Identification and pathogenicity of Fusarium spp. associated with tea wilt in Zhejiang Province, China.

BACKGROUND: Tea is one of the most widely consumed beverages in the world, with significant economic and cultural value. However, tea production faces many challenges due to various biotic and abiotic stresses, among which fungal diseases are particularly devastating. RESULTS: To understand the identity and pathogenicity of isolates recovered from tea plants with symptoms of wilt, phylogenetic analyses and pathogenicity assays were conducted. Isolates were characterized to the species level by sequencing the ITS, tef-1α, tub2 and rpb2 sequences and morphology. Four Fusarium species were identified: Fusarium fujikuroi, Fusarium solani, Fusarium oxysporum, and Fusarium concentricum. The pathogenicity of the Fusarium isolates was evaluated on 1-year-old tea plants, whereby F. fujikuroi OS3 and OS4 strains were found to be the most virulent on tea. CONCLUSIONS: To the best of our knowledge, this is the first report of tea rot caused by F. fujikuroi in the world. This provides the foundation for the identification and control of wilt disease in tea plants.

Transforming primary human hepatocytes into hepatocellular carcinoma with genetically defined factors.

Our understanding of human hepatocellular carcinoma (HCC) development and progression has been hampered by the lack of in vivo models. We performed a genetic screen of 10 oncogenes and genetic mutations in Fah-ablated immunodeficient mice in which primary human hepatocytes (PHHs) are used to reconstitute a functional human liver. We identified that MYC, TP53R249S , and KRASG12D are highly expressed in induced HCC (iHCC) samples. The overexpression of MYC and TP53R249S transform PHHs into iHCC in situ, though the addition of KRASG12D significantly increases the tumorigenic efficiency. iHCC, which recapitulate the histological architecture and gene expression characteristics of clinical HCC samples, reconstituted HCC after serial transplantations. Transcriptomic analysis of iHCC and PHHs showed that MUC1 and FAP are expressed in iHCC but not in normal livers. Chimeric antigen receptor (CAR) T cells against these two surface markers efficiently lyse iHCC cells. The properties of iHCC model provide a biological basis for several clinical hallmarks of HCC, and iHCC may serve as a model to study HCC initiation and to identify diagnostic biomarkers and targets for cellular immunotherapy.

First worldwide report of Exserohilum rostratum causing leaf spot on strawberry (Fragaria × ananassa Duch.).

