Genetic characterization and molecular epidemiological analysis of enterovirus C99 in China.

Enterovirus C99 (EV-C99) is a newly identified EV serotype within the species Enterovirus C. Few studies on EV-C99 have been conducted globally. More information and research on EV-C99 are needed to assess its genetic characteristics, phylogenetic relationships, and associations with enteroviral diseases. Here, the phylogenetic characteristics of 11 Chinese EV-C99 strains have been reported. The full-length genomic sequences of these 11 strains show 79.4-80.5% nucleotide identity and 91.7-94.3% amino acid (aa) identity with the prototype EV-C99. A maximum likelihood phylogenetic tree constructed based on the entire VP1 coding region identified 13 genotypes (A-M), revealing a high degree of variation among the EV-C99 strains. Phylogeographic analysis showed that the Xinjiang Uygur Autonomous Region is an important source of EV-C99 epidemics in various regions of China. Recombination analysis revealed inter-serotype recombination events of 16 Chinese EV-C99 strains in 5' untranslated regions and 3D regions, resulting in the formation of a single recombination form. Additionally, the Chinese strain of genotype J showed rich aa diversity in the P1 region, indicating that the genotype J of EV-C99 is still going through variable dynamic changes. This study contributes to the global understanding of the EV-C99 genome sequence and holds substantial implications for the surveillance of EV-C99.

Identification and genetic characterization of a recently identified enterovirus C116 in China.

Enterovirus C116 (EV-C116) is a new member of the enterovirus C group which is closely associated with several infectious diseases. Although sporadic studies have detected EV-C116 in clinical samples worldwide, there is currently limited information available. In this study, two EV-C-positive fecal specimens were detected in apparently healthy children, which harbored low abundance, through meta-transcriptome sequencing. Based on the prototypes of several EV-Cs, two lineages were observed. Lineage 1 included many types that could not cause EV-like cytopathic effect in cell culture. Three genogroups of EV-C116 were divided in the maximum likelihood tree, and the two strains in this study (XZ2 and XZ113) formed two different lineages, suggesting that EV-C116 still diffuses worldwide. Obvious inter-type recombination events were observed in the XZ2 strain, with CVA22 identified as a minor donor. However, another strain (XZ113) underwent different recombination situations, highlighting the importance of recombination in the formation of EV-Cs biodiversity. The EV-C116 strains could propagate in rhabdomyosarcoma cell cultures at low titer; however, EV-like cytopathic effects were not observed. HEp-2, L20B, VERO, and 293T cell lines did not provide an appropriate environment for EV-C116 growth. These results challenge the traditional recognition of the uncultured nature of EV-C116 strains and explain the difficulty of clinical detection.

PDGFRA exhibits potential as an indicator of angiogenesis within the tumor microenvironment and is up-regulated in BLCA.

Bladder cancer (BLCA) is a common type of urogenital malignancy worldwide. The recurrence and metastasis of bladder cancer are closely related to angiogenesis, but the underlying mechanisms are unclear. In this study, we developed a method to predict survival outcomes among BLCA patients, which could be used to guide immunotherapy and chemotherapy. We obtained patient data from The Cancer Genome Atlas (TCGA) and identified angiogenesis-related genes from the GeneCards database. First, we used differential expression analysis and univariate Cox analysis to identify angiogenesis-related genes and used correlation analysis to generate molecular subtypes based on M2 macrophages. Next, we constructed a prognostic signature consisting of four genes (ECM1, EFEMP1, SLIT2, and PDGFRΑ), which was found to be an independent prognostic factor. Higher risk scores were associated with worse overall survival and higher expression of immune checkpoints. We also evaluated immune cell infiltration using the CIBERSORT and ssGSEA algorithms. Additionally, we performed stratification analyses, constructed a nomogram, and predicted chemotherapeutic responses based on the risk signature. Finally, we validated our findings by using qRT-PCR as well as IHC data to detect the expression levels of the four genes at mRNA and protein levels in BLCA patients and obtained results that were consistent with our predictions. Our study demonstrates the utility of a four-gene prognostic signature for prognostication in bladder cancer patients and designing personalized treatments, which could provide new avenues for personalized management of these patients.

