Development of Genome-Wide Intron Length Polymorphism (ILP) Markers in Tea Plant (Camellia sinensis) and Related Applications for Genetics Research.

The market value of tea is largely dependent on the tea species and cultivar. Therefore, it is important to develop efficient molecular markers covering the entire tea genome that can be used for the identification of tea varieties, marker-assisted breeding, and mapping important quantitative trait loci for beneficial traits. In this study, genome-wide molecular markers based on intron length polymorphism (ILP) were developed for tea trees. A total of 479, 1393, and 1342 tea ILP markers were identified using the PCR method in silico from the 'Shuchazao' scaffold genome, the chromosome-level genome of 'Longjing 43', and the ancient tea DASZ chromosome-level genome, respectively. A total of 230 tea ILP markers were used to amplify six tea tree species. Among these, 213 pairs of primers successfully characterize products in all six species, with 112 primer pairs exhibiting polymorphism. The polymorphism rate of primer pairs increased with the improvement in reference genome assembly quality level. The cross-species transferability analysis of 35 primer pairs of tea ILP markers showed an average amplification rate of 85.17% through 11 species in 6 families, with high transferability in Camellia reticulata and tobacco. We also used 40 pairs of tea ILP primers to evaluate the genetic diversity and population structure of C. tetracocca with 176 plants from Puan County, Guizhou Province, China. These genome-wide markers will be a valuable resource for genetic diversity analysis, marker-assisted breeding, and variety identification in tea, providing important information for the tea industry.

The association of genetic polymorphisms in protocadherin 15 with sudden sensorineural hearing loss in a Chinese population.

BACKGROUND: Sudden sensorineural hearing loss (SSNHL) is a multifactorial disease, and its etiology is still unknown. SSNHL may be caused by environmental factors and genetic changes. PCDH15 is associated with susceptibility to hearing loss. The relationship between PCDH15 and SSNHL remains unknown. METHODS: In this study, the potential association between PCDH15 polymorphism and SSNHL in Chinese population was evaluated. Two single nucleotide polymorphisms PCDH15-rs7095441 and rs11004085 in 195 SSNHL patients and 182 healthy controls were determined by TaqMan technology. RESULTS: In Chinese population, the TT genotype and T allele of rs7095441 are associated with increased susceptibility to SSNHL. The relationships between rs7095441 and the degree of hearing loss were analyzed, and TT genotype increased the risk of hearing loss. Among SSNHL patients, patients with TT genotype of rs7095441 have an increased risk of vertigo. CONCLUSION: This study found that the TT genotype of SNP rs7095441 can increase the risk of SSNHL in Chinese population.

Managing erosion and deposition to stabilize a silt-laden river.

Human regulations are involved in the hydrogeomorphic processes of silt-laden rivers with unprecedented intensity, and further, affect the structures and functions of the riverine social-ecosystem. The braided reach (BR) of the lower Yellow River is one of the world's most sediment-rich and dynamic rivers. In the recent twenty years, the Xiaolangdi Reservoir constructed upstream and the growing river training works have deeply changed the conditions of the BR, however, the behaviors of the fluvial system under multiple human influences and their mechanisms remain unexplored. Here we systematically analyze the changes in the BR in the past four decades from the view of a coupled human and natural system. We find that compared with the pre-dam period, the channel of the BR in the post-dam period is 60 % narrower and 122 % deeper. Meanwhile, the lateral erosion rate and lateral accretion rate have decreased by 164 m yr-1 and 236 m yr-1, and the flood transport capacity has increased by nearly 79 %. These changes were mainly caused by anthropic flow regime changes and boundary modifications, whose relative contributions were 71 ± 10 % and 29 ± 10 %, respectively. The interactions among channel morphology change, regional flood risk and human activities underpinned the evolution of the fluvial system by shifting the human-river relationship. Reach-scale stabilization of a silt-laden river needs the effective management of erosion and deposition processes, which calls for integrated management of soil conservation, dam regulation, and floodplain governance at a basin scale. Lessons from the lower Yellow River have important implications for other rivers faced with siltation problems, especially in the Global South.

Streamflow and sediment load changes from China's large rivers: Quantitative contributions of climate and human activity factors.

