Explore the vast range of titles available.

Until recently the total synthesis of insulin, with its characteristic heterodimeric structure crosslinked by two interchain and one intrachain disulfide (SS) bridge, remained largely an unsolved challenge. By optimizing the synthesis and directed disulfide crosslinking of the two chains, and by applying biomimetic monocomponent proinsulin approaches, efficient insulin syntheses have been realized. Here we report the optimization and characterisation of an alternative strategy, oxidative native chain assembly. In this method unprotected A- and B-chains assemble oxidatively under thermodynamic control to afford bovine pancreatic insulin in 39% yield. Folding is found to proceed predominantly via structured 1SS* and 2SS* intermediates with a common interchain Cys A20 ‒Cys B19 disulfide. These results suggest that native chain assembly, long considered inefficient, may represent a reasonable strategy to access insulin variants. This is supported by the synthesis of human insulin and human type-II relaxin in yields of up to 49 and 47%, respectively, although the application to human insulin Val A16 variant is unsuccessful. The synthesis and folding pathways of insulin and related proteins are of wide interest. Here the authors characterise the major two-chain oxidative folding pathways of bovine pancreatic insulin, and develop synthetic conditions applicable to related foldable insulin variants

A water-soluble selenoxide (DHSox) having a five-membered ring structure enables rapid and selective conversion of cysteinyl SH groups in a polypeptide chain into SS bonds in a wide pH and temperature range. It was previously demonstrated that the second-order rate constants for the SS formation with DHSox would be proportional to the number of the free SH groups present in the substrate if there is no steric congestion around the SH groups. In the present study, kinetics of the SS formation with DHSox was extensively studied at pH 4–10 and 25 °C by using reduced ribonuclease A, recombinant hirudin variant (CX-397), insulin A- and B-chains, and relaxin A-chain, which have two to eight cysteine residues, as polythiol substrates. The obtained rate constants showed stochastic SS formation behaviors under most conditions. However, the rate constants for CX-397 at pH 8.0 and 10.0 were not proportional to the number of the free SH groups, suggesting that the SS intermediate ensembles possess densely packed structures under weakly basic conditions. The high two-electron redox potential of DHSox (375 mV at 25 °C) compared to l-cystine supported the high ability of DHSox for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the pKa value of the cysteinyl SH groups. The SS formation velocity was slightly decreased by addition of a denaturant due probably to the interaction between the denaturant and the peptide. The stochastic behaviors as well as the absolute values of the second-order rate constants in comparison to dithiothreitol (DTTred) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains. [Display omitted] ▸ DHSox was applied as a SS-forming reagent for five polythiol peptides and DTTred. ▸ The SS formation velocities depend on the kind of substrate and solvent conditions. ▸ The SS-formation rate constants are proportional to the number of free SH groups. ▸ Folded structures and SH pKa modify the stochastic nature and absolute values of the rate constants. ▸ DHSox is a useful probe of chemical reactivity and intermediate structures in oxidative protein folding.

Adoptive T‐cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen‐specific stem cell memory T (TSCM) cells is expected to overcome this shortcoming as TSCM cells are close to naïve T cells, but are also highly proliferative, long‐lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into TSCM‐like cells (iTSCM) by coculturing with OP9 cells expressing Notch ligand, Delta‐like 1 (OP9‐hDLL1). Here we show the methodological parameters of human CD8+ iTSCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti‐CD3/CD28 antibodies or by antigen‐presenting cells, human iTSCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by coculture with OP9‐hDLL1 cells, interleukin (IL)‐7 and IL‐15 (but not IL‐2 or IL‐21) could efficiently generate iTSCM cells. Epstein–Barr virus‐specific iTSCM cells showed much stronger antitumor potentials than conventionally activated T cells in humanized Epstein–Barr virus transformed‐tumor model mice. Thus, adoptive T‐cell therapy with iTSCM offers a promising therapeutic strategy for cancer immunotherapy. Adoptive T cell therapy is an effective strategy for cancer immunotherapy. Adoptive transfer of tumor antigen‐specific stem cell memory T (TSCM) cells is expected to overcome this shortcoming since TSCM cells are close to naïve T cells, but are also highly proliferative, long‐lived, and produce a large number of effector T cells in response to antigen stimulation. Here we show the methodological parameters of human CD8+ iTSCM cell generation and their application to adoptive cancer immunotherapy.

