The tricarboxylic acid cycle is inhibited under acute stress from carbonate alkalinity in the gills of Eriocheir sinensis.

Owing to population growth and environmental pollution, freshwater aquaculture has been rapidly shrinking in recent years. Aquaculture in saline-alkaline waters is a crucial strategy to meet the increasing demand for aquatic products. The Chinese mitten crab is an important economic food in China, but the molecular mechanism by which it tolerates carbonate alkalinity (CA) in water remains unclear. Here, we found that enzyme activities of the tricarboxylic acid (TCA) cycle in the gills, such as citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase, were markedly reduced under CA stress induced by 40 mM NaHCO3. Secondly, the TCA cycle in the gills is inhibited under acute CA stress, according to proteomic and metabolomic analyses. The expressions of six enzymes, namely aconitate hydratase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, dihydrolipoyl dehydrogenase, succinate-CoA ligase, and malate dehydrogenase, were downregulated, resulting in the accumulation of phosphoenolpyruvic acid, citric acid, cis-aconitate, and α-ketoglutaric acid. Finally, we testified that if the TCA cycle is disturbed by malonate, the survival rate increases in CA water. To our knowledge, this is the first study to show that the TCA cycle in the gills is inhibited under CA stress. Overall, the results provide new insights into the molecular mechanism of tolerance to saline-alkaline water in crabs, which helped us expand the area for freshwater aquaculture and comprehensively understand the physiological characteristics of crab migration.

Effects of Thermal Exposure on the Microstructure and Mechanical Properties of a Ti-48Al-3Nb-1.5Ta Alloy via Powder Hot Isostatic Pressing.

Research on how thermal exposure affects the microstructure and mechanical properties of the Ti-48Al-3Nb-1.5Ta (at. %) alloy, which is prepared via powder hot isostatic pressing (P-HIP), is essential since this low-density alloy shows promise for use in high-temperature applications, particularly for aero-engines, which require long-term stable service. In this study, a P-HIP Ti-48Al-3Nb-1.5Ta (at. %) alloy was exposed to high temperatures for long durations. The phase, microstructure and mechanical properties of the P-HIP Ti-48Al-3Nb-1.5Ta alloy after thermal exposure under different conditions were analyzed using XRD, SEM, EBSD, EPMA, TEM, nanomechanical testing and tensile testing. The surface scale is composed of oxides and nitrides, primarily Al2O3, TiO2, and TiN, among which Al2O3 is preferentially generated and then covered by rapidly growing TiO2 as the thermal exposure duration increases. The nitrides appear later than the oxides and exist between the oxides and the substrate. With increasing exposure temperature and duration, the surface scale becomes more continuous, TiO2 particles grow larger, and the oxide layer thickens or even falls off. The addition of Ta and Nb can improve the oxidation resistance because Ta5+ and Nb5+ replace Ti4+ in the rutile lattice and weaken O diffusion. Compared with the P-HIP Ti-48Al-3Nb-1.5Ta alloy, after thermal exposure, the grain size does not increase significantly, and the γ phase increases slightly (by less than 3%) with the decomposition of the α2 phase. With increasing thermal exposure duration, the γ phase exhibits discontinuous coarsening (DC). Compared with the P-HIP Ti-48Al-3Nb-1.5Ta alloy, the hardness increases by about 2 GPa, the tensile strength increases by more than 50 MPa, and the fracture strain decreases by about 0.1% after thermal exposure. When the depth extends from the edge of the thermally exposed specimens, the hardness decreases overall.

Glutamate-pantothenate pathway promotes antibiotic resistance of Edwardsiella tarda.

Although cellular metabolic states have been shown to modulate bacterial susceptibility to antibiotics, the interaction between glutamate (Glu) and chloramphenicol (CAP) resistance remains unclear because of the specificity of antibiotics and bacteria. We found that the level of Glu was upregulated in the CAP-resistant strain of Edwardsiella tarda according to a comparative metabolomics approach based on LC-MS/MS. Furthermore, we verified that exogenous metabolites related to Glu, the tricarboxylic acid (TCA) cycle, and glutathione (GSH) metabolism could promote CAP resistance in survival assays. If GSH metabolism or the TCA cycle is inhibited by L-buthionine sulfoximine or propanedioic acid, the promotion of CAP resistance by Glu in the corresponding pathway disappears. According to metabolomic analysis, exogenous Glu could change pantothenate metabolism, affecting GSH biosynthesis and the TCA cycle. These results showed that the glutamate-pantothenate pathway could promote CAP resistance by being involved in the synthesis of GSH, entering the TCA cycle by direct deamination, or indirectly affecting the metabolism of the two pathways by pantothenate. These results extend our knowledge of the effect of Glu on antibiotic resistance and suggest that the potential effect, which may aggravate antibiotic resistance, should be considered before Glu and GSH administration in the clinic.

Metabolites in the TCA Cycle Promote Resistance to Chloramphenicol of Edwardsiella tarda.

Antibiotic-resistant bacteria are a serious threat to human and animal health. Metabolite-enabled eradication of drug-resistant pathogens is an attractive strategy, and metabolite adjuvants, such as fumarate, are used for restoring the bactericidal ability of antibiotics. However, we show that metabolites in the TCA cycle increase the viability of Edwardsiella tarda against chloramphenicol (CAP), based on the survival assay of differential metabolites identified by LC-MS/MS. Furthermore, NADPH promotes CAP resistance in the CAP-resistant strain, while oxidants restore the bactericidal ability. Finally, we show that the intracellular redox state determines the sensitivity to CAP, and the total antioxidative capacity is decreased significantly in the antibiotic-resistant strain. Considering that the metabolites promote CAP resistance, metabolite adjuvants should be applied very cautiously. Overall, our research expands on the knowledge that the redox state is related to the bactericidal ability of CAP.

Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

Retroviral transfer of a dominant TCR prevents surface expression of a large proportion of the endogenous TCR repertoire in human T cells.

The latent membrane protein-2 (LMP2) of Epstein-Barr virus is a potential target for T-cell receptor (TCR) gene therapy of Hodgkin lymphoma and nasopharyngeal carcinoma. Here, we modified a human leukocyte antigen-A2-restricted, LMP2-specific TCR to achieve efficient expression following retroviral TCR gene transfer. The unmodified TCR was poorly expressed in primary human T cells, suggesting that it competed inefficiently with endogenous TCR chains for cell surface expression. In order to improve this TCR, we replaced the human constant region with murine sequences, linked the two TCR genes using a self-cleaving 2A sequence and finally, codon optimized the TCR-alpha-2A-beta cassette for efficient translation in human cells. Retroviral transfer of the modified TCR resulted in efficient surface expression and HLA-A2/LMP2 pentamer binding. The transduced cells showed peptide-specific interferon-gamma and interleukin-2 production and killed target cells displaying the LMP2 peptide. Importantly, the introduced LMP2-TCR suppressed the cell surface expression of a large proportion of endogenous TCR combinations present in primary human T cells. The design of dominant TCR is likely to improve TCR gene therapy by reducing the risk of potential autoreactivity of endogenous and mispaired TCR combinations.