Riverine water and sediment discharge drive global material circulation and energy transfer, and they are crucial to the biogeochemical cycle. We investigated the changes in water-sediment fluxes in six major rivers from north to south in China from the mid-1950s to 2020 under the influence of climate change and human activities, and quantified the contributions of these specific influencing factors to water-sediment flux changes. Results showed that streamflow of the Songhua, Liao and Yellow rivers decreased significantly (p < 0.05). The sediment load of all rivers reduced significantly (p < 0.01) except the Songhua River. Streamflow or sediment fluxes to the oceans have increased or stabilized since around 2000, and the terrestrial sediment yielding center in China has shifted southward from the Yellow River to the Yangtze and Pearl rivers. The contribution of precipitation to the streamflow and sediment load changes decreased from north to south across the six rivers. From the mid-1950s to 2020, the underlying land surface change was the dominant contributor (>70 %) to reducing streamflow in the Songhua and Yellow rivers, while climate change (>50 %) was responsible for decreased streamflow in the Liao and Huai rivers. The sediment load reduction of the six rivers was attributed mainly to human activities. Among them, dam construction, human water consumption and catchment land surface change have reduced the total sediment load into the sea by 49 %, 25 % and 19 %, respectively. These results highlight that north-south variability in water and sediment flux are driven by both natural and anthropogenic forcing agents.

Screening, Expression, and Identification of Nanobody against SARS-CoV-2 Spike Protein.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an infectious disease that has become a serious burden on global public health. This study screened and yielded specific nanobodies (Nbs) against SARS-CoV-2 spike protein receptor binding domain (RBD), following testing its basic characteristics. A nanobody phage library was established by immunizing a camel with RBD protein. After three rounds of panning, the positive colonies were screened by enzyme-linked immunosorbent assay (ELISA). By sequencing, four different sequences of nanobody gene fragments were selected. The four nanobody fusion proteins were expressed and purified, respectively. The specificity and affinity of the four nanobodies were identified by ELISA. Our results showed that an immune phage display library against SARS-CoV-2 has been successfully constructed with a library capacity of which was 4.7 × 108 CFU. The four purified nanobodies showed specific high-affinity binding SARS-CoV-2 S-RBD. Among these, the antigen binding affinity of Nb61 was more comparable to that of commercial rabbit anti-SARS-CoV-2 S-RBD antibodies. In sum, our study has obtained four nanobody strains against SARS-CoV-2 S-RBD with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of SARS-CoV-2.

Prognostic Gene Expression Signature for Age-Related Hearing Loss.

Background: Our study aimed to determine the pathological mechanism of presbycusis at the molecular level, and determine potential biomarkers for the same. Methods: Differentially expressed genes (DEGs) for presbycusis were obtained by analyzing the microarray data sets (GSE6045 and GSE49543) downloaded from the Gene Expression Omnibus (GEO). Gene ontology (GO), Kyoto Encyclopedia of Genes and Genome (KEGG) pathway, and protein-protein interaction (PPI) network analyses, and Gene Set Enrichment Analysis (GSEA) were performed to analyze the biological functions, molecular pathways, autophagy-related molecular markers, and the immune microenvironment of the DEGs in presbycusis. Then the prognostic roles of the hub genes were analyzed and verified in vivo. Results: In the old mild hearing loss group (27.7 ± 3.4 months old), 27 down-regulated and 99 up-regulated genes were significantly differentially expressed compared with those in the young control group (3.5 ± 0.4 months old). In the old severe hearing loss group (30.6 ± 1.9 months old), 131 down-regulated and 89 up-regulated genes were significantly differentially expressed compared with those in the young control group. The results of the GO, GSEA, KEGG pathway, and immune infiltration analyses showed that the enrichment terms were mainly focused on immune response in mild presbycusis, and immune response and cell death in severe presbycusis. In the PPI network, autophagy-related genes ATG5, ATG7 showed the highest node scores in mild presbycusis; whereas MTOR, BECN1 showed the highest scores in severe presbycusis. In the GSE49543 data set, four genes (Ywhag, Mapre2, Fgf1, Acss2) were used to construct the prognostic model, and those four genes were significantly up-regulated in the rat model of presbycusis. Conclusion: Our study is the first to report the difference in autophagy factors and immune microenvironment among different degrees of hearing loss in presbycusis. Furthermore, we provide the prognostic gene expression signature for age-related hearing loss, intending to develop preventative therapies.

