Pre-Evaluated Safe Human iPSC-Derived Neural Stem Cells Promote Functional Recovery after Spinal Cord Injury in Common Marmoset without Tumorigenicity

Murine and human iPSC-NS/PCs (induced pluripotent stem cell-derived neural stem/progenitor cells) promote functional recovery following transplantation into the injured spinal cord in rodents. However, for clinical applicability, it is critical to obtain proof of the concept regarding the efficacy of grafted human iPSC-NS/PCs (hiPSC-NS/PCs) for the repair of spinal cord injury (SCI) in a non-human primate model. This study used a pre-evaluated “safe” hiPSC-NS/PC clone and an adult common marmoset (Callithrix jacchus) model of contusive SCI. SCI was induced at the fifth cervical level (C5), followed by transplantation of hiPSC-NS/PCs at 9 days after injury. Behavioral analyses were performed from the time of the initial injury until 12 weeks after SCI. Grafted hiPSC-NS/PCs survived and differentiated into all three neural lineages. Furthermore, transplantation of hiPSC-NS/PCs enhanced axonal sparing/regrowth and angiogenesis, and prevented the demyelination after SCI compared with that in vehicle control animals. Notably, no tumor formation occurred for at least 12 weeks after transplantation. Quantitative RT-PCR showed that mRNA expression levels of human neurotrophic factors were significantly higher in cultured hiPSC-NS/PCs than in human dermal fibroblasts (hDFs). Finally, behavioral tests showed that hiPSC-NS/PCs promoted functional recovery after SCI in the common marmoset. Taken together, these results indicate that pre-evaluated safe hiPSC-NS/PCs are a potential source of cells for the treatment of SCI in the clinic.     

Corynebacterium ulcerans 0102 carries the gene encoding diphtheria toxin on a prophage different from the C. diphtheriae NCTC 13129 prophage

Background

Two important plant pathogenic bacteria Acidovorax oryzae and Acidovorax citrulli are closely related and often not easy to be differentiated from each other, which often resulted in a false identification between them based on traditional methods such as carbon source utilization profile, fatty acid methyl esters, and ELISA detection tests. MALDI-TOF MS and Fourier transform infrared (FTIR) spectra have recently been successfully applied in bacterial identification and classification, which provide an alternate method for differentiating the two species.

Results

Characterization and comparison of the 10 A. oryzae strains and 10 A. citrulli strains were performed based on traditional bacteriological methods, MALDI-TOF MS, and FTIR spectroscopy. Our results showed that the identity of the two closely related plant pathogenic bacteria A. oryzae and A. citrulli was able to be confirmed by both pathogenicity tests and species-specific PCR, but the two species were difficult to be differentiated based on Biolog and FAME profile as well as 16?S rRNA sequence analysis. However, there were significant differences in MALDI-TOF MS and FTIR spectra between the two species of Acidovorax. MALDI-TOF MS revealed that 22 and 18 peaks were specific to A. oryzae and A. citrulli, respectively, while FTIR spectra of the two species of Acidovorax have the specific peaks at 1738, 1311, 1128, 1078, 989?cm^-1 and at 1337, 968, 933, 916, 786?cm^-1, respectively.

Conclusions

This study indicated that MALDI-TOF MS and FTIR spectra may give a new strategy for rapid bacterial identification and differentiation of the two closely related species of Acidovorax.     

Predicting subsite interactions of plasmin with substrates and inhibitors through computational docking analysis

Plasmin plays important roles in various physiological systems. The identification of inhibitors controlling its regulation represents a promising drug-discovery challenge. To develop selective inhibitors of plasmin, structural information of the binding modes is crucial. Here, a computational docking study was conducted to provide structural insight into plasmin subsite interactions with substrates/inhibitors. Predicted binding modes of two peptide-substrates (D/L-Ile-Phe-Lys), and potent and weak inhibitors (YO-2 and PKSI-527) suggested non-prime and prime subsite interactions relevant to recognition by plasmin. Predicted binding modes also correlated well with the experimental structure-activity relationships for plasmin substrates/inhibitors, namely the differences of K(M) values between the D- and L-peptide-substrates and inhibitory potencies of YO-2 and PKSI-527. In particular, interaction observed at a hydrophobic pocket near S2 and at a tunnel-shaped hydrophobic S1' was strongly suggested to be significantly involved in tight binding of inhibitors to plasmin. Our present findings may aid in the design of potent and selective plasmin inhibitors.     

Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A

The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A.     

Species differences between human and rat in the substrate specificity of cathepsin K

Cathepsin K is known to play an important role in bone resorption, and it has the P2 specificity for proline. Rat cathepsin K has 88% identity with the human enzyme. However, it has been reported that its enzymatic activity for a Cbz-Leu-Arg-MCA substrate is lower than that of human cathepsin K, and that the rat enzyme is not well inhibited by human cathepsin K inhibitors. For this study, we prepared recombinant enzyme to investigate the substrate specificity of rat cathepsin K. Cleavage experiments using the fragment of type I collagen and peptidic libraries demonstrated that rat cathepsin K preferentially hydrolyses the substrates at the P2 Hyp position. Comparison of the S2 site between rat and human cathepsin K sequences indicated that two S2 residues at Ser134 and Val160 in rat are varied to Ala and Leu, respectively, in the human enzyme. Cleavage experiments using two single mutants, S134A and V160L, and one double mutant, S134A/V160L, of rat cathepsin K showed that all the rat mutants lost the P2 Hyp specificity. The information obtained from our comparative studies on rat and human cathepsin K should make a significant impact on developing specific inhibitors of human cathepsin K since rat is usually used as test species.     

Evolutionary conservation and changes in insect TRP channels

Background  

TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts.     

New chemotypes for cathepsin K inhibitors

Cyano pyrimidine acetylene and cyano pyrimidine t-amine, which belong to a new chemical class, were prepared and tested for inhibitory activities against cathepsin K and the highly homologous cathepsins L and S. The use of novel chemotypes in the development of cathepsin K inhibitors has been demonstrated by derivatives of compounds 1 and 8.     

Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 μM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.     

Immunohistochemical demonstration of c-fos protein in neurons of the medulla oblongata of the musk shrew (Suncus murinus) after veratrine administration.

We subcutaneously injected 0.5 mg/kg veratrine into the musk shrew (Suncus murinus), observed the presence or absence, latency, and the incidence of vomiting in each animal for 90 min, and selected animals that frequently vomited (FV group) and those that did not vomit (NV group). Subsequently, animal brains were removed, and the induction of c-fos protein (Fos) was immunohistochemically examined to evaluate neuronal activity in the medulla oblongata. The distribution of Fos-positive neurons in the medulla oblongata was similar between FV and NV groups, with numerous neurons along the entire length of the nucleus of the solitary tract and in the ventrolateral reticular formation. Both veratrine-injected groups showed higher numbers of positive neurons than the saline administered group. However, while the FV group showed a high concentration of positive neurons in the dorsal-dorsomedial reticular formation of the nucleus ambiguus in the rostral medulla, the NV group showed few positive neurons in this area. Fos activity in neurons in this area appeared to be higher in animals with a higher incidence of vomiting.     

Role of multiple efflux pumps in Escherichia coli in indole expulsion

Escherichia coli chromosome encodes several multidrug transporters. Despite their protective function against antibacterial agents, the specific physiological actions of these transporters are not fully understood. E. coli produces indole, a metabolite of tryptophan, under physiological conditions. Defined inactivation of the acrEF gene, the product of which is known as an energy-dependent multiple drug efflux pump, decreased indole excretion while reintroduction of the acrEF gene restored it. A DeltaacrEF mutant accumulated more intracellular indole than the parent. This mutant was more susceptible to the growth-inhibitory effect of indole than the parent. These results indicate that the AcrEF system plays a significant role in indole efflux.