Improved efficacy and safety of zanubrutinib versus ibrutinib in patients with relapsed/refractory chronic lymphocytic leukemia (R/R CLL) in China: a subgroup of ALPINE.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) has different epidemiology in Chinese vs. Western patients, but there are few studies of CLL/SLL in large populations of Chinese patients. ALPINE is a global phase 3 trial investigating Bruton tyrosine kinase inhibitors zanubrutinib vs. ibrutinib to treat relapsed/refractory (R/R) CLL/SLL. Here we report results from the subgroup of Chinese patients. Adults with R/R CLL/SLL were randomized 1:1 to receive zanubrutinib (160 mg twice-daily) or ibrutinib (420 mg once-daily) until disease progression or unacceptable toxicity. Endpoints included overall response rate (ORR), progression-free survival (PFS), overall survival (OS), and safety. Data were analyzed descriptively. Ninety patients were randomized in China (zanubrutinib, n = 47; ibrutinib, n = 43). Baseline characteristics were balanced between groups, with fewer male patients in the zanubrutinib vs. ibrutinib group (55.3% vs. 69.8%). Median age was 60.5 years, 11% had del(17p) mutation, and 32% had tumor protein 53 (TP53) mutation. With median 25.3 months follow-up, ORR was 80.9% with zanubrutinib vs. 72.1% with ibrutinib. PFS was improved with zanubrutinib vs. ibrutinib (HR = 0.34 [95% CI, 0.15, 0.77]), and the HR for OS was 0.45 (95% CI, 0.14, 1.50). Rates of Grade ≥ 3 treatment-emergent adverse events (TEAEs; 64.4% vs. 72.1%), AEs leading to discontinuation (6.4% vs. 14.0%), and serious TEAEs (35.6% vs. 51.2%) were lower with zanubrutinib vs. ibrutinib. Zanubrutinib demonstrated improved ORR, PFS, and OS vs. ibrutinib and a more favorable safety profile in patients with R/R CLL/SLL in China. These results are consistent with the full global population of ALPINE. ClinicalTrials.gov: NCT03734016, registered November 7, 2018.

Factors predicting depressive symptoms in parents of children with autism spectrum disorder in eastern China.

BACKGROUND: Parents of children with autism spectrum disorder (ASD) are at a higher risk of depression than parents of typically developing children and those of children with other developmental disorders. Depression affects the well-being and quality of life of parents of children with ASD and has serious consequences for the long-term health outcomes of children with ASD. Therefore, this study explored the current status of depressive symptoms in parents of children with ASD in eastern China and further analyzed multiple aspects of the predictors of depressive symptoms. METHODS: A multicenter cross-sectional survey was conducted among parents of children with ASD in the rehabilitation department of a large specialized hospital and 10 rehabilitation centers for children with special needs in Lianyungang, Jiangsu Province, Eastern China. A structured questionnaire that focused on child-related factors, parent-related factors, depressive symptoms, courtesy stigma, and social support was used to obtain data. Binary logistic regression was used to identify the independent predictors of depressive symptoms in parents of children with ASD. RESULTS: A total of 409 parents of children with ASD were recruited, of whom 18.8% had depressive symptoms. Parents of children with ASD who raised a child who spoke few to no words (odds ratio [OR]: 2.747, 95% confidence interval [CI]: 1.026-7.357), claimed a high economic burden (OR: 3.215, 95% CI: 1.234-8.379), reported no change or increased severity of ASD in their children (OR: 2.518, 95% CI: 1.108-5.720), and those with a higher courtesy stigma score (OR: 1.189, 95% CI: 1.093-1.294) were more likely to have depressive symptoms. Conversely, parents of children with ASD who were employed (OR: 0.427, 95% CI: 0.201-0.907), satisfied with their current marital status (OR: 0.429, 95% CI: 0.221-0.834), and those with a higher social support score (OR: 0.973, 95% CI: 0.950-0.996) were less likely to have depressive symptoms. CONCLUSIONS: Depressive symptoms are common in parents of children with ASD in eastern China. Therefore, screening and intervention for depressive symptoms in parents of children with ASD is necessary, especially for those with high-risk factors.