During April 2022, leaf spot was observed on strawberry (Fragaria × ananassa Duch.) with a disease incidence of approximately 45% among 100 plants. Strawberry was cultivated in a nursery at Huzhou University (30.87゜N, 120.13゜E), Zhejiang Province, China. In the strawberry greenhouse, the average temperature was 15-18 degrees, 40%-60% humidity. Early symptoms appeared as dark brown or black spotted necrotic lesions, which expanded from 2 to 6 mm in diameter. Dark brown spots with yellow halos occupied half of the leaf area and eventually developed leaf blight with large yellow halos. To isolate the causal agent, 0.5 cm x 0.5 cm fragments were cut from three symptomatic leaves, and were surface sterilized with 75% ethanol for 30 s and then rinsed three times with sterilized water. The airdried leaf fragments were placed on PDA with 50 µg/ml ampicillin and incubated in the dark at 25℃ for two days. Isolates were obtained by transferring hyphal plugs of 1 mm in diameter onto PDA. The colony morphology was circular and dark brown on the upperside and black on the underside, with cottony mycelium and an large amount of gray aerial mycelium. Conidia were large, light olive-brown to dark olive-brown and light olive-black and septate. The typical conidia were oval or rod-shaped, rarely curved, and dark septa defined the basal and apical cells. In the two typical forms of conidia, the average size of oval conidia was approximately 18.77 × 54.92 µm (11.99 to 26.97 × 35.13 to 74.59 µm, n = 20), and the average size of the rod-shaped conidia was approximately 14.80 × 103.24 µm (11.24 to 24.64 × 73.11 to 131.51 µm, n = 20). The morphological characteristics matched well with previous descriptions of Exserohilum rostratum (Sharma et al. 2014; Liu et al. 2021). The identity of C1-L and C1-S from symptomatic tissues was confirmed by means of multi-locus gene sequencing. Genomic DNA was extracted from the mycelium using the CTAB (cetyltrimethylammonium bromide) method (Griffith & Shaw 1998). Molecular identification was conducted by sequencing the internal transcribed spacer (ITS) rDNA region, partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, partial actin (ACT) gene, and partial beta-tubulin 2 (TUB2), using the primers ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT512F/ACT783R (Carbone and Kohn 1999), T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson, 1995). The obtained sequences of C1-L and C1-S were the same. Moreover, the sequences have been deposited in GenBank under accession numbers ON982516 (ITS), ON996915 (GAPDH), ON996916 (ACT), and ON996917 (TUB2). The results of Basic Local Alignment Search Tool (BLAST) analysis revealed that the ITS, GAPDH, and ACT had 100% identity with the sequences of E. rostratum (GenBank Accession No. LT837834, LT883550, and LT837672, respectively), the TUB2 had 99.61% similarity with BLAST sequences of E. rostratum (LT899391). These morphological characteristics and molecular analyses allowed the identification of the pathogen as E. rostratum. Koch's postulates were performed with five healthy detached strawberry leaves with three inoculations per leaf of the 'Akihime' strawberry variety. Surface-sterilized leaves were wounded with an aseptic needle, and inoculated with 2 mm diameter mycelial plugs from 5-day-old cultures of E. rostratum. Control leaves were also wounded with the aseptic needle, and inoculated with a sterile PDA agar plug. The leaves were incubated at 25℃ in Petri plates with petioles wrapped in moist sterile cotton. The diseased symptoms included black spots on the epidermis of the wounded leaves within 5, 10, and 20 days after inoculation. Mock-inoculated controls remained asymptomatic, and three biological repetitions were conducted. The fungus reisolated from the diseased leaves was confirmed as E. rostratum by sequencing. Abundant reports have shown that E. rostratum can infect many economically important crops such as maize, rice, and pineapple (Sun et al. 2021; Kabore et al. 2022; Luo et al. 2012). To the best of our knowledge, this is the first report of E. rostratum on strawberry in China and worldwide.

Integrated next-generation sequencing and comparative transcriptomic analysis of leaves provides novel insights into the ethylene pathway of Chrysanthemum morifolium in response to a Chinese isolate of chrysanthemum virus B.

BACKGROUND: Chrysanthemum virus B (CVB), a key member of the genus Carlavirus, family Betaflexiviridae, causes severe viral diseases in chrysanthemum (Chrysanthemum morifolium) plants worldwide. However, information on the mechanisms underlying the response of chrysanthemum plants to CVB is scant. METHODS: Here, an integrated next-generation sequencing and comparative transcriptomic analysis of chrysanthemum leaves was conducted to explore the molecular response mechanisms of plants to a Chinese isolate of CVB (CVB-CN) at the molecular level. RESULTS: In total, 4934 significant differentially expressed genes (SDEGs) were identified to respond to CVB-CN, of which 4097 were upregulated and 837 were downregulated. Gene ontology and functional classification showed that the majority of upregulated SDEGs were categorized into gene cohorts involved in plant hormone signal transduction, phenylpropanoid and flavonoid biosynthesis, and ribosome metabolism. Enrichment analysis demonstrated that ethylene pathway-related genes were significantly upregulated following CVB-CN infection, indicating a strong promotion of ethylene biosynthesis and signaling. Furthermore, disruption of the ethylene pathway in Nicotiana benthamiana, a model plant, using virus-induced gene silencing technology rendered them more susceptible to cysteine-rich protein of CVB-CN induced hypersensitive response, suggesting a crucial role of this pathway in response to CVB-CN infection. CONCLUSION: This study provides evidence that ethylene pathway has an essential role of plant in response to CVB and offers valuable insights into the defense mechanisms of chrysanthemum against Carlavirus.

Infection mechanisms and putative effector repertoire of the mosquito pathogenic oomycete Pythium guiyangense uncovered by genomic analysis.

