LncRNA NONRATT021972 involved the pathophysiologic processes mediated by P2X<Subscript>7</Subscript> receptors in stellate ganglia after myocardial ischemic injury

Adenosine triphosphate (ATP) acts on P2X receptors to initiate signal transmission. P2X₇ receptors play a role in the pathophysiological process of myocardial ischemic injury. Long noncoding RNAs (lncRNAs) participate in numerous biological functions independent of protein translation. LncRNAs are implicated in nervous system diseases. This study investigated the effects of NONRATT021972 small interference RNA (siRNA) on the pathophysiologic processes mediated by P2X₇ receptors in stellate ganglia (SG) after myocardial ischemic injury. Our results demonstrated that the expression of NONRATT021972 in SG was significantly higher in the myocardial ischemic (MI) group than in the control group. Treatment of MI rats with NONRATT021972 siRNA, the P2X₇ antagonist brilliant blue G (BBG), or P2X₇ siRNA improved the histology of injured ischemic cardiac tissues and decreased the elevated concentrations of serum myocardial enzymes, creatine kinase (CK), CK isoform MB (CK-MB), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) compared to the MI rats. NONRATT021972 siRNA, BBG, or P2X₇ siRNA treatment in MI rats decreased the expression levels of P2X₇ immunoreactivity, P2X₇ messenger RNA (mRNA), and P2X₇ protein, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) in the SG compared to MI rats. NONRATT021972 siRNA treatment prevented the pathophysiologic processes mediated by P2X₇ receptors in the SG after myocardial ischemic injury.     

Detection and characterization of diverse alpha- and betacoronaviruses from bats in China

Bats have been implicated as important reservoir hosts of alpha- and betacoronaviruses. In this study, diverse coronaviruses (CoVs) were detected in 50 of 951 (positive rate 5.3%) intestinal specimens of eight bat species collected in four provinces and the Tibet Autonomous Region of China by pan-coronavirus RT-PCR screening. Based on 400-nt RNA-dependent RNA polymerase (RdRP) sequence analysis, eight belonged to genus Alphacoronavirus and 42 to Betacoronavirus. Among the 50 positive specimens, thirteen gave rise to CoV full-length RdRP gene amplification for further sequence comparison, of which three divergent sequences (two from a unreported province) were subjected to full genome sequencing. Two complete genomes of betacoronaviruses (JTMC15 and JPDB144) and one nearly-complete genome of alphacoronavirus (JTAC2) were sequenced and their genomic organization predicted. The present study has identified additional numbers of genetically diverse bat-borne coronaviruses with a wide distribution in China. Two new species of bat CoV, identified through sequence comparison and phylogenetic analysis, are proposed.

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A Questionnaire Survey of the Type of Support Required by <Emphasis Type="Italic">Yogo</Emphasis> Teachers to Effectively Manage Students Suspected of Having an Eating Disorder

Background

Many studies have focused on the decreasing age of onset of eating disorders (EDs). Because school-age children with EDs are likely to suffer worse physical effects than adults, early detection and appropriate support are important. The cooperation of Yogo teachers is essential in helping these students to find appropriate care. To assist Yogo teachers, it is helpful to clarify the encounter rates (the proportion of Yogo teachers who have encountered ED students) and kinds of requested support (which Yogo teachers felt necessary to support ED students). There are no studies that have surveyed the prevalence rates of ED children by ED type as defined by the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5), nor were we able to find any quantitative study surveying the kinds of support Yogo teachers feel helpful to support ED students.

Methods

A questionnaire survey was administered to 655 Yogo teachers working at elementary/junior high/senior high/special needs schools in Chiba Prefecture. The questionnaire asked if the respondents had encountered students with each of the ED types described in DSM-5 (anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED), avoidant/restrictive food intake disorder (ARFID), and other types of EDs (Others)), and the kinds of support they felt necessary to support these students. The encounter rates and the kinds of requested were obtained and compared, taking their confidence intervals into consideration.

Results

The encounter rates for AN, BN, BED, ARFID, and Others were 48.4, 14.0, 8.4, 10.7, and 4.6 %, respectively. When classified by school type, AN, BN, BED, and ARFID had their highest encounter rates in senior high schools. Special needs schools had the highest rate for Others. The support most required for all ED types was “a list of medical/consultation institutions.”

Conclusions

Our results have clarified how to support Yogo teachers in the early detection and support of ED students. We found that the encounter rate of AN was the highest, and that it is effective to offer “a list of medical/consultation institutions” to junior and senior high schools where the encounter rates for AN are high.