A new PD-1-specific nanobody enhances the antitumor activity of T-cells in synergy with dendritic cell vaccine.

Despite the many successes and opportunities presented by PD-1 blockade in cancer therapies, anti-PD-1 monoclonal antibodies still face multiple challenges. Herein we report a strategy based on a nanobody (Nb) to circumvent these obstacles. A new PD-1-blocking Nb (PD-1 Nb20) in combination with tumor-specific dendritic cell (DC)/tumor-fusion cell (FC) vaccine that aims to improve the activation, proliferation, cytokine secretion, and tumor cell cytotoxicity of CD8+ T-cells. This combination was found to effectively enhance the in vitro cytotoxicity of CD8+ T-cells to kill human non-small cell lung cancer (NSCLC) HCC827 cells, hepatocellular carcinoma (HCC) HepG2 cells, and tongue squamous cell carcinoma (TSCC) Tca8113 cells. Moreover, CD8+ T-cells pre-treated with PD-1 Nb20 and tumor-specific DC/tumor-FCs significantly suppressed the growth of NSCLC-, HCC- and TSCC-derived xenograft tumors and prolonged the survival of tumor-bearing mice, through promoting T-cell infiltration to kill tumor cells and inhibiting tumor angiogenesis. These data demonstrate that PD-1 Nb20 in synergy with DC/tumor-FC vaccine augment the broad spectrum of antitumor activity of CD8+ T-cells, providing an alternative and promising immunotherapeutic strategy for tumor patients who are T-cell-dysfunctional or not sensitive to anti-PD-1 therapy.

Nanobody: A Small Antibody with Big Implications for Tumor Therapeutic Strategy.

The development of monoclonal antibody treatments for successful tumor-targeted therapies took several decades. However, the efficacy of antibody-based therapy is still confined and desperately needs further improvement. Nanobodies are the recombinant variable domains of heavy-chain-only antibodies, with many unique properties such as small size (~15kDa), excellent solubility, superior stability, ease of manufacture, quick clearance from blood, and deep tissue penetration, which gain increasing acceptance as therapeutical tools and are considered also as building blocks for chimeric antigen receptors as well as for targeted drug delivery. Thus, one of the promising novel developments that may address the deficiency of monoclonal antibody-based therapies is the utilization of nanobodies. This article provides readers the significant factors that the structural and biochemical properties of nanobodies and the research progress on nanobodies in the fields of tumor treatment, as well as their application prospect.

Nanobody-based chimeric antigen receptor T cells designed by CRISPR/Cas9 technology for solid tumor immunotherapy.

Chimeric antigen receptor-based T-cell immunotherapy is a promising strategy for treatment of hematological malignant tumors; however, its efficacy towards solid cancer remains challenging. We therefore focused on developing nanobody-based CAR-T cells that treat the solid tumor. CD105 expression is upregulated on neoangiogenic endothelial and cancer cells. CD105 has been developed as a drug target. Here we show the generation of a CD105-specific nanobody, an anti-human CD105 CAR-T cells, by inserting the sequences for anti-CD105 nanobody-linked standard cassette genes into AAVS1 site using CRISPR/Cas9 technology. Co-culture with CD105+ target cells led to the activation of anti-CD105 CAR-T cells that displayed the typically activated cytotoxic T-cell characters, ability to proliferate, the production of pro-inflammatory cytokines, and the specific killing efficacy against CD105+ target cells in vitro. The in vivo treatment with anti-CD105 CAR-T cells significantly inhibited the growth of implanted CD105+ tumors, reduced tumor weight, and prolonged the survival time of tumor-bearing NOD/SCID mice. Nanobody-based CAR-T cells can therefore function as an antitumor agent in human tumor xenograft models. Our findings determined that the strategy of nanobody-based CAR-T cells engineered by CRISPR/Cas9 system has a certain potential to treat solid tumor through targeting CD105 antigen.

A Novel Prognostic Index Based on the Analysis of Glycolysis-Related Genes in Head and Neck Squamous Cell Carcinomas.

