Amphibians serve as reliable indicators of ecosystem health and are the most threatened group of vertebrates. Studies on their spatial distribution pattern and threats are crucial to formulate conservation strategies. Gongga Mountains, with a peak at 7509 m a.s.l. and running latitudinally, are in the center of the Hengduan Mountains Range and at the eastern steep edge of the Qinghai–Xizang Plateau, providing heterogeneous habitats and varied niches for amphibians. In this study, we combined 83 days of field work with information from 3894 museum specimens that were collected over the past 80 years, and identified twenty amphibian species belonging to seven families and twelve genera by morphology. Of these species, seven were listed in the threatened categories of the Red List of China’s Biodiversity and thirteen were endemic to China. Ten species were found on the plateau side (western slope) and eleven species were found on the other side close to the Sichuan Basin (eastern slope). Only one species was found on both sides, indicating different community structures horizontally. The species richness was unimodal vertically and peaking at mid elevation on both sides, with the maximum number (ten vs. nine) of species occurring at 3300–3700 vs. 1700–1900 m a.s.l. and in different types of vegetation. The elevation span and body length of species distributed on both slopes did not show significant differences. These findings help to understand the horizontal and vertical distribution pattern of amphibian diversity, laying a foundation for future biogeographical and conservation research in this area.

Size evolution among gigantic genomes involves gain and loss of many gigabases of transposable elements (TEs), sequences that parasitize host genomes. Animals suppress TEs using piRNA and KRAB-ZFP pathways. TEs and hosts coevolve in an arms race, where suppression strength reflects TE fitness costs. In enormous genomes, additional TE costs become miniscule. How, then, do TEs and host suppression invoke further addition of massive DNA amounts? We analyzed TE proliferation histories, deletion rates, and community diversities in six salamander genomes (21.3 - 49.9 Gb), alongside gonadal expression of TEs and suppression pathways. TE activity is higher in testes than ovaries, attributable to lower KRAB-ZFP suppression. Unexpectedly, genome size/expansion is uncorrelated with TE deletion rate, proliferation history, expression, and host suppression. Also, TE community diversity increases with genome size, contrasting theoretical predictions. TE/host antagonism in gigantic genomes likely produces stochastic TE accumulation, determined by noisy intermolecular interactions in huge genomes/cells. ### Competing Interest Statement The authors have declared no competing interest.

Transposable elements (TEs) and the silencing machinery of their hosts are engaged in a germline arms-race dynamic that shapes TE accumulation and, therefore, genome size. In animal species with extremely large genomes (>10 Gb), TE accumulation has been pushed to the extreme, prompting the question of whether TE silencing also deviates from typical conditions. To address this question, we characterize TE silencing via two pathways—the piRNA pathway and KRAB-ZFP transcriptional repression—in the male and female gonads of Ranodon sibiricus, a salamander species with a ∼21 Gb genome. We quantify 1) genomic TE diversity, 2) TE expression, and 3) small RNA expression and find a significant relationship between the expression of piRNAs and TEs they target for silencing in both ovaries and testes. We also quantified TE silencing pathway gene expression in R. sibiricus and 14 other vertebrates with genome sizes ranging from 1 to 130 Gb and find no association between pathway expression and genome size. Taken together, our results reveal that the gigantic R. sibiricus genome includes at least 19 putatively active TE superfamilies, all of which are targeted by the piRNA pathway in proportion to their expression levels, suggesting comprehensive piRNA-mediated silencing. Testes have higher TE expression than ovaries, suggesting that they may contribute more to the species’ high genomic TE load. We posit that apparently conflicting interpretations of TE silencing and genomic gigantism in the literature, as well as the absence of a correlation between TE silencing pathway gene expression and genome size, can be reconciled by considering whether the TE community or the host is currently “on the attack” in the arms race dynamic.

为了探究水域三氯生(TCS)污染对峨眉林蛙Rana omeimontis蝌蚪生长发育的影响,本研究以峨眉林蛙蝌蚪(G22期)为试验对象,进行了96 h急性毒性实验和21 d慢性毒性实验.急性毒性实验中每隔24 h记录蝌蚪的死亡数;慢性毒性实验分别于饲养0d、7d、14d、21d测定蝌蚪的体质量、全长、头体长、尾长、吻长和眼间距.结果表明:96 h-LC50为266μg·L?1、96 h-SC为89μg·L?1,可知TCS对峨眉林蛙蝌蚪为高毒化合物,强烈影响其生长发育.慢性毒性实验暴露21 d,30μg·L?1、60μg·L?1、90μg·L?1、120μg·L?1浓度的TCS极显著抑制蝌蚪的体质量增长,60μg·L?1、90μg·L?1、120μg·L?1浓度的TCS极显著抑制蝌蚪的吻长增长.建议减少TCS污水排放,控制水体中TCS浓度.