BackgroundAntitumor effect of chimeric antigen receptor (CAR)-T cells against solid tumors is limited due to various factors, such as low infiltration rate, poor expansion capacity, and exhaustion of T cells within the tumor. NR4A transcription factors have been shown to play important roles in T-cell exhaustion in mice. However, the precise contribution of each NR4a factor to human T-cell differentiation remains to be clarified.MethodsIn this study, we deleted NR4A family factors, NR4A1, NR4A2, and NR4A3, in human CAR-T cells recognizing human epidermal growth factor receptor type 2 (HER2) by using the CRISPR/Cas9 system. We induced T-cell exhaustion in these cells in vitro through repeated co-culturing of CAR-T cells with Her2+A549 lung adenocarcinoma cells and evaluated cell surface markers such as memory and exhaustion phenotypes, proliferative capacity, cytokine production and metabolic activity. We validated the antitumor toxicity of NR4A1/2/3 triple knockout (TKO) CAR-T cells in vivo by transferring CAR-T cells into A549 tumor-bearing immunodeficient mice.ResultsHuman NR4A-TKO CAR-T cells were resistant against exhaustion induced by repeated antigen stimulation in vitro, and maintained higher tumor-killing activity both in vitro and in vivo compared with control CAR-T cells. A comparison of the effectiveness of NR4A single, double, and TKOs demonstrated that triple KO was the most effective in avoiding exhaustion. Furthermore, a strong enhancement of antitumor effects by NR4A TKO was also observed in T cells from various donors including aged persons. Mechanistically, NR4A TKO CAR-T cells showed enhanced mitochondrial oxidative phosphorylation, therefore could persist for longer periods within the tumors.ConclusionsNR4A factors regulate CAR-T cell persistence and stemness through mitochondrial gene expression, therefore NR4A is a highly promising target for the generation of superior CAR-T cells against solid tumors.

Early detection of perinatal depression is an urgent issue. Our study aimed to examine the construct validity and factor structure of the Japanese version of the Edinburgh Postnatal Depression Scale (EPDS) from a prospective cohort study from pregnancy to postpartum. A total of 1075 women completed all items of the EPDS at four time points: early pregnancy, late pregnancy, 5 days postpartum and 1 month postpartum. The participants were randomly divided into two sample sets. The first sample set (n = 304) was used for exploratory factor analysis, and the second sample set (n = 771) was used for confirmatory factor analysis. As a result, the Cronbach’s alpha coefficients of the EPDS items were 0.762, 0.740, 0.765 and 0.772 at the four time points. From the confirmatory factor analysis of the EPDS in a sample set of Japanese women from pregnancy to postpartum, the following three factors were detected: depression (items 7, 9), anxiety (items 4, 5) and anhedonia (items 1, 2). In conclusion, the EPDS is a useful rating scale, and its factor structure is consistently stable during the whole peripartum period.

Although the association between social support and postpartum depression has been previously investigated, its causal relationship remains unclear. Therefore, we examined prospectively whether social support during pregnancy affected postpartum depression. Social support and depressive symptoms were assessed by Japanese version of Social Support Questionnaire (J-SSQ) and Edinburgh Postnatal Depression Scale (EPDS), among 877 pregnant women in early pregnancy and at one month postpartum. First, J-SSQ was standardized among peripartum women. The J-SSQ was found to have a two-factor structure, with Number and Satisfaction subscales, by exploratory and confirmatory factor analyses. Analysis of covariance was performed to examine how EPDS and J-SSQ scores during pregnancy affected the EPDS score at postpartum. Significant associations were found between postpartum EPDS score and both EPDS and total scores on the Number subscales during pregnancy (β = 0.488 and -0.054, ps < 0.001). Specifically, this negative correlation was stronger in depressive than non-depressive groups. Meanwhile, total score on Satisfaction subscales was not significantly associated with postpartum EPDS score. These results suggest that having a larger number of supportive persons during pregnancy helps protect against postpartum depression and that this effect is greater in depressive than non-depressive pregnant women. This finding is expected to be vitally important in preventive interventions.