Pythium guiyangense, an oomycete from a genus of mostly plant pathogens, is an effective biological control agent that has wide potential to manage diverse mosquitoes. However, its mosquito-killing mechanisms are almost unknown. In this study, we observed that P. guiyangense could utilize cuticle penetration and ingestion of mycelia into the digestive system to infect mosquito larvae. To explore pathogenic mechanisms, a high-quality genome sequence with 239 contigs and an N50 contig length of 1,009 kb was generated. The genome assembly is approximately 110 Mb, which is almost twice the size of other sequenced Pythium genomes. Further genome analysis suggests that P. guiyangense may arise from a hybridization of two related but distinct parental species. Phylogenetic analysis demonstrated that P. guiyangense likely evolved from common ancestors shared with plant pathogens. Comparative genome analysis coupled with transcriptome sequencing data suggested that P. guiyangense may employ multiple virulence mechanisms to infect mosquitoes, including secreted proteases and kazal-type protease inhibitors. It also shares intracellular Crinkler (CRN) effectors used by plant pathogenic oomycetes to facilitate the colonization of plant hosts. Our experimental evidence demonstrates that CRN effectors of P. guiyangense can be toxic to insect cells. The infection mechanisms and putative virulence effectors of P. guiyangense uncovered by this study provide the basis to develop improved mosquito control strategies. These data also provide useful knowledge on host adaptation and evolution of the entomopathogenic lifestyle within the oomycete lineage. A deeper understanding of the biology of P. guiyangense effectors might also be useful for management of other important agricultural pests.

Pythium myriotylum Is Recovered Most Frequently from Pythium Soft Rot-Infected Ginger Rhizomes in China.

Pythium soft rot is a major soilborne disease of crops such as ginger (Zingiber officinale). Our objective was to identify which Pythium species were associated with Pythium soft rot of ginger in China, where approximately 20% of global ginger production is located. Oomycetes infecting ginger rhizomes from seven provinces were investigated using two molecular markers, the internal transcribed spacer, and cytochrome c oxidase subunit II (CoxII). In total, 81 isolates were recovered; approximately 95% of the isolates were identified as Pythium myriotylum, and the other isolates were identified as either P. aphanidermatum or P. graminicola. Notably, the P. myriotylum isolates from China did not contain the single nucleotide polymorphism in the CoxII sequence found previously in the P. myriotylum isolates infecting ginger in Australia. A subset of 36 isolates was analyzed repeatedly by temperature-dependent growth, severity of disease on ginger plants, and aggressiveness of colonization on ginger rhizome sticks. In the pathogenicity assays, 32 of 36 isolates were able to significantly infect and cause severe disease symptoms on the ginger plants. A range of temperature-dependent growth, disease severity, and aggressiveness in colonization was found, with a significant moderate positive correlation between growth and aggressiveness of colonization of the ginger sticks. This study identified P. myriotylum as the major oomycete pathogen in China from infected ginger rhizomes and suggested that P. myriotylum should be a key target to control soft rot of ginger disease.

The association of metabolic syndrome and cognitive impairment in Jidong of China: a cross-sectional study.

BACKGROUND: Metabolic syndrome (Mets) is prevalent in the general population and has been reported to be an independent risk factor for cognitive impairment. This study aimed to investigate the association of Mets with the risk of cognitive impairment. METHODS: We studied 5854 participants from the Jidong community. Cognitive function was assessed by the Mini-Mental State of Examination (MMSE) scale. Mets was diagnosed according to the International Diabetes Federation criteria. We used logistic regression analysis to investigate the association of metabolic syndrome with the risk of cognitive impairment. RESULT: Among the 5854 adults included in the study, the age mean (SD) of age was 44 (13.57) years, and 2916 (50.34%) were male. There was a higher (56.03%) cognitive impairment incidence rate among participants with Mets than among those without Mets. In addition, there was a significant association between Mets and cognitive impairment (OR: 2.39, 95% CI: 2.00-2.86, P < 0.05) after adjusting for potential confounders, including age, gender, education level, marital status, smoking and alcohol consumption status. Regarding the 5 Mets components, abdominal obesity and elevated blood pressure were associated with the risk of Mets (OR: 1.36, 95% CI: 1.09-1.70, P < 0.001; OR: 1.32, 95% CI: 1.07-1.63, P < 0.05). Moreover, the strongest statistical correlation (adjusted OR: 1.86, 95% CI: 1.22-2.83, P < 0.05) was found when the number of Mets components was three. CONCLUSION: Our study suggested that Mets was associated with cognitive impairment and that abdominal obesity and hypertension were associated with an increased risk of cognitive impairment.