Chimeric antigen receptor (CAR) T cell therapy has been successful in treating hematological malignancy, but solid tumors remain refractory. Here, we demonstrated that knocking out transcription factor IKZF3 in HER2specific CAR T cells targeting breast cancer cells did not affect CAR expression or CAR T cell differentiation, but markedly enhanced killing of the cancer cells in vitro and in a xenograft model, which was associated with increased T cell activation and proliferation. Furthermore, IKZF3 KO had similar effects on the CD133-specific CAR T cells targeting glioblastoma cells. AlphaLISA and RNA-seq analyses indicate that IKZF3 KO increased the expression of genes involved in cytokine signaling, chemotaxis and cytotoxicity. Our results suggest a general strategy for enhancing CAR T efficacy on solid tumors.

Chimeric antigen receptor (CAR) T-cell therapy is mostly limited to hematological malignancies and has a poor effect on solid tumors. CAR T cells as a kind of immune cell may be affected by some immunomodulatory drugs such as pomalidomide, so the use of pomalidomide may improve the effect of CART cells on solid tumors. In this study, CD133-or HER2-specific CART cells were chosen to investigate whether pomalidomide can regulate the function of CAR T cells in vitro. We found that pomalidomide can significantly enhance the ability of CD133-CAR T cells and HER2-CAR T cells to kill tumor cells and increase the cytokine secretion of CD133-CAR T cells and HER2-CAR T cells. Also, pomalidomide was shown to induce down-regulation of protein levels of IL-2 transcriptional repressors Aiolos and Ikaros in CART cells. This study suggests that the combination of pomalidomide and CAR T cells may be a new strategy for the treatment of solid tumors.

Using T-cell chimeric antigen receptors (CAR-T) to activate and redirect T cells to tumors expressing the cognate antigen represents a powerful approach in cancer therapy. However, normal tissues with low expression of tumor-associated antigens (TAAs) can be mistargeted, resulting in severe side effects. An approach using a collection of T cells expressing a diverse, 106-member combinatorial cellular library of CARs, in which members can be specifically enriched based on avidity for cell membrane antigens, is reported. Using CD38 as the target antigen, an efficient and effective selection of CARs specifically recognizing CD38+ tumor cells is demonstrated. These selected CAR-T's produce cytokines known to be associated with T cell activation in a CD38 expression-dependent manner. This avidity-based selection endows the engineered T cells with minimal off-tumor effects, while retaining robust antitumor efficacy both in vitro and in vivo. The described method may facilitate the application of CAR-T therapy to TAAs previously considered undruggable.

Chimeric antigen receptor (CAR) T cells are powerful in eradicating hematological malignancies, but their efficacy is limited in treating solid tumors. One of the barriers is the immunosuppressive response induced by immunomodulatory signaling pathways. Pharmacological targeting of these immunosuppressive pathways may be a simple way to improve the efficacy of CAR T cells. In this study, anti-CD133 and anti-HER2 CAR T cells were generated from healthy donors, and combination therapy using CAR T cells and small molecules targeting adenosine receptors was performed in vitro and in vivo with the goal of probing for potential synergistic antitumor activities. The adenosine A2b receptor agonist, BAY 60-6583, was found to significantly increase cytokine secretion of CD133-or HER2-specific CAR T cells when co-cultured with the respective target tumor cells. The in vitro cytotoxicity and proliferation of CAR T cells were also enhanced when supplied with BAY 60-6583. Furthermore, the combination with this small molecule facilitated the anti-HER2 CAR T cell-mediated elimination of tumor cells in a xenograft mouse model. However, the enhanced antitumor activities could not be suppressed by knockout of the adenosine A2b receptor in CAR T cells. Furthermore, mass spectrometry and computational methods were used to predict several potential alternative targets. Four potential targets (pyruvate kinase M (PKM), Talin-1, Plastin-2, and lamina-associated polypeptide 2) were captured by a photo-affinity probe, of which PKM and Talin-1 were predicted to interact with BAY 60-6583. Overall, our data suggest that BAY 60-6583 upregulates T cell functions through a mechanism independent of the adenosine A2b receptor.

