Modelling, Cloning, and Expression of the J domain of C. elegans Rme-8 Protein

Rme-8 is a J domain containing plasma membrane protein that is required for endocytosis in various cells The J domain is a characteristic structural motif found mainly in heat shock protein 40 (Hsp 40 or DnaJ) and other proteins such as Rme-8 Within the J domain is a tripeptide, the HPD motif, that is required by the J domain protein to interact with and stimulate the ATPase activity of Hsp70 a major cellular chaperone Rme-8 protein in C elegans CeRme-8 has not been identified with a particular Hsp70 partner CeHsp70 1 is the only cytosolic Hsp70 in C elegans therefore, we hypothesize that it is the binding partner for the J domain of CeRme-8 To test this hypothesis, we first need to express and purify the J domain of CeRme-8 We report herein the successful cloning and expression of the J domain of CeRme-8 Computer modelling revealed that the amino acid sequence of the J domain of CeRme-8 folds into the canonical J domain conformation, containing the HPD tripeptide Complementary DNA of the J domain of CeRme-8 was cloned into the pGEX-Tev-KG plasmid, in frame with the gene for glutathione S transferase ( to yield a GST CeRme-8 fusion protein IPTG induced expression of the expected 37 kilodalton fusion protein was confirmed by both SDS-PAGE and western blotting using antibody against GST Work is ongoing to develop a protocol for purification of both GST tagged and untagged J domains of CeRme-8 Future work will involve testing the effect of the J domain protein on the ATPase activity of CeHsp70-

Terrace-like structure in the above-threshold ionization spectrum of an atom in an IR+XUV two-color laser field

Based on the frequency-domain theory, we investigate the above-threshold ionization (ATI) process of an atom in a two-color laser field with infrared (IR) and extreme ultraviolet (XUV) frequencies, where the photon energy of the XUV laser is close to or larger than the atomic ionization threshold. By using the channel analysis, we find that the two laser fields play different roles in an ionization process, where the XUV laser determines the ionization probability by the photon number that the atom absorbs from it, while the IR laser accelerates the ionized electron and hence widens the electron kinetic energy spectrum. As a result, the ATI spectrum presents a terrace-like structure. By using the saddle-point approximation, we obtain a classical formula which can predict the cutoff of each plateau in the terrace-like ATI spectrum. Furthermore, we find that the difference of the heights between two neighboring plateaus in the terrace-like structure of the ATI spectrum increases as the frequency of the XUV laser increases

The Effect of Safflower Yellow on Spinal Cord Ischemia Reperfusion Injury in Rabbits

Safflower yellow (SY) is the safflower extract and is the one of traditional Chinese medicine. The aim of the present work was to investigate the effect of SY on spinal cord ischemia reperfusion injury (SCIRI) in rabbits. The models of spinal cord ischemia reperfusion (SI/R) were constructed, and the degree of the post-ischemic injury was assessed by means of the neurological deficit scores and plasma levels of lipid peroxidation reactioin and neuronal morphologic changes. SCIRI remarkably affected the functional activities of the hind limbs and activated lipid peroxidation reaction. SY could attenuate apoptosis and SCIRI by enhancing Bcl-2 expression and inhibiting Bax and caspase-3 activation

A TLR4 agonist synergizes with dendritic cell-directed lentiviral vectors for inducing antigen-specific immune responses

TLR4 agonists can be used as adjuvants to trigger innate immune responses of antigen-presenting cells (APCs) such as dendritic cells (DCs) to enhance vaccine-specific immunity. Adjuvant effects of TLR4 agonists are mediated by downstream signaling controlled by both MyD88 and TRIF adapter proteins. In this study, we investigated the adjuvanting capacity of glucopyranosyl lipid A (GLA), a chemically synthesized TLR4 agonist, to boost antigen-specific immunity elicited by DC-directed lentiviral vectors (DC-LV). We found that stimulation by this agonist in vitro can activate DCs in a TLR4-dependent manner. The agonist can significantly boost DC-LV-induced humoral and cellular immune responses, resulting in better antitumor reactions in response to tumor challenges. We observed that the adjuvant-mediated enhancement of cytotoxic CD8+ T cell responses is CD4+ T cell-dependent and determined that in vitro the agonist stimulation involves the participation of both MyD88 and TRIF pathways to activate DCs. In vivo immunization study however revealed that adjuvant effects depend more on the MyD88 signaling as TRIF^(−/−) mice but not MyD88^(−/−) mice were able to maintain the enhanced CD8+ T cell responses upon DC-LV immunization. Thus, our study supports the use of this TLR4 agonist as a potent adjuvant candidate for boosting DC-LV immunization

Complete mitochondrial genome sequences of Physogyra lichtensteini (Milne Edwards & Haime, 1851) and Plerogyra sinuosa (Dana, 1846) (Scleractinia, Plerogyridae): characterisation and phylogenetic analysis