Comparative study of treatment for striae alba stage striae gravidarum: 1565-nm non-ablative fractional laser versus fractional microneedle radiofrequency.

Striae gravidarum is a common dermatologic condition for females caused by multiple factors during pregnancy. It remains a therapeutic challenge especially in the striae alba (SA) stage, generating psychological and emotional distress to those affected. This study aims to compare the efficacy and safety of 1565-nm non-ablative fractional laser (NAFL) and fractional microneedle radiofrequency (MRF) for treatment of SA striae gravidarum. Fourteen Chinese women with SA striae gravidarum were included in this study. Patient abdomens were randomly divided into NAFL and MRF treatment sides, treated three times at 6-week intervals. Treatment efficacy was evaluated by subjective (clinical assessments, patient satisfaction rating, adverse effects assessment) assessments and objective (skin melanin index measurement, histological study) assessments. Clinical assessment suggested MRF was more effective (P = 0.0143) for improving the appearance of SA striae gravidarum. Both NAFL and MRF demonstrated effective improvement (P = 0.0082 and P = 0.0158, respectively), with no significant difference according to patient satisfaction ratings and changes in melanin index (P = 0.5900). Both treatments induced limited adverse reactions, but MRF treatment caused significant pain compared with the more moderate NAFL treatment (P = 0.0003). MRF treatment increased neocollagen and elastic fibers more significantly than NAFL, based on histological assessments (P = 0.0298 and P = 0.0048, respectively). MRF treatment improved collagen regeneration in SA striae gravidarum more than NAFL but caused considerable pain during treatment. Corresponding treatment or therapeutic strategies should be applied according to clinical scenario.

First report of Colletotrichum fructicola causing anthracnose on cherry (Prunus avium) in China.

Cherry (Prunus avium) has become an important economical fruit in China. In October 2020, a leaf spot disease was found on cherry in the orchard of Taizhou Academy of Agriculture Sciences, Zhejiang, China. The symptoms appeared as small, water-soaked spots on the leaves, which later became larger, dark brown, and necrotic lesions of 1 cm to 3 cm in width, 4 cm to 8 cm in length. Disease incidences of approximately 60% of the leaves were observed by sampling five locations. To isolate the causing agent, small fragments from five target symptomatic leaves were surface-sterilized with 1.0% sodium hypochlorite solution for 1 min and then rinsed three times with sterilized water. Afterwards the leaf fragments were air-dried, plated onto potato dextrose agar (PDA) medium, and incubated at 25 ℃ in the dark for 2 days. The pure cultures were obtained by transferring hyphal plug of 2 mm in diameter onto PDA, which followed single spore isolation. The colony morphology showed light to dark gray, cottony mycelium, with the underside of the culture became brownish after 7 days. Conidia (n = 28) were hyaline, smooth-walled, cylindrical, aseptate, broadly rounded ends, and average size around 3.84 × 12.82 µm (2.99 to 4.87 × 10.27 to 15.68 µm). Appressoria (n = 27) were mostly brown, ovoid and slightly irregular in shape, and average size around 8.04 × 9.68 µm (6.29 to 9.67 × 9.32 to 12.06 µm). Perithecia average size is 106.25 µm, textura angularis, thick-walled. Asci 26.35-49.18 × 5.00-12.03 µm (average size 37.44 × 7.80 µm, n = 17), unitunicate, thin-walled, clavate or cymbiform. Ascospores 13.69-20.93 × 3.86-6.69 µm (average size 16.00 × 5.42 µm, n = 30), one-celled, hyaline, one or two large guttulate at the centre, slightly rounded ends. The morphological characteristics matched well with previous descriptions of Colletotrichum species of C. gloeosporioides species complex, including C. fructicola (Prihastuti et al. 2009; Fu et al. 2019). The identity of two representative isolates (cf2-3 and cf4-4) from different leaves was confirmed by means of multi-locus gene sequencing. To this end, genomic DNA was extracted by the Plant Direct PCR kit (Vazyme Biotech Co., Ltd, China). Molecular identification was conducted by sequencing the internal transcribed spacer (ITS) rDNA region, partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, partial actin (ACT) gene, partial beta-tubulin 2 gene (TUB2), and partial chitin synthase gene (CHS). The obtained sequences have been deposited in GenBank under accession numbers MW581851 and MW581852 (ITS), MW590586 and MW590587 (GAPDH), MW616561 and MW616562 (ACT), MW729380 and MW729381 (TUB2), MW729378 and MW729379 (CHS). The results of Basic Local Alignment Search Tool (BLAST) analysis revealed that the ITS, GAPDH, ACT, TUB2 and CHS sequences of both isolates matched with 100% identity to Colletotrichum fructicola culture collection sequences in GenBank database (JX010165, JX009998, JX009491, JX010405, and JX009866 respectively). These morphological characteristics and molecular analyses allowed the identification of the pathogen as C. fructicola. Koch's postulates were performed with healthy detached cherry leaves of cultivar namely 'HongMi' from Taizhou Academy of Agriculture Sciences. Surface-sterilized leaves were inoculated with five-day-old cultures of C. fructicola mycelial discs of 2 mm in diameter after being wounded with a needle or non-wounded. Control leaves were inoculated with discs of same size PDA agar. Treated leaves were incubated at 25 ℃ in the dark at high relative humidity. Anthracnose symptoms appeared within 3 days both on non-wounded and wounded inoculation approaches. Mock-inoculated controls remained asymptomatic. Biological repetitions were carried out three times. The fungus was reisolated from infected leaves and confirmed as C. fructicola following the methods described above. Until recently, it has been found that C. fructicola can infect tea, apple, pear, Pouteria campechiana in China (Fu et al. 2014; Li et al. 2013; Shi et al. 2018; Yang et al. 2020). To the best of our knowledge, this is the first report of C. fructicola on cherry in China.