Chimeric antigen receptor (CAR) T cell therapy still faces the challenge of immunosuppression when treating solid tumors. TGF-β is one of the critical factors in the tumor microenvironment to help tumors escape surveillance by the immune system. Here we tried using the combination of a small molecule inhibitor of TGF-β receptor I, Galunisertib, and CAR T cells to explore whether Galunisertib could enhance CAR T cell function against solid tumor cells. In vitro experiments showed Galunisertib could significantly enhance the specific cytotoxicity of both CD133- and HER2-specific CAR T cells. However, Galunisertib had no direct killing effect on target cells. Galunisertib significantly increased the cytokine secretion of CAR T cells and T cells that do not express CAR (Nontransfected T cells). Galunisertib did not affect the proliferation of T cells, the antigen expression on target cells and CD69 on CAR T cells. We found that TGF-β was secreted by T cells themselves upon activation, and Galunisertib could reduce TGF-β signaling in CAR T cells. Our findings can provide the basis for further preclinical and clinical studies of the combination of Galunisertib and CAR T cells in the treatment of solid tumors.

Chimeric antigen receptor (CAR) T-cell immunotherapy still faces many challenges in the treatment of solid tumors, one of which is T-cell dysfunction or exhaustion. Immunomodulator lenalidomide may improve CAR T-cell function. In this study, the effects of lenalidomide on CAR T-cell functions (cytotoxicity, cytokine secretion, and cell proliferation) were investigated. Two different CAR T cells (CD133-specific CAR and HER2-specific CAR) were prepared, and the corresponding target cells including human glioma cell line U251 CD133-OE that overexpress CD133 and human breast cancer cell line MDA-MB-453 were used for functional assay. We found that lenalidomide promoted the killing of U251 CD133-OE by CD133-CAR T cells, the cytokine secretion, and the proliferation of CD133-CAR T cells. Lenalidomide also enhanced the cytotoxicity against MDA-MB-453 and the cytokine secretion of HER2-CAR T cells but did not affect their proliferation significantly. Furthermore, lenalidomide may regulate the function of CAR T cells by inducing the degradation of transcription factors Ikaros and Aiolos.

Next-generation sequencing has produced a large quantity of DNA or RNA sequences related to the processes occurring within tumors and their microenvironment in a reasonable time and cost. These data have been used to guide the identification of neoantigens and to determine their specific T-cell receptors. Furthermore, adoptive T-cell therapy targeting neoantigens is under development for cancer treatment. In this review, we first provide an overview of sequencing technologies and the updated findings concerning neoantigens related to adoptive T-cell therapy and then summarize the methods and principles underlying the development of next-generation sequencing-based neoantigen-reactive T-cell therapy for cancer.

Next-generation sequencing has produced a large quantity of DNA or RNA sequences related to the processes occurring within tumors and their microenvironment in a reasonable time and cost. These data have been used to guide the identification of neoantigens and to determine their specific T-cell receptors. Furthermore, adoptive T-cell therapy targeting neoantigens is under development for cancer treatment. In this review, we first provide an overview of sequencing technologies and the updated findings concerning neoantigens related to adoptive T-cell therapy and then summarize the methods and principles underlying the development of next-generation sequencing-based neoantigen-reactive T-cell therapy for cancer.

CRISPR/Cas9-mediated programmed cell death protein 1 (PD-1) disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in an immunosuppressive tumor microenvironment. In most of the reported cases, Cas9 was delivered into T cells by way of electroporation with RNA or protein. However, transient expression of Cas9 by transfection with a plasmid encoding its gene is apparently simpler, as it avoids the steps of in vitro transcription of DNA or protein production. This study tried nucleofection into human primary T cells of plasmids encoding both CRISPR/Cas9 for disrupting the PD-1 gene and the piggyBac transposon system for expressing CD133-specific CAR in one reaction. Based on drug selection, CD133-specific CAR T cells were obtained in which, on average, 91.5% of the PD-1 gene sites were disrupted, but almost no Cas9 gene expression was found in the final engineered CAR T cells. The PD-1-deficient CD133-specific CAR T cells showed similar levels of cytokine secretion and improved proliferation and cytotoxicity in vitro, and enhanced inhibition of tumor growth in an orthotopic mouse model of glioma, compared to conventional CD133-CAR T cells. The described method could be useful for the production of PD-1-deficient CAR T cells for cancer immunotherapy.