AIMS: The preferential dependence on glycolysis as a pathway of energy metabolism is a hallmark of cancer cells. However, the prognostic significance of glycolysis-related genes in head and neck squamous cell carcinoma (HNSCC) remains obscure. The purpose of this study was to identify glycolysis-related genes of prognostic value in HNSCC. RESULTS: Transcriptional and clinical data of 544 HNSCC samples were obtained from The Cancer Genome Atlas (TCGA) dataset. By gene set enrichment analysis (GSEA) and by employing a univariate and subsequently a stepwise multivariate Cox proportional regression model, eight glycolysis-related genes of prognostic significance in HNSCC (KIF2A, JMJD8, HMMR, STC2, HK1, EXT2, GPR8, and STC1) were identified. The patients were clustered into two groups (high and low risk) based on the expression of these genes. High-risk patients had significantly a shorter overall survival than low-risk patients. Furthermore, a new prognostic indicator based on selected glycolysis-related genes was developed by multivariate Cox analysis that proved to be a better predictor of patient outcome compared to other clinical factors. CONCLUSION: Our findings provide new insights into the role of glycolysis in HNSCC. The identified genes predict the patient prognosis and might substantially contribute to the development of individualized treatments.

Single-Domain Antibody-Based TCR-Like CAR-T: A Potential Cancer Therapy.

Retargeting the antigen-binding specificity of T cells to intracellular antigens that are degraded and presented on the tumor surface by engineering chimeric antigen receptor (CAR), also named TCR-like antibody CAR-T, remains limited. With the exception of the commercialized CD19 CAR-T for hematological malignancies and other CAR-T therapies aiming mostly at extracellular antigens achieving great success, the rareness and scarcity of TCR-like CAR-T therapies might be due to their current status and limitations. This review provides the probable optimized initiatives for improving TCR-like CAR-T reprogramming and discusses single-domain antibodies administered as an alternative to conventional scFvs and secreted by CAR-T cells, which might be of great value to the development of CAR-T immunotherapies for intracellular antigens.

Gene Expression, Network Analysis, and Drug Discovery of Neurofibromatosis Type 2-Associated Vestibular Schwannomas Based on Bioinformatics Analysis.

Neurofibromatosis Type 2- (NF2-) associated vestibular schwannomas (VSs) are histologically benign tumors. This study aimed to determine disease-related genes, pathways, and potential therapeutic drugs associated with NF2-VSs using the bioinformatics method. Microarray data of GSE108524 were downloaded from the Gene Expression Omnibus (GEO) database, and differentially expressed genes (DEGs) were screened using GEO2R. The functional enrichment and pathway enrichment of DEGs were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes Genomes (KEGG). Furthermore, the STRING and Cytoscape were used to analyze the protein-protein interaction (PPI) network of all differentially expressed genes and identify hub genes. Finally, the enriched gene sets belonging to the identified pathways were queried against the Drug-Gene Interaction database to find drug candidates for topical use in NF2-associated VSs. A total of 542 DEGs were identified, including 13 upregulated and 329 downregulated genes, which were mainly enriched in terms of focal adhesion, PI3K-Akt signaling pathway, ECM-receptor interaction, Toll-like receptor signaling pathway, Rap1 signaling pathway, and regulation of actin cytoskeleton. 28 hub genes were identified based on the subset of PPI network, and 31 drugs were selected based on the Drug-Gene Interaction database. Drug discovery using bioinformatics methods facilitates the identification of existing or potential therapeutic drugs to improve NF2 treatment.

Study on the Drug Targets and Molecular Mechanisms of Rhizoma Curcumae in the Treatment of Nasopharyngeal Carcinoma Based on Network Pharmacology.