The glycoside hydrolase 18 family chitinases are associated with development and virulence in the mosquito pathogen Pythium guiyangense.

Chitinases, the enzymes responsible for the biological degradation of chitin, participate in numerous physiological processes such as nutrition, parasitism, morphogenesis and immunity in various organisms. However, the genome-wide distribution, evolution and biological functions of chitinases are rarely reported in oomycetes. This study systematically investigated the glycoside hydrolase 18 (GH18) family of chitinases from the mosquito pathogenic oomycete, Pythium guiyangense using bioinformatics and experimental assays. A total of 3 pairs of GH18 chitinase genes distributed in three distinct phylogenic clusters were identified from P. guiyangense genome, which is consistent with the ones in plant pathogenic oomycetes. Further transcriptional analysis revealed that Pgchi1/2 was highly expressed at the development stages, while Pgchi3/4 and Pgchi5/6 were up-regulated at the infection stages. The biological function analysis of chitinase genes using genetic transformation silencing method showed that silencing of Pgchi1/2 resulted in reduced zoospore production, without affecting the virulence. However, attenuation of Pgchi3/4 and Pgchi5/6 genes regulated not only oxidative stress responses, but also led to decreased infection rates to mosquito larvae. Taken together, this study provides a comprehensive overview of P. guiyangense chitinase family and reveals their diverse roles in the development, stress response, and virulence, which would elucidate insightful information on the molecular mechanism of chitinase in entomopathogenic pathogens.

Genome-wide and functional analyses of tyrosine kinase-like family genes reveal potential roles in development and virulence in mosquito pathogen Pythium guiyangense.

