Nanomechanics of wild-type and mutant dimers of the inner-ear tip-link protein protocadherin 15.

Mechanical force controls the opening and closing of mechanosensitive ion channels atop the hair bundles of the inner ear. The filamentous tip link connecting transduction channels to the tallest neighboring stereocilium modulates the force transmitted to the channels and thus changes their probability of opening. Each tip link comprises four molecules: a dimer of protocadherin 15 (PCDH15) and a dimer of cadherin 23, all of which are stabilized by Ca2+ binding. Using a high-speed optical trap to examine dimeric PCDH15, we find that the protein's mechanical properties are sensitive to Ca2+ and that the molecule exhibits limited unfolding at a physiological Ca2+ concentration. PCDH15 can therefore modulate its stiffness without undergoing large unfolding events under physiological conditions. The experimentally determined stiffness of PCDH15 accords with published values for the stiffness of the gating spring, the mechanical element that controls the opening of mechanotransduction channels. When PCDH15 exhibits a point mutation, V507D, associated with nonsyndromic hearing loss, unfolding events occur more frequently under tension and refolding events occur less often than for the wild-type protein. Our results suggest that the maintenance of appropriate tension in the gating spring is critical to the appropriate transmission of force to transduction channels, and hence to hearing.

Cooperative regulation of coupled oncoprotein synthesis and stability in triple-negative breast cancer by EGFR and CDK12/13.

Evidence has long suggested that epidermal growth factor receptor (EGFR) may play a prominent role in triple-negative breast cancer (TNBC) pathogenesis, but clinical trials of EGFR inhibitors have yielded disappointing results. Using a candidate drug screen, we identified that inhibition of cyclin-dependent kinases 12 and 13 (CDK12/13) dramatically sensitizes diverse models of TNBC to EGFR blockade. This combination therapy drives cell death through the 4E-BP1-dependent suppression of the translation and translation-linked turnover of driver oncoproteins, including MYC. A genome-wide CRISPR/Cas9 screen identified the CCR4-NOT complex as a major determinant of sensitivity to the combination therapy whose loss renders 4E-BP1 unresponsive to drug-induced dephosphorylation, thereby rescuing MYC translational suppression and promoting MYC stability. The central roles of CCR4-NOT and 4E-BP1 in response to the combination therapy were further underscored by the observation of CNOT1 loss and rescue of 4E-BP1 phosphorylation in TNBC cells that naturally evolved therapy resistance. Thus, pharmacological inhibition of CDK12/13 reveals a long-proposed EGFR dependence in TNBC that functions through the cooperative regulation of translation-coupled oncoprotein stability.

Genome-wide association study, population structure, and genetic diversity of the tea plant in Guizhou Plateau.

BACKGROUND: Guizhou Plateau, as one of the original centers of tea plant, has a profound multi-ethnic cultural heritage and abundant tea germplasm resources. However, the impact of indigenous community factors on the genetic diversity, population structure and geographical distribution of tea plant is still unclear. RESULTS: Using the genotyping-by-sequencing (GBS) approach, we collected 415 tea plant accessions from the study sites, estimated genetic diversity, developed a core collection, and conducted a genome-wide association study (GWAS) based on 99,363 high-quality single-nucleotide polymorphisms (SNPs). A total of 415 tea accessions were clustered into six populations (GP01, GP02, GP03, GP04, GP05 and GP06), and the results showed that GP04 and GP05 had the highest and lowest genetic diversity (Pi = 0.214 and Pi = 0.145, respectively). Moreover, 136 tea accessions (33%) were selected to construct the core set that can represent the genetic diversity of the whole collection. By analyzing seven significant SNP markers associated with the traits such as the germination period of one bud and two leaves (OTL) and the germination period of one bud and three leaves (OtL), four candidate genes possibly related to OTL and OtL were identified. CONCLUSIONS: This study revealed the impact of indigenous communities on the population structure of 415 tea accessions, indicating the importance of cultural practices for protection and utilization of tea plant genetic resources. Four potential candidate genes associated with the OTL and OtL of tea plant were also identified, which will facilitate genetic research, germplasm conservation, and breeding.