Mass spectrometry-based metabolomics reveals metabolism of molnupiravir may lead to metabolic disorders and hepatotoxicity.

Molnupiravir (MO) is a pyrimidine nucleoside anti-SARS-CoV-2 drug. MO treatment could cause mild liver injury. However, the underlying mechanism of MO-induced liver injury and the metabolic pathway of MO in vivo are unclear. In this study, metabolomics analysis and molecular biology methods were used to explore these issues. Through metabolomics analysis, it was found that the homeostasis of pyrimidine, purine, lysophosphatidylcholine (LPC), and amino acids in mice was destroyed after MO treatment. A total of 80 changed metabolites were detected. Among these changed metabolites, 4-ethylphenyl sulfate, dihydrouracil, and LPC 20:0 was related to the elevation of alkaline phosphatase (ALP), interleukin-6 (IL6), and nuclear factor kappa-B (NF-κB). The levels of 4-ethylphenyl sulfate, dihydrouracil, and LPC 20:0 in plasma were positively correlated with their levels in the liver, suggesting that these metabolites were associated with MO-induced liver injury. MO treatment could increase NHC and cytidine levels, activate cytidine deaminase (CDA), and increase LPC levels. CDA and LPC could increase the mRNA expression level of toll-like receptor (TLR). The current study indicated that the elevation of hepatic TLR may be an important reason for MO leading to the liver injury.

Novel KMT5B variant associated with neurodevelopmental disorder in a Chinese family: A case report.

Background: We report here the clinical and genetic features of KMT5B-related neurodevelopmental disorder caused by a novel heterozygous frameshift variant in KMT5B in a Chinese family. Case presentation: A 7-year-old Chinese boy with mild-to-moderate intellectual disability, significant language impairment, motor disability, and coordination difficulties presented to our hospital because he "could not speak and did not look at others." He was diagnosed with autism spectrum disorder previously owing to developmental delays in cognition, language expression, and understanding. The child also had variable nonspecific features including macrocephaly, wide button-hole space and nasal bridge, low ear, social behavior disorder, and foot deformities. Exome sequencing (ES) revealed that both the proband and his younger brother had inherited a novel heterozygous frameshift variant c.438_439ins[ASD; KT192064.1:1_310] of the KMT5B gene from their father. Bioinformatics analysis showed that the novel mutation affected the structure of the KMT5B pre-SET domain, mainly in the α-helix region. According to the American College of Medical Genetics and Genomics (ACMG) guidelines, this type of variant was eventually determined to be likely pathogenic (PVS1+PM2_P). Conclusions: Our investigation expands the mutation spectrum of KMT5B to help us to better understand KMT5B-related neurodevelopmental disorder.

Upconversion-based chiral nanoprobe for highly selective dual-mode sensing and bioimaging of hydrogen sulfide in vitro and in vivo.

Chiral assemblies have become one of the most active research areas due to their versatility, playing an increasingly important role in bio-detection, imaging and therapy. In this work, chiral UCNPs/CuxOS@ZIF nanoprobes are prepared by encapsulating upconversion nanoparticles (UCNPs) and CuxOS nanoparticles (NPs) into zeolitic imidazolate framework-8 (ZIF-8). The novel excited-state energy distribution-modulated upconversion nanostructure (NaYbF4@NaYF4: Yb, Er) is selected as the fluorescence source and energy donor for highly efficient fluorescence resonance energy transfer (FRET). CuxOS NP is employed as chiral source and energy acceptor to quench upconversion luminescence (UCL) and provide circular dichroism (CD) signal. Utilizing the natural adsorption and sorting advantages of ZIF-8, the designed nanoprobe can isolate the influence of other common disruptors, thus achieve ultra-sensitive and highly selective UCL/CD dual-mode quantification of H2S in aqueous solution and in living cells. Notably, the nanoprobe is also capable of in vivo intra-tumoral H2S tracking. Our work highlights the multifunctional properties of chiral nanocomposites in sensing and opens a new vision and idea for the preparation and application of chiral nanomaterials in biomedical and biological analysis.