In this study, the whole mitochondrial genomes of Physogyra lichtensteini and Plerogyra sinuosa have been sequenced for the first time. The length of their assembled mitogenome sequences were 17,286 bp and 17,586 bp, respectively, both including 13 protein-coding genes, two tRNAs, and two rRNAs. Their mitogenomes offered no distinct structure and their gene order were the same as other typical scleractinians. Based on 13 protein-coding genes, a maximum likelihood phylogenetic analysis showed that Physogyra lichtensteini and Plerogyra sinuosa are clustered in the family Plerogyridae, which belongs to the “Robust” clade. The 13 tandem mitogenome PCG sequences used in this research can provide important molecular information to clarify the evolutionary relationships amongst stony corals, especially at the family level. On the other hand, more advanced markers and more species need to be used in the future to confirm the evolutionary relationships of all the scleractinians

Long-Range Ordered Carbon Clusters: A Crystalline Material with Amorphous Building Blocks

Solid-state materials can be categorized by their structures into crystalline (having periodic translation symmetry), amorphous (no periodic and orientational symmetry), and quasi-crystalline (having orientational but not periodic translation symmetry) phases. Hybridization of crystalline and amorphous structures at the atomic level has not been experimentally observed. We report the discovery of a long-range ordered material constructed from units of amorphous carbon clusters that was synthesized by compressing solvated fullerenes. Using x-ray diffraction, Raman spectroscopy, and quantum molecular dynamics simulation, we observed that, although carbon-60 cages were crushed and became amorphous, the solvent molecules remained intact, playing a crucial role in maintaining the long-range periodicity. Once formed, the high-pressure phase is quenchable back to ambient conditions and is ultra-incompressible, with the ability to indent diamond

The Murine Reg3a Stimulated by Lactobacillus casei Promotes Intestinal Cell Proliferation and Inhibits the Multiplication of Porcine Diarrhea Causative Agent in vitro

Lactobacillus casei (L. casei), a normal resident of the gastrointestinal tract of mammals, has been extensively studied over the past few decades for its probiotic properties in clinical and animal models. Some studies have shown that some bacterium of Lactobacillus stimulate the production of antimicrobial peptides in intestinal cells to clear enteric pathogens, however, which antimicrobial peptides are produced by L. casei stimulation and its function are still not completely understood. In this study, we investigated the changes of antimicrobial peptides’ expression after intragastric administration of L. casei to mice. The bioinformatics analysis revealed there were nine genes strongly associated with up-regulated DEGs. But, of these, only the antimicrobial peptide mReg3a gene was continuously up-regulated, which was also confirmed by qRT-PCR. We found out the mReg3a expressed in engineering E.coli promoted cell proliferation and wound healing proved by CCK-8 assay and wound healing assay. Moreover, the tight junction proteins ZO-1 and E-cadherin in mReg3a treatment group were significantly higher than that in the control group under the final concentration of 0.2 mg/ml both in Porcine intestinal epithelial cells (IPEC-J2) and Mouse intestinal epithelial cells (IEC-6) (p < 0.05). Surprisingly, the recombinant mReg3a not only inhibited Enterotoxigenic Escherichia coli (ETEC), but also reduced the copy number of the piglet diarrheal viruses, porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus (PoRV), indicating the antimicrobial peptides mReg3a may be feed additives to resist the potential of the intestinal bacterial and viral diarrhea disease

Exosomes from embryonic mesenchymal stem cells alleviate osteoarthritis through balancing synthesis and degradation of cartilage extracellular matrix

Abstract Background Mesenchymal stem cell therapy for osteoarthritis (OA) has been widely investigated, but the mechanisms are still unclear. Exosomes that serve as carriers of genetic information have been implicated in many diseases and are known to participate in many physiological processes. Here, we investigate the therapeutic potential of exosomes from human embryonic stem cell-induced mesenchymal stem cells (ESC-MSCs) in alleviating osteoarthritis (OA). Methods Exosomes were harvested from conditioned culture media of ESC-MSCs by a sequential centrifugation process. Primary mouse chondrocytes treated with interleukin 1 beta (IL-1β) were used as an in vitro model to evaluate the effects of the conditioned medium with or without exosomes and titrated doses of isolated exosomes for 48 hours, prior to immunocytochemistry or western blot analysis. Destabilization of the medial meniscus (DMM) surgery was performed on the knee joints of C57BL/6 J mice as an OA model. This was followed by intra-articular injection of either ESC-MSCs or their exosomes. Cartilage destruction and matrix degradation were evaluated with histological staining and OARSI scores at the post-surgery 8 weeks. Results We found that intra-articular injection of ESC-MSCs alleviated cartilage destruction and matrix degradation in the DMM model. Further in vitro studies illustrated that this effect was exerted through ESC-MSC-derived exosomes. These exosomes maintained the chondrocyte phenotype by increasing collagen type II synthesis and decreasing ADAMTS5 expression in the presence of IL-1β. Immunocytochemistry revealed colocalization of the exosomes and collagen type II-positive chondrocytes. Subsequent intra-articular injection of exosomes derived from ESC-MSCs successfully impeded cartilage destruction in the DMM model. Conclusions The exosomes from ESC-MSCs exert a beneficial therapeutic effect on OA by balancing the synthesis and degradation of chondrocyte extracellular matrix (ECM), which in turn provides a new target for OA drug and drug-delivery system development