Unlocking PD-1 antibody resistance: The MUC1 DNA vaccine augments CD8+ T cell infiltration and attenuates tumour suppression.

In light of increasing resistance to PD1 antibody therapy among certain patient populations, there is a critical need for in-depth research. Our study assesses the synergistic effects of a MUC1 DNA vaccine and PD1 antibody for surmounting PD1 resistance, employing a murine CT26/MUC1 colon carcinoma model for this purpose. When given as a standalone treatment, PD1 antibodies showed no impact on tumour growth. Additionally, there was no change observed in the intra-tumoural T-cell ratios or in the functionality of T-cells. In contrast, the sole administration of a MUC1 DNA vaccine markedly boosted the cytotoxicity of CD8+ T cells by elevating IFN-γ and granzyme B production. Our compelling evidence highlights that combination therapy more effectively inhibited tumour growth and prolonged survival compared to either monotherapy, thus mitigating the limitations intrinsic to single-agent therapies. This enhanced efficacy was driven by a significant alteration in the tumour microenvironment, skewing it towards pro-immunogenic conditions. This assertion is backed by a raised CD8+/CD4+ T-cell ratio and a decrease in immunosuppressive MDSC and Treg cell populations. On the mechanistic front, the synergistic therapy amplified expression levels of CXCL13 in tumours, subsequently facilitating T-cell ingress into the tumour setting. In summary, our findings advocate for integrated therapy as a potent mechanism for surmounting PD1 antibody resistance, capitalizing on improved T-cell functionality and infiltration. This investigation affords critical perspectives on enhancing anti-tumour immunity through the application of innovative therapeutic strategies.

Screening and Constructing of Novel Angiotensin I-Converting Enzyme Inhibiting Peptides from Walnut Protein Isolate and Their Mechanisms of Action: A Merged In Silico and In Vitro Study.

Angiotensin I-converting enzyme (ACE)-inhibiting peptides were isolated from walnut protein isolate (WPI) using ultrasound-assisted extraction. This study aimed to assess the impact of ultrasonic pretreatment on the physicochemical properties of WPI. The optimal extraction conditions for WPI were determined as a 15-min ultrasonic treatment at 400 W. Subsequently, the hydrolysate exhibiting the highest in vitro ACE-inhibiting activity underwent further processing and separation steps, including ultrafiltration, ion exchange chromatography, liquid chromatography-tandem mass spectrometry, ADMET screening, and molecular docking. As a result of this comprehensive process, two previously unidentified ACE-inhibiting peptides, namely Tyr-Ile-Gln (YIQ) and Ile-Tyr-Gln (IYQ), were identified. In addition, a novel peptide, Ile-Lys-Gln (IKQ), was synthesized, demonstrating superior ACE-inhibiting activity and temperature stability. In silico analysis estimated an in vivo utilization rate of 21.7% for IKQ. These peptides were observed to inhibit ACE through an anti-competitive mechanism, with molecular docking simulations suggesting an interaction mechanism involving hydrogen bonding. Notably, both IYQ and IKQ peptides exhibited no discernible toxicity to HUVECs cells and promoted nitric oxide (NO) generation. These findings underscore the potential of ultrasonicated WPI in the separation of ACE-inhibiting peptides and their utility in the development of novel ACE inhibitors for functional food applications.

Revealing the impact of CD70 expression on the manufacture and functions of CAR-70 T-cells based on single-cell transcriptomics.