The tyrosine kinase-like (TKL) gene family is widely existed in most eukaryotes and participates in many biological processes, however, has been rarely studied in oomycetes. In this study we performed bioinformatic and experimental analyses to characterize TKLs in Pythium guiyangense, a promising mosquito biological control agent. Our results revealed that TKLs were widely distributed in all the detected oomycetes, but were largely expanded in P. guiyangense in a species-specific expansion manner. The expansion was mostly driven by whole-genome duplication and tandem duplication. Domain distributions and exon-intron structures were highly conserved in the same group while diverse in different groups, suggesting of functional divergence. Transcriptional analysis revealed that over one fourth of TKLs were differentially expressed after infection of mosquito larvae, implying that these genes might participate in the infection process. Furthermore, subgroup A TKLs were functionally investigated using genetic transformation silencing method. Our findings demonstrated that subgroup A TKLs were up-regulated at the early infection stages and silencing of subgroup A TKLs led to reduced mycelia growth, zoospore production and alteration of stress responses. Pathogenicity assays also revealed that silencing of subgroup A TKLs reduced P. guiyangense virulence to mosquito larvae. Taken together, this study provides a comprehensive overview of P. guiyangense TKL family and reveals their potential roles in growth, development, stress response, and especially virulence.

MiR-491 attenuates cancer stem cells-like properties of hepatocellular carcinoma by inhibition of GIT-1/NF-κB-mediated EMT.

Hepatocellular carcinoma (HCC) is the most common liver malignancy. Current standard practices for treatment of HCC are less than satisfactory because of CSCs-mediated recurrence. For this reason, targeting CSCs, or cancer cells with CSCs-like properties, is a new approach for HCC treatment. As we reported previously, microRNA-491 (miR-491) is lower expressed in poorly differentiated HCC tissues relative to well-differentiated HCC tissues. Here, we further evaluate the effects of miR-491 on the CSCs-like properties by using HCC cell lines and HCC tissue samples. Our data showed that miR-491 had a negative relationship with CSCs-like properties both in cell lines and tissue samples of HCC. Further, miR-491 levels of non-recurrence HCC tissues were higher than those of recurrence HCC tissues. In HCC cell lines, nuclear factor kappa B (NF-κB)/snail pathway was involved in the epithelial to mesenchymal transition and the maintenance of CSCs-like properties. Overexpression of miR-491 targeted G-protein-coupled receptor kinase-interacting protein 1 (GIT-1), which blocked the activation of NF-κB by the inhibition of extracellular signal-regulated kinases (ERKs). Such process attenuated the CSCs-like properties in HCC cells. Our results point to a previously undefined mechanism by which miR-491 decreases CSCs-like properties and help to identify potential targets for the therapy of HCCs.

Bispecific CAR-T cells targeting FAP and GPC3 have the potential to treat hepatocellular carcinoma.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated robust efficacy against hematological malignancies, but there are still some challenges regarding treating solid tumors, including tumor heterogeneity, antigen escape, and an immunosuppressive microenvironment. Here, we found that SNU398, a hepatocellular carcinoma (HCC) cell line, exhibited high expression levels of fibroblast activation protein (FAP) and Glypican 3 (GPC3), which were negatively correlated with patient prognosis. The HepG2 HCC cell line highly expressed GPC3, while the SNU387 cell line exhibited high expression of FAP. Thus, we developed bispecific CAR-T cells to simultaneously target FAP and GPC3 to address tumor heterogeneity in HCC. The anti-FAP-GPC3 bispecific CAR-T cells could recognize and be activated by FAP or GPC3 expressed by tumor cells. Compared with anti-FAP CAR-T cells or anti-GPC3 CAR-T cells, bispecific CAR-T cells achieved more robust activity against tumor cells expressing FAP and GPC3 in vitro. The anti-FAP-GPC3 bispecific CAR-T cells also exhibited superior antitumor efficacy and significantly prolonged the survival of mice compared with single-target CAR-T cells in vivo. Overall, the use of anti-FAP-GPC3 bispecific CAR-T cells is a promising treatment approach to reduce tumor recurrence caused by tumor antigen heterogeneity.

CD318 is a target of chimeric antigen receptor T cells for the treatment of colorectal cancer.

