Superoxide constricts rat pulmonary arteries via Rho-kinase-mediated Ca2+ sensitization

Reactive oxygen species play a key role in vascular disease, pulmonary hypertension, and hypoxic pulmonary vasoconstriction. We investigated contractile responses, intracellular Ca²⁺ ([Ca²⁺]_i), Rho-kinase translocation, and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light chain (MLC₂₀) in response to LY83583, a generator of superoxide anion, in small intrapulmonary arteries (IPA) of rat. LY83583 caused concentration-dependent constrictions in IPA and greatly enhanced submaximal PGF_2α-mediated preconstriction. In small femoral or mesenteric arteries of rat, LY83583 alone was without effect, but it relaxed a PGF₂α-mediated preconstriction. Constrictions in IPA were inhibited by superoxide dismutase and tempol, but not catalase, and were endothelium and guanylate cyclase independent. Constrictions were also inhibited by the Rho-kinase inhibitor Y27632 and the Src-family kinase inhibitor SU6656. LY83583 did not raise [Ca²⁺]_i, but caused a Y27632-sensitive constriction in α-toxin-permeabilized IPA. LY83583 triggered translocation of Rho-kinase from the nucleus to the cytosol in pulmonary artery smooth muscle cells and enhanced phosphorylation of MYPT-1 at Thr-855 and of MLC₂₀ at Ser-19 in IPA. This enhancement was inhibited by superoxide dismutase and abolished by Y27632. Hydrogen peroxide did not activate Rho-kinase. We conclude that in rat small pulmonary artery, superoxide triggers Rho-kinase-mediated Ca²⁺ sensitization and vasoconstriction independent of hydrogen peroxide.     

Structural and energetic profiling of SARS-CoV-2 receptor binding domain antibody recognition and the impact of circulating variants

The SARS-CoV-2 pandemic highlights the need for a detailed molecular understanding of protective antibody responses. This is underscored by the emergence and spread of SARS-CoV-2 variants, including Alpha (B.1.1.7) and Delta (B.1.617.2), some of which appear to be less effectively targeted by current monoclonal antibodies and vaccines. Here we report a high resolution and comprehensive map of antibody recognition of the SARS-CoV-2 spike receptor binding domain (RBD), which is the target of most neutralizing antibodies, using computational structural analysis. With a dataset of nonredundant experimentally determined antibody-RBD structures, we classified antibodies by RBD residue binding determinants using unsupervised clustering. We also identified the energetic and conservation features of epitope residues and assessed the capacity of viral variant mutations to disrupt antibody recognition, revealing sets of antibodies predicted to effectively target recently described viral variants. This detailed structure-based reference of antibody RBD recognition signatures can inform therapeutic and vaccine design strategies.     

The CNS in inbred transgenic models of 4-repeat Tauopathy develops consistent tau seeding capacity yet focal and diverse patterns of protein deposition

Background

MAPT mutations cause neurodegenerative diseases such as frontotemporal dementia but, strikingly, patients with the same mutation may have different clinical phenotypes.

Methods

Given heterogeneities observed in a transgenic (Tg) mouse line expressing low levels of human (2 N, 4R) P301L Tau, we backcrossed founder stocks of mice to C57BL/6Tac, 129/SvEvTac and FVB/NJ inbred backgrounds to discern the role of genetic versus environmental effects on disease-related phenotypes.

Results

Three inbred derivatives of a TgTau^P301L founder line had similar quality and steady-state quantity of Tau production, accumulation of abnormally phosphorylated 64–68 kDa Tau species from 90 days of age onwards and neuronal loss in aged Tg mice. Variegation was not seen in the pattern of transgene expression and seeding properties in a fluorescence-based cellular assay indicated a single “strain” of misfolded Tau. However, in other regards, the aged Tg mice were heterogeneous; there was incomplete penetrance for Tau deposition despite maintained transgene expression in aged animals and, for animals with Tau deposits, distinctions were noted even within each subline. Three classes of rostral deposition in the cortex, hippocampus and striatum accounted for 75% of pathology-positive mice yet the mean ages of mice scored as class I, II or III were not significantly different and, hence, did not fit with a predictable progression from one class to another defined by chronological age. Two other patterns of Tau deposition designated as classes IV and V, occurred in caudal structures. Other pathology-positive Tg mice of similar age not falling within classes I-V presented with focal accumulations in additional caudal neuroanatomical areas including the locus coeruleus. Electron microscopy revealed that brains of Classes I, II and IV animals all exhibit straight filaments, but with coiled filaments and occasional twisted filaments apparent in Class I. Most strikingly, Class I, II and IV animals presented with distinct western blot signatures after trypsin digestion of sarkosyl-insoluble Tau.

Conclusions

Qualitative variations in the neuroanatomy of Tau deposition in genetically constrained slow models of primary Tauopathy establish that non-synchronous, focal events contribute to the pathogenic process. Phenotypic diversity in these models suggests a potential parallel to the phenotypic variation seen in P301L patients.