BACKGROUND: Chimeric antigen receptor-modified T cells (CAR T-cells) have shown exhilarative clinical efficacy for hematological malignancies. However, a shared antigen pool between healthy and malignant T-cells remains a concept to be technically and clinically explored for CAR T-cell therapy in T-cell cancers. No guidelines for engineering CAR T-cells targeting self-expressed antigens are currently available. METHOD: Based on anti-CD70 CAR (CAR-70) T-cells, we constructed CD70 knock-out and wild-type CAR (CAR-70KO and CAR-70WT) T-cells and evaluated their manufacturing and anti-tumor capability. Single-cell RNA sequencing and TCR sequencing were performed to further reveal the underlying differences between the two groups of CAR T-cells. RESULTS: Our data showed that the disruption of target genes in T-cells before CAR transduction advantaged the expansion and cell viability of CAR T-cells during manufacturing periods, as well as the degranulation, anti-tumor efficacy, and proliferation potency in response to tumor cells. Meanwhile, more naïve and central memory phenotype CAR+ T-cells, with higher TCR clonal diversity, remained in the final products in KO samples. Gene expression profiles revealed a higher activation and exhaustion level of CAR-70WT T-cells, while signaling transduction pathway analysis identified a higher level of the phosphorylation-related pathway in CAR-70KO T-cells. CONCLUSION: This study evidenced that CD70 stimulation during manufacturing process induced early exhaustion of CAR-70 T-cells. Knocking-out CD70 in T-cells prevented the exhaustion and led to a better-quality CAR-70 T-cell product. Our research will contribute to good engineering CAR T-cells targeting self-expressed antigens.

Genome-wide association study of leaf-related traits in tea plant in Guizhou based on genotyping-by-sequencing.

BACKGROUND: Studying the genetic characteristics of tea plant (Camellia spp.) leaf traits is essential for improving yield and quality through breeding and selection. Guizhou Plateau, an important part of the original center of tea plants, has rich genetic resources. However, few studies have explored the associations between tea plant leaf traits and single nucleotide polymorphism (SNP) markers in Guizhou. RESULTS: In this study, we used the genotyping-by-sequencing (GBS) method to identify 100,829 SNP markers from 338 accessions of tea germplasm in Guizhou Plateau, a region with rich genetic resources. We assessed population structure based on high-quality SNPs, constructed phylogenetic relationships, and performed genome-wide association studies (GWASs). Four inferred pure groups (G-I, G-II, G-III, and G-IV) and one inferred admixture group (G-V), were identified by a population structure analysis, and verified by principal component analyses and phylogenetic analyses. Through GWAS, we identified six candidate genes associated with four leaf traits, including mature leaf size, texture, color and shape. Specifically, two candidate genes, located on chromosomes 1 and 9, were significantly associated with mature leaf size, while two genes, located on chromosomes 8 and 11, were significantly associated with mature leaf texture. Additionally, two candidate genes, located on chromosomes 1 and 2 were identified as being associated with mature leaf color and mature leaf shape, respectively. We verified the expression level of two candidate genes was verified using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and designed a derived cleaved amplified polymorphism (dCAPS) marker that co-segregated with mature leaf size, which could be used for marker-assisted selection (MAS) breeding in Camellia sinensis. CONCLUSIONS: In the present study, by using GWAS approaches with the 338 tea accessions population in Guizhou, we revealed a list of SNPs markers and candidate genes that were significantly associated with four leaf traits. This work provides theoretical and practical basis for the genetic breeding of related traits in tea plant leaves.

Combination of novel oncolytic herpesvirus with paclitaxel as an efficient strategy for breast cancer therapy.