     

Crystal structure of human S100A8 in complex with zinc and calcium

Background

S100 proteins are a large family of calcium binding proteins present only in vertebrates. They function intra- and extracellularly both as regulators of homeostatic processes and as potent effectors during inflammation. Among these, S100A8 and S100A9 are two major constituents of neutrophils that can assemble into homodimers, heterodimers and higher oligomeric species, including fibrillary structures found in the ageing prostate. Each of these forms assumes specific functions and their formation is dependent on divalent cations, notably calcium and zinc. In particular, zinc appears as a major regulator of S100 protein function in a disease context. Despite this central role, no structural information on how zinc bind to S100A8/S100A9 and regulates their quaternary structure is yet available.

Results

Here we report two crystallographic structures of calcium and zinc-loaded human S100A8. S100A8 binds two zinc ions per homodimer, through two symmetrical, all-His tetracoordination sites, revealing a classical His-Zn binding mode for the protein. Furthermore, the presence of a (Zn)₂-cacodylate complex in our second crystal form induces ligand swapping within the canonical His₄ zinc binding motif, thereby creating two new Zn-sites, one of which involves residues from symmetry-related molecules. Finally, we describe the calcium-induced S100A8 tetramer and reveal how zinc stabilizes this tetramer by tightening the dimer-dimer interface.

Conclusions

Our structures of Zn²⁺/Ca²⁺-bound hS100A8 demonstrate that S100A8 is a genuine His-Zn S100 protein. Furthermore, they show how zinc stabilizes S100A8 tetramerization and potentially mediates the formation of novel interdimer interactions. We propose that these zinc-mediated interactions may serve as a basis for the generation of larger oligomers in vivo.

     

Nanosuspensions Containing Oridonin/HP-β-Cyclodextrin Inclusion Complexes for Oral Bioavailability Enhancement via Improved Dissolution and Permeability

Chemotherapy via oral route of anticancer drugs offers much convenience and compliance to patients. However, oral chemotherapy has been challenged by limited absorption due to poor drug solubility and intestinal efflux. In this study, we aimed to develop a nanosuspension formulation of oridonin (Odn) using its cyclodextrin inclusion complexes to enhance oral bioavailability. Nanosuspensions containing Odn/2 hydroxypropyl-β-cyclodextrin inclusion complexes (Odn-CICs) were prepared by a solvent evaporation followed by wet media milling technique. The nanosuspensions were characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and dissolution. The resulting nanosuspensions were approximately 313.8 nm in particle size and presented a microcrystal morphology. Nanosuspensions loading Odn-CICs dramatically enhanced the dissolution of Odn. Further, the intestinal effective permeability of Odn was markedly enhanced in the presence of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and poloxamer. Bioavailability studies showed that nanosuspensions with Odn-CICs can significantly promote the oral absorption of Odn with a relative bioavailability of 213.99% (Odn suspensions as reference). Odn itself possesses a moderate permeability and marginal intestinal metabolism. Thus, the enhanced bioavailability for Odn-CIC nanosuspensions can be attributed to improved dissolution and permeability by interaction with absorptive epithelia and anti-drug efflux. Nanosuspensions prepared from inclusion complexes may be a promising approach for the oral delivery of anticancer agents.     

Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli

Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.     

Dynamic Alterations to α-Actinin Accompanying Sarcomere Disassembly and Reassembly during Cardiomyocyte Mitosis

Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk) disassembly precedes that of titin (M-line), suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble) and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1) induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.     

Rosin Surfactant QRMAE Can Be Utilized as an Amorphous Aggregate Inducer: A Case Study of Mammalian Serum Albumin

Quaternary amine of diethylaminoethyl rosin ester (QRMAE), chemically synthesized biocompatible rosin based cationic surfactant, has various biological applications including its use as a food product additive. In this study, we examined the amorphous aggregation behavior of mammalian serum albumins at pH 7.5, i.e., two units above their isoelectric points (pI ~5.5), and the roles played by positive charge and hydrophobicity of exogenously added rosin surfactant QRMAE. The study was carried out on five mammalian serum albumins, using various spectroscopic methods, dye binding assay, circular dichroism and electron microscopy. The thermodynamics of the binding of mammalian serum albumins to cationic rosin modified surfactant were established using isothermal titration calorimetry (ITC). It was observed that a suitable molar ratio of protein to QRMAE surfactant enthusiastically induces amorphous aggregate formation at a pH above two units of pI. Rosin surfactant QRMAE-albumins interactions revealed a unique interplay between the initial electrostatic and the subsequent hydrophobic interactions that play an important role towards the formation of hydrophobic interactions-driven amorphous aggregate. Amorphous aggregation of proteins is associated with varying diseases, from the formation of protein wine haze to the expansion of the eye lenses in cataract, during the expression and purification of recombinant proteins. This study can be used for the design of novel biomolecules or drugs with the ability to neutralize factor(s) responsible for the aggregate formation, in addition to various other industrial applications.