     

Low frequency mechanical stimulation inhibits adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

Oscillatory mechanical stimulation at relatively high frequencies (0.1 Hz) has been shown to inhibit adipogenic and promote osteogenic differentiation of mesenchymal stem cells. However, for physiological interpretations and ease of implementation it is of interest to know whether different rates of mechanical stimulation can produce similar results. We hypothesized that relatively low frequency mechanical stimulation (0.01 Hz) can inhibit adipogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells, even in a potent adipogenic differentiation medium. C3H10T1/2 cells were cultured in adipogenic medium under control (non-mechanically stimulated) conditions and under oscillatory surface stretch with 10% amplitude and 0.01 Hz frequency for 6h per day for up to 5 days. Cell population was assessed by counting and adipogenic differentiation was assessed by real-time quantitative PCR (qPCR) analysis of peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid binding protein 4 (FABP4) after 3 and 5 days. Involvement of the ERK signaling pathway was assessed by Western blot. Low frequency mechanical stimulation significantly decreased expression of PPARγ after 3 days and FABP4 after 3 and 5 days versus non-stimulated culture. ERK signaling was decreased in mechanically-stimulated culture, indicating a role in the inhibition of adipogenic differentiation. Application of this study: Low frequency mechanical stimulation may provide a technically simple means for control of mesenchymal stem cell differentiation in cell-based therapies, particularly for inhibition of differentiation toward undesired adipogenic lineages.     

Identification of MAMDC1 as a Candidate Susceptibility Gene for Systemic Lupus Erythematosus (SLE)

Background

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with multiple susceptibility genes. We have previously reported suggestive linkage to the chromosomal region 14q21-q23 in Finnish SLE families.

Principal Findings

Genetic fine mapping of this region in the same family material, together with a large collection of parent affected trios from UK and two independent case-control cohorts from Finland and Sweden, indicated that a novel uncharacterized gene, MAMDC1 (MAM domain containing glycosylphosphatidylinositol anchor 2, also known as MDGA2, MIM 611128), represents a putative susceptibility gene for SLE. In a combined analysis of the whole dataset, significant evidence of association was detected for the MAMDC1 intronic single nucleotide polymorphisms (SNP) rs961616 (P –value = 0.001, Odds Ratio (OR) = 1.292, 95% CI 1.103–1.513) and rs2297926 (P –value = 0.003, OR = 1.349, 95% CI 1.109–1.640). By Northern blot, real-time PCR (qRT-PCR) and immunohistochemical (IHC) analyses, we show that MAMDC1 is expressed in several tissues and cell types, and that the corresponding mRNA is up-regulated by the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) in THP-1 monocytes. Based on its homology to known proteins with similar structure, MAMDC1 appears to be a novel member of the adhesion molecules of the immunoglobulin superfamily (IgCAM), which is involved in cell adhesion, migration, and recruitment to inflammatory sites. Remarkably, some IgCAMs have been shown to interact with ITGAM, the product of another SLE susceptibility gene recently discovered in two independent genome wide association (GWA) scans.

Significance

Further studies focused on MAMDC1 and other molecules involved in these pathways might thus provide new insight into the pathogenesis of SLE.     

Analysis of Micro-RNA-144 Expression Profile in Patients with Multiple Sclerosis in Comparison with Healthy Individuals.

Background:Etiology of multiple sclerosis is non-clarified. It seems that environmental factors impact epigenetic in this disease. Micro-RNAs (MIR) as epigenetic factors are one of the most important factors in non-genetically neurodegenerative diseases. It has been found MIR-144 plays a main role in the regulation of many processes in the central nervous system. Here, we aimed to investigation of MIR-144 expression alteration in Multiple sclerosis (MS) patients.Methods:In this study 32 healthy and 32 MS patient''s blood sample were analyzed by quantitative Real-Time PCR method and obtained data analyzed by REST 2009 software.Results:Analysis of Real-Time PCR data revealed that miR-144 Increase significantly in MS patients compared to healthy controls.Conclusion:The increase of MIR-144 expression in MS patients is obvious. MIR-144 can be used as a biomarker of MS and help to early diagnosis and treatment of this disease.Key Words: MicroRNA (miRNA), MiRNA-144, Multiple Sclerosis (MS)     

Enhancing compost quality by using whey-grown biosurfactant-producing bacteria as inocula

Wild screening of bacterial strains from compost materials was performed. Several biosurfactant-producer strains were isolated and then cultured in whey as a low-cost medium for biosurfactant production. Two strains, identified as Bacillus sp. and Streptomyces sp., were the best biosurfactant producers and were selected for determination of compost quality enhancing. The effect of cell biomass, cell-free supernatant, and a consortium of these two strains on compost quality were determined and specific parameters of compost were analyzed. The results showed that using these bacteria (or supernatants) in compost processing have slight stimulatory effect on bacterial population (8.08 log₁₀ CFU/g), surface tension reduction (to 42.6 mN/m at 24 h), and heavy metal bioremediation (>50 % in most treatments), speeding up the decomposition rate of organic matter (42.3 % OM at the end of experiment), accelerating the stabilization process by reduction of NH ₄ ⁺ to NO ₃ ^? ratio (reduced from 0.2 to 0.026), decreasing the biotoxicity (tested by seed germination and root length of germinated seed), and also reduction of pathogens (reduced from 2100 to 120 MPN/g in fecal coliform).