AIM: To analyse the target of Rhizoma Curcumae in nasopharyngeal carcinoma by using network pharmacological techniques and to explore the associated molecular mechanism. METHODS: The targets of nasopharyngeal carcinoma were retrieved from the GeneCards database. At the same time, the drug therapeutic targets of Rhizoma Curcumae were obtained from the TCMSP and SymMap databases. The data were imported into the STRING database and Cytoscape 3.7.1 to construct a network of "Chinese medicine component-target-disease" interactions; then, the intersection was screened as the core Rhizoma Curcumae antinasopharyngeal cancer targets. Through GO target function and KEGG pathway enrichment analyses of the core targets, we predicted the biological processes and key signalling pathways involved in the Rhizoma Curcumae treatment of nasopharyngeal carcinoma. RESULTS: Twenty-five core targets of Rhizoma Curcumae in nasopharyngeal carcinoma were mined: TP53, BCL2 ICAM1 RXRA, TLR3 and TLR9, TNF, PTGS2, IL-6, CTSD, MMP2, MMP9, MMP14, TIMP2, ABCC1, ABCB1, ABCG2, and so on. The results of visual analysis showed that the Rhizoma Curcumae treatment of nasopharyngeal carcinoma mainly involves leukocyte adhesion to vascular endothelial cells, positive regulation of NF-κB import into the nucleus, regulation of the reactive oxygen species biosynthetic and metabolic process, regulation of the chemokine biosynthetic and metabolic process, various cancer-related signalling pathways, and a variety of cytokine signal transduction pathways, such as the NF-κB, TLR, IL-17, and TNF signalling pathways. CONCLUSION: The core targets predicted by our research can be used as molecular markers for the treatment and prediction of nasopharyngeal carcinoma. The mechanism of Rhizoma Curcumae treatment in NPC may be related to immune regulatory pathways, the inhibition of cancer cell proliferation, metastasis, and angiogenesis, as well as the regulation of tumour microenvironment. Combined with the prediction of its associated mechanism of action, the core targets can provide targeted reference value for subsequent drug development related to Curcuma.

A Nanobody Against Cytotoxic T-Lymphocyte Associated Antigen-4 Increases the Anti-Tumor Effects of Specific CD8+ T Cells.

Adoptive cell-based immunotherapy typically utilizes cytotoxic T lymphocytes (CTLs), expanding these cells ex vivo. Such expansion is traditionally accomplished through the use of autologous APCs that are capable of interactions with T cells. However, incidental inhibitory program such as CTLA-4 pathway can impair T cell proliferation. We therefore designed a nanobody which is specific for CTLA-4 (CTLA-4 Nb 16), and we then used this molecule to assess its ability to disrupt CTLA-4 signaling and thereby overcome negative costimulation of T cells. With CTLA-4 Nb16 stimulation, dendritic cell/hepatocellular carcinoma fusion cells (DC/HepG2-FCs) enhanced autologous CD8+ T cell proliferation and production of IFN-γ in vitro, thereby leading to enhanced killing of tumor cells. Using this approach in the context of adoptive CD8+ immunotherapy led to a marked suppression of tumor growth in murine NOD/SCID hepatocarcinoma or breast cancer xenograft models. We also observed significantly increased tumor cell apoptosis, and corresponding increases in murine survival. These findings thus demonstrate that in response to nanobody stimulation, DC/tumor cells-FC-induced specific CTLs exhibit superior anti-tumor efficacy, making this a potentially valuable means of achieving better adoptive immunotherapy outcomes in cancer patients.

Opportunities and Challenges for Antibodies against Intracellular Antigens.

Therapeutic antibodies are one most significant advances in immunotherapy, the development of antibodies against disease-associated MHC-peptide complexes led to the introduction of TCR-like antibodies. TCR-like antibodies combine the recognition of intracellular proteins with the therapeutic potency and versatility of monoclonal antibodies (mAb), offering an unparalleled opportunity to expand the repertoire of therapeutic antibodies available to treat diseases like cancer. This review details the current state of TCR-like antibodies and describes their production, mechanisms as well as their applications. In addition, it presents an insight on the challenges that they must overcome in order to become commercially and clinically validated.

Highly sensitive detection of CTLA-4-positive T-cell subgroups based on nanobody and fluorescent carbon quantum dots.

The detection of cytotoxic T-lymphocyte antigen-4-positive (CTLA-4+) T-cell subgroups in peripheral blood samples and tumor tissues is of great significance. In the present study, a rapid, succinct and efficient method was designed for the detection of CTLA-4+ human T cells using a CTLA-4-specific nanobody-fluorescent carbon quantum dots complex (QDs-Nb36). QDs-Nb36 was used for high sensitivity detection of CTLA-4+ T cells by flow cytometry or immumofluorescent staining. The present study demonstrated that the novel technique was more specific and effective in the detection of CTLA-4+ T-cell ratio in the peripheral blood and tumor tissues compared with a traditional monoclonal antibody approach. Furthermore, no significant toxicity was identified in vitro and in vivo, thus suggesting that the method may have broad applications for the detection of certain lowly expressed targets.