Causal relationships between perinatal bonding failure, depression, and social support among mothers remain unclear. A total of 494 women (mean age 32.4 ± 4.5 years) completed the Mother-Infant Bonding Questionnaire (MIBQ), the Edinburgh Postnatal Depression Scale (EPDS), and the Japanese version of the Social Support Questionnaire in early pregnancy before week 25 (T1) and 1 month after delivery (T2). Our model of recursive structured equation modeling (SEM) showed acceptable fit (CMIN/ df  = 2.2, CFI = 0.97, and RMSEA = 0.05). It was revealed that: (1) a lower number of supportive persons at T1 significantly predicted both MIBQ and EPDS scores at T1 and T2; (2) at T1, poorer satisfaction with the social support received significantly predicted EPDS scores; (3) both MIBQ and EPDS scores at T1 significantly predicted their respective scores at T2. Out cohort study indicates that the number of individuals who are available to provide social support and the degree of satisfaction with the level of social support received during pregnancy have a great influence on bonding failure and depression in the postpartum period. These findings suggest that psychosocial interventions that focus on these two aspects of social support during pregnancy are effective in preventing bonding failure and depression in the postpartum period.

The Edinburgh Postnatal Depression Scale (EPDS) is a widely used screening tool for postpartum depression (PPD). Although the reliability and validity of EPDS in Japanese has been confirmed and the prevalence of PPD is found to be about the same as Western countries, the factor structure of the Japanese version of EPDS has not been elucidated yet. 690 Japanese mothers completed all items of the EPDS at 1 month postpartum. We divided them randomly into two sample sets. The first sample set (n = 345) was used for exploratory factor analysis, and the second sample set was used (n = 345) for confirmatory factor analysis. The result of exploratory factor analysis indicated a three-factor model consisting of anxiety, depression and anhedonia. The results of confirmatory factor analysis suggested that the anxiety and anhedonia factors existed for EPDS in a sample of Japanese women at 1 month postpartum. The depression factor varies by the models of acceptable fit. We examined EPDS scores. As a result, \"anxiety\" and \"anhedonia\" exist for EPDS among postpartum women in Japan as already reported in Western countries. Cross-cultural research is needed for future research.

Oceanic island ecosystems are vulnerable to the introduction of alien species, and they provide a habitat for many endangered species. Knowing the diet of an endangered animal is important for appropriate nature restoration efforts on oceanic islands because introduced species may be a major component of the diets of some endangered species. DNA barcoding techniques together with next‐generation sequencing may provide more detailed information on animal diets than other traditional methods. We performed a diet analysis using 48 fecal samples from the critically endangered red‐headed wood pigeon that is endemic to the Ogasawara Islands based on chloroplast trnL P6 loop sequences. The frequency of each detected plant taxa was compared with a microhistological analysis of the same sample set. The DNA barcoding approach detected a much larger number of plants than the microhistological analysis. Plants that were difficult to identify by microhistological analysis after being digested in the pigeon stomachs were frequently identified only by DNA barcoding. The results of the barcoding analysis indicated the frequent consumption of introduced species, in addition to several native species, by the red‐headed wood pigeon. The rapid eradication of specific introduced species may reduce the food resources available to this endangered bird; thus, balancing eradication efforts with the restoration of native food plants should be considered. Although some technical problems still exist, the trnL approach to next‐generation sequencing may contribute to a better understanding of oceanic island ecosystems and their conservation. We performed diet analysis of critically endangered red‐headed wood pigeon using next‐generation sequencer. The results indicated frequent use of introduced species, not only native species, by the pigeon.

A history of major depressive disorder before pregnancy is one risk factor for peripartum depression. Therefore, the purpose of the present study was to examine the validation and factor structure of the Japanese version of the Inventory to Diagnose Depression, Lifetime version (IDDL) for pregnant women. The study participants were 556 pregnant women. Factor analysis was performed to identify the factor structure, construct validity was examined based on the results of the factor analysis, and reliability was examined using Cronbach's [alpha] coefficient. Based on the results of the factor analysis of the IDDL, a bifactor model composed of a single general dimension along with the following five factors was extracted: (1) depression, anxiety, and irritability (items 1, 2, 8-10, and 19-21); (2) retardation, decreased concentration, indecisiveness, and insomnia (items 4, 11, 12, and 17); (3) decrease in appetite/significant weight loss (items 13 and 14); (4) increase in appetite/significant weight gain (items 15 and 16); and (5) diminished interest, pleasure, and libido (items 5-7). Cronbach's [alpha] coefficients for these five factors were as follows: 0.910, 0.815, 0.780, 0.683, and 0.803, respectively. The reliability, construct validity, and factor structure of the Japanese version of the IDDL were confirmed in pregnant women.