vitro/In Vivo Study

Gliclazide is a second generation of hypoglycemic sulfonylurea and acts selectively on pancreatic β cell to control diabetes mellitus. The objective of this study was to produce a controlled release system of gliclazide using chitosan beads. Chitosan beads were produced by dispersion technique using tripolyphosphate (TPP) as gelating agent. The effects of process variables including chitosan molecular weight, concentration of chitosan and TPP, pH of TPP, and cross-linking time after addition of chitosan were evaluated by Taguchi design on the rate of drug release, mean release time (MRT), release efficiency (RE₈%), and particle size of the beads. The blood glucose lowering effect of the beads was studied in normal and streptozotocin-diabetic rats. The optimized formulation CL₂T₅P₂t₁₀ with about 31% drug loading, 2.4 h MRT, and 69.16% RE₈% decreased blood glucose level in normal rats for 24 h compared to pure powder of gliclazide that lasted for just 10 h.     

Hamsters Running on Time: Is the Lateral Habenula a Part of the Clock?

Previous anatomical and physiological studies have implicated the lateral habenula, and especially its medial division (LHbM), as a candidate component of the circadian timing system in rodents. We assayed lateral habenula rhythmicity in rodents using c‐FOS immunohistochemistry and found a robust rhythm in immunoreactive cell counts in the LHbM, with higher counts during the dark phase of a light‐dark (LD) cycle and during subjective night in constant darkness. We have also observed an obvious asymmetry of c‐FOS expression in the LHbM of behaviorally “split” hamsters in constant light, but only during their active phase (when they were running in wheels). Locomotor activity rhythms appear to be regulated by the suprachiasmatic nucleus (SCN) via multiple output pathways, one of which might be diffusible while the other might be neural, involving the lateral habenula.     

Mesenchymal stromal cells; a new horizon in regenerative medicine

In recent decades, mesenchymal stromal cells (MSCs) biomedical utilizing has attracted worldwide growing attention. After the first report of the human MSCs obtaining from the bone marrow (BM) tissue, these cells were isolated from wide types of the other tissues, ranging from adipose tissue to dental pulp. Their specific characteristics, comprising self-renewality, multipotency, and availability accompanied by their immunomodulatory properties and little ethical concern denote their importance in the context of regenerative medicine. Considering preclinical studies, MSCs can modify immune reactions during tissue repair and restoration, providing suitable milieu for tissue recovery; on the other hand, they can be differentiated into comprehensive types of the body cells, such as osteoblast, chondrocyte, hepatocyte, cardiomyocyte, fibroblast, and neural cells. Though a large number of studies have investigated MSCs capacities in regenerative medicine in varied animal models, the oncogenic capability of unregulated MSCs differentiation must be more assessed to enable their application in the clinic. In the current review, we provide a brief overview of MSCs sources, isolation, and expansion as well as immunomodulatory activities. More important, we try to collect and discuss recent preclinical and clinical research and evaluate current challenges in the context of the MSC-based cell therapy for regenerative medicine.