Anti-Tumor Activity of Mannose-CpG-Oligodeoxynucleotides-Conjugated and Hepatoma Lysate-Loaded Nanoliposomes for Targeting Dendritic Cells In Vivo.

Dendritic cell (DC)-based tumor vaccines are a promising immunotherapeutic method of cancer treatment. However, their therapeutic applications are significantly limited by their weak immunogenicity, costly culturing steps, and easily degradable properties. Thus, the anti-tumor activity for the vaccines should be improved. In this study, a novel lipid nanoparticle (M/CpG-ODN-H22-Lipo) was developed, which was conjugated with synthetic CpG oligodeoxynucleotides (CpG-ODN) and mannose and then loaded with H22 hepatoma lysate. Our data corroborate that M/CpG-ODN-H22-Lipo selectively targeted DCs and significantly increased their induced-maturation. Besides, the vaccine halted tumor growth and extended survival of mice with hepatocellular carcinoma. Moreover, M/CpG-ODN-H22-Lipo treatment reduced the percentages of myeloid-derived suppressor cells (in the tumor and bone marrow) and regulatory T cells (Treg) in the spleen. In contrast, the number of IFN-gamma-positive cells in the spleen along with the serum IgG levels were up-regulated. Moreover, tumor angiogenesis and tumor-cell proliferation were halted by the treatment of M/CpG-ODN-H22-Lipo, whereas tumor cell apoptosis was up-regulated. Our data revealed that CD8 + T cells and NK cells were vital to mediate the anti-tumor immunity of M/CpG-ODN-H22-Lipo treatment. In sum, the results here proved M/CpG-ODN-H22-Lipo vaccine a safe, specific and effective DC-based anti-tumor immunotherapy with great potential for clinical applications.

Fusion of Dendritic Cells and Cancer-Associated Fibroblasts for Activation of Anti-Tumor Cytotoxic T Lymphocytes.

Here we explored the fusion of dendritic cells (DCs), potent antigen-presenting cells that initiate primary immune responses, with cancer-associated fibroblasts (CAFs), which are a stromal component needed for tumor progression, with the aim of stimulating T cells to inhibit tumor growth. Dendritic cells from the bone marrow of BALB/c mice were co-cultured with CAFs from H22 mouse hepatoma cells. CAFs were found to express fibroblast activation protein and α-smooth muscle actin by flow cytometry, Western blotting and immunofluorescence. Polyethylene glycol was added to the co-culture medium to encourage fusion, and the ability of the resulting fusion cells to produce TNF-α, IL-1ß, IL-6, and IL-12p70 was confirmed using ELISA. These fusion cells efficiently stimulated T lymphocytes in vitro, causing them to generate IFN-α and IFN-γ. T cells activated by DC/CAF fusion cells led to strong CTL response against CAFs in vitro. The activated T cells also inhibited growth of H22 xenografts in vivo. These results indicate that DC/CAF fusion cells show potential for stimulating T cells as a novel anti-tumor vaccine.

Association between the V Leiden G1691A mutation and sudden sensorineural hearing loss in Italian population: a meta-analysis.

Epidemiological studies have reported inconsistent findings on the association between the V Leiden G1691A mutation and sudden sensorineural hearing loss (SSNHL) in Italian population. The aim of this meta-analysis was to clarify this association. PubMed, Embase, and the China National Knowledge Infrastructure (CNKI) were searched up to April 1, 2015. We used STATA12.0 to calculate summary odds ratios (ORs) with 95 % confidence intervals (CIs). Four studies including 958 patients were identified. Pooled data showed no significant association between V Leiden G1691A mutation and risk of SSNHL in Italian population: A vs. G (OR = 1.660, 95 % CI 0.428-6.446, P OR = 0.464) and AG vs. GG (OR = 1.680, 95 % CI 0.422-6.688, P OR = 0.462). The present meta-analysis suggests that V Leiden G1691A mutation is not significantly associated with increased risk of SSNHL disease in Italian population. Further large and well-designed studies are needed to confirm this association.