Colorectal cancer (CRC) currently has a poor prognosis with a 6.9-year median survival time; to relieve this malignant cancer, we proposed to establish CRC xenografts that can be used to evaluate the cytotoxicity of adoptive chimeric antigen receptor (CAR)-T cells and accelerate the clinical translation of CAR-T cells for use against CRC. We first verified that CD318 had a higher expression level in primary human CRC tissues than in normal tissues based on hundreds of clinical samples. Then, we redirected CAR-T cells containing anti-CD318 single-chain variable fragment (anti-CD318 scFv), CD3ζ, CD28, and Toll-like receptor 2 (TLR2) domains. Next, we evaluated the function of these CAR-T cells in vitro in terms of surface phenotype changes, cytotoxicity and cytokine secretion when they encountered CD318+ CRC cells. Finally, we established two different xenograft mouse models to assess in vivo antitumor activity. The results showed that CAR318 T cells were significantly activated and exhibited strong cytotoxicity and cytokine-secreting abilities against CRC cells in vitro. Furthermore, CAR318 T cells induced CRC regression in different xenograft mouse models and suppressed tumors compared with CAR19 T cells. In summary, our work demonstrates that CAR318 T cells possess strong antitumor capabilities and represent a promising therapeutic approach for CRC.

The onco-embryonic antigen ROR1 is a target of chimeric antigen T cells for colorectal cancer.

Colorectal cancer is globally ranked second in both incidence and mortality rate. It usually develops during the middle or late stages of diagnosis, and is characterized by easy metastasis, poor prognosis, and a significant decline in postoperative quality of life. ROR1 is an excellent oncoembryonic antigen in numerous immunotherapy treatments for tumors. Additionally, it is overexpressed in colorectal cancer. To fill the void in CRC treatment with ROR1 as a target of CAR-T immunotherapy, we designed and prepared antiROR1-CART. This third-generation CAR-T cell can effectively inhibit the growth of colorectal cancer in vitro and in vivo.

Disruption of CISH promotes the antitumor activity of human T cells and decreases PD-1 expression levels.

Tumor cells and the immunosuppressive tumor microenvironment suppress the antitumor activity of T cells through immune checkpoints, including the PD-L1/PD-1 axis. Cytokine-inducible SH2-containing protein (CISH), a member of the suppressor of cytokine signaling (SOCS) family, inhibits JAK-STAT and T cell receptor (TCR) signaling in T and natural killer (NK) cells. However, its role in the regulation of immune checkpoints in T cells remains unclear. In this study, we ablated CISH in T cells with CRISPR-Cas9 and found that the sensitivity of T cells to TCR and cytokine stimulation was increased. In addition, chimeric antigen receptor T cells with CISH deficiency exhibited longer survival and higher cytokine secretion and antitumor activity. Notably, PD-1 expression was decreased in activated CISH-deficient T cells in vitro and in vivo. The level of FBXO38, a ubiquitination-regulating protein that reduces PD-1 expression, was elevated in activated T cells after CISH ablation. Hence, this study reveals a mechanism by which CISH promotes PD-1 expression by suppressing the expression of FBXO38 and proposes a new strategy for augmenting the therapeutic effect of CAR-T cells by inhibiting CISH.

T lymphocytes expressing the switchable chimeric Fc receptor CD64 exhibit augmented persistence and antitumor activity.

Chimeric antigen receptor (CAR) T cell therapy lacks persistent efficacy with "on-target, off-tumor" toxicities for treating solid tumors. Thus, an antibody-guided switchable CAR vector, the chimeric Fc receptor CD64 (CFR64), composed of a CD64 extracellular domain, is designed. T cells expressing CFR64 exert more robust cytotoxicity against cancer cells than CFR T cells with high-affinity CD16 variant (CD16v) or CD32A as their extracellular domains. CFR64 T cells also exhibit better long-term cytotoxicity and resistance to T cell exhaustion compared with conventional CAR T cells. With trastuzumab, the immunological synapse (IS) established by CFR64 is more stable with lower intensity induction of downstream signaling than anti-HER2 CAR T cells. Moreover, CFR64 T cells exhibit fused mitochondria in response to stimulation, while CARH2 T cells contain predominantly punctate mitochondria. These results show that CFR64 T cells may serve as a controllable engineered T cell therapy with prolonged persistence and long-term antitumor activity.

[Exploration of the talent training mode of biology majors driven by high-quality employments in higher education--a case study of Huzhou University].