BACKGROUND: New strategies are needed to improve the treatment of patients with breast cancer (BC). Oncolytic virotherapy is a promising new tool for cancer treatment but still has a limited overall durable antitumor response. A novel replicable recombinant oncolytic herpes simplex virus type 1 called VG161 has been developed and has demonstrated antitumor effects in several cancers. Here, we explored the efficacy and the antitumor immune response of VG161 cotreatment with paclitaxel (PTX) which as a novel oncolytic viral immunotherapy for BC. METHODS: The antitumor effect of VG161 and PTX was confirmed in a BC xenograft mouse model. The immunostimulatory pathways were tested by RNA-seq and the remodeling of tumor microenvironment was detected by Flow cytometry analysis or Immunohistochemistry. Pulmonary lesions were analyzed by the EMT6-Luc BC model. RESULTS: In this report, we demonstrate that VG161 can significantly represses BC growth and elicit a robust antitumor immune response in a mouse model. The effect is amplified when combined with PTX treatment. The antitumor effect is associated with the infiltration of lymphoid cells, including CD4+ T cells, CD8+ T cells, and NK cells (expressing TNF and IFN-γ), and myeloid cells, including macrophages, myeloid-derived suppressor cells, and dendritic cell cells. Additionally, VG161 cotreatment with PTX showed a significant reduction in BC lung metastasis, which may result from the enhanced CD4+ and CD8+ T cell-mediated responses. CONCLUSIONS: The combination of PTX and VG161 is effective for repressing BC growth by inducing proinflammatory changes in the tumor microenvironment and reducing BC pulmonary metastasis. These data will provide a new strategy and valuable insight for oncolytic virus therapy applications in primary solid or metastatic BC tumors.

Synergistic Multisystem Photocatalytic Degradation of Anionic and Cationic Dyes Using Graphitic Phase Carbon Nitride.

Graphitic phase carbon nitride (g-C3N4) is a promising photocatalytic environmental material. For this study, the graphitic phase carbon nitride was prepared using a thermal polymerization method. The characteristic peaks, structures, and morphologies were determined using Fourier-transform infrared spectroscopy (FT-IR), X-ray diffractometry (XRD), and scanning electron microscopy (SEM), respectively. Under the synergetic visible light catalysis of H2O2 and Na2S2O8, the degradation effects of g-C3N4 on the anionic dye methyl orange (MO) and the cationic dye rhodamine b (Rhb) were investigated. The effects of adding different volumes of H2O2 and Na2S2O8 were likewise tested. The results showed that the above two synergistic systems increased the degradation rates of MO and Rhb by 2.5 and 3.5 times, respectively, compared with pure g-C3N4, and that the degradation rates of both MO and Rhb reached 100% within 120 min and 90 min, respectively, in accordance with the primary reaction kinetics. When H2O2 and Na2S2O8 were added dropwise at 10 mL each, the degradation rates of MO and Rhb were 82.22% and 99.81%, respectively, after 30 min of open light. The results of experiments upon both zeta potential and radical quenching showed that ·OH and ·O2- were the main active radicals for dye degradation in our synergistic system. In addition, stability tests showed that the photocatalysts in the synergistic system still had good reusability. Therefore, the use of a synergistic system can effectively reduce the photogenerated electron-hole pair complexation rate, representing a significant improvement in both photocatalytic degradation and for stability levels.

Recovery of Li and Co in Waste Lithium Cobalt Oxide-Based Battery Using H1.6Mn1.6O4.

H1.6Mn1.6O4 lithium-ion screen adsorbents were synthesized by soft chemical synthesis and solid phase calcination and then applied to the recovery of metal Li and Co from waste cathode materials of a lithium cobalt oxide-based battery. The leaching experiments of cobalt and lithium from cathode materials by a citrate hydrogen peroxide system and tartaric acid system were investigated. The experimental results showed that under the citrate hydrogen peroxide system, when the temperature was 90 °C, the rotation speed was 600 r·min-1 and the solid-liquid ratio was 10 g·1 L-1, the leaching rate of Co and Li could reach 86.21% and 96.9%, respectively. Under the tartaric acid system, the leaching rates of Co and Li were 90.34% and 92.47%, respectively, under the previous operating conditions. The adsorption results of the lithium-ion screen showed that the adsorbents were highly selective for Li+, and the maximum adsorption capacities were 38.05 mg·g-1. In the process of lithium removal, the dissolution rate of lithium was about 91%, and the results of multiple cycles showed that the stability of the adsorbent was high. The recovery results showed that the purity of LiCl, Li2CO3 and CoCl2 crystals could reach 93%, 99.59% and 87.9%, respectively. LiCoO2 was regenerated by the sol-gel method. XRD results showed that the regenerated LiCoO2 had the advantages of higher crystallinity and less impurity.