In recent years, driven by the support of national policies and societal needs for employments, talents in biology majors have been growing rapidly. To foster high-calibre biology talents for the society in the context of the "double world-class initiative" in higher education, this study analyzed the opinion of biology undergraduates in Huzhou University on employment and their professional recognition of biology majors. The aim of this study was to propose a high-quality employments-driven talent training mode for undergraduates in biology majors, so as to serve as a reference for the reform in training modes of other relevant majors.

Long-term fertilization lowers the alkaline phosphatase activity by impacting the phoD-harboring bacterial community in rice-winter wheat rotation system.

PhoD-harboring bacteria and the secreted alkaline phosphatases (ALP) are crucial in the regulation of soil phosphorus (P) cycling. However, the influential factors of these crucial indicators and their internal interactions remain controversial. Here, a long-term field experiment containing different fertilization regimes was conducted (chemical, organic, and no fertilizer applied). The results indicated that the richness and diversity of phoD-harboring bacterial community were significantly decreased after long-term fertilization. The applied fertilizer promoted the growth of competitive species, while phoD-harboring bacteria lost the advantage to outcompete other microorganisms after long-term fertilization. The decreased ALP activity was caused by the declined phoD gene abundance, which is attributed to the comprehensive effects of soil organic C (SOC), total nitrogen (TN), and various forms of P. The random forest models identified SOC, TN, and available P (AP) to be the dominant environmental factors in shaping the phoD-harboring bacterial community. In addition, some other forms of P such as organic P (Po), inorganic P (Pi) or total P (TP) also exerted significant effects. Different fertilization regimes changed the keystone genera that contributed significantly to soil ALP activities, while Pseudolabrys and Pseudomonas were predicted to be the most important genera regardless of different fertilization regimes. This study extends the understanding of the main process and mechanisms of P mobilization in response to different fertilization regimes.

Association Between Immunoglobulin G N-glycosylation and Vascular Cognitive Impairment in a Sample With Atherosclerosis: A Case-Control Study.

BACKGROUND: Atherosclerosis is considered a crucial component in the pathogenesis of decreased cognitive function, as occurs in vascular cognitive impairment (VCI). Inflammation and the immune response play a significant role in the development of many chronic diseases. Immunoglobulin G (IgG) N-glycosylation has been implicated in the development of a variety of diseases by affecting the anti-inflammatory and proinflammatory responses of IgG. This study aimed to investigate the association between IgG N-glycosylation and VCI in a sample of patients with atherosclerosis through a case-control study. METHOD: We recruited a total of 330 patients with atherosclerosis to participate in this case-control study, including 165 VCI patients and 165 sex- and age-matched participants with normal cognitive function. The plasma IgG N-glycans of participants were separated by ultrahigh-performance liquid chromatography. An enzyme-linked immunosorbent assay (ELISA) kit was used to determine the corresponding serum inflammatory factors. Atherosclerosis was diagnosed by carotid ultrasound, and the diagnosis of VCI was based on the "Guidelines for the Diagnosis and Treatment of Vascular Cognitive Impairment in China (2019)". A multivariate logistic regression model was used to explore the association between IgG N-glycans and VCI. We also analyzed the relationship between IgG N-glycans and the inflammatory state of VCI through canonical correlation analysis (CCA). RESULTS: Through the multivariate logistic regression analysis, 8 glycans and 13 derived traits reflecting decreased sialylation and galactosylation and increased bisecting GlcNAc significantly differed between the case and control groups after adjusting for confounding factors (P < 0.05, q < 0.05). Similarly, the differences in TNF-α, IL-6, and IL-10 were statistically significant between the case and control groups after adjusting for the effects of confounding factors (P < 0.05, q < 0.05). The CCA results showed that VCI-related initial N-glycans were significantly correlated with VCI-related inflammatory factors (r = 0.272, P = 0.004). The combined AUC value (AUC combined = 0.885) of 7 initial glycans and inflammatory factors was higher than their respective values (AUC initial glycans = 0.818, AUC inflammatory factors = 0.773). CONCLUSION: The findings indicate that decreased sialylation and galactosylation and increased bisecting GlcNAc reflected by IgG N-glycans might affect the occurrence of VCI in patients with atherosclerosis though promoting the proinflammatory function of IgG. IgG N-glycans may serve as potential biomarkers to distinguish VCI in individuals with atherosclerosis.