DNA demethylase Tet2 promotes the terminal maturation of natural killer cells.

The cytotoxicity feature to eliminate malignant cells makes natural killer (NK) cells a candidate for tumor immunotherapy. However, this scenario is currently hampered by inadequate understanding of the regulatory mechanisms of NK cell development. Ten-Eleven-Translocation 2 (Tet2) is a demethylase whose mutation was recently shown to cause phenotypic defects in NK cells. However, the role of Tet2 in the development and maturation of NK cells is not entirely clear. Here we studied the modulatory role of Tet2 in NK cell development and maturation by generating hematopoietic Tet2 knockout mice and mice with Tet2 conditional deletion in NKp46+ NK cells. The results showed that both hematopoietic and NK cell conditional deletion of Tet2 had no effect on the early steps of NK cell development, but impaired the terminal maturation of NK cells defined by CD11b, CD43, and KLRG1 expression. In the liver, Tet2 deletion not only prevented the terminal maturation of NK cells, but also increased the proportion of type 1 innate lymphoid cells (ILC1s) and reduced the proportion of conventional NK cells (cNK). Moreover, hematopoietic deletion of Tet2 lowered the protein levels of perforin in NK cells. Furthermore, hematopoietic deletion of Tet2 downregulated the protein levels of Eomesodermin (Eomes), but not T-bet, in NK cells. In conclusion, our results demonstrate that Tet2 plays an important role in the terminal maturation of NK cells, and the Eomes transcription factor may be involved.

Ti2C3 MXene-based nanocomposite as an intelligent nanoplatform for efficient mild hyperthermia treatment.

Photothermal therapy (PTT) has attracted much attention due to its less invasive, controllable and highly effective nature. However, PTT also suffers from intrinsic cancer resistance mediated by cell survival pathways. These survival pathways are regulated by a variety of proteins, among which heat shock protein (HSP) triggers thermotolerance and protects tumor cells from hyperthermia-induced apoptosis. Confronted by this challenge, we propose and validate here a novel MXene-based HSP-inhibited mild photothermal platform, which significantly enhances the sensitivity of tumor cells to heat-induced stress and thus improves the PPT efficacy. The Ti3C2@Qu nanocomposites are constructed by utilizing the high photothermal conversion ability of Ti3C2 nanosheets in combination with quercetin (Qu) as an inhibitor of HSP70. Qu molecules are loaded onto the nanoplatform in a pH-sensitive controlled release manner. The acidic environment of the tumor causes the burst-release of Qu molecules, which deplete the level of heat shock protein 70 (HSP70) in tumor cells and leave the tumor cells out from the protection of the heat-resistant survival pathway in advance, thus sensitizing the hyperthermia efficacy. The nanostructure, photothermal properties, pH-responsive controlled release, synergistic photothermal ablation of tumor cells in vitro and in vivo, and hyperthermia effect on subcellular structures of the Ti3C2@Qu nanocomposites were systematically investigated.

Myeloid Cell Trim59 Deficiency Worsens Experimental Ischemic Stroke and Alters Cerebral Proteomic Profile.

Background: Tripartite motif containing 59 (TRIM59) is a ubiquitin ligase and is involved in the pathogenesis of various diseases, including cancers, sepsis, and other immune-related diseases. However, it has not been defined whether TRIM59 plays a role in ischemic stroke in mice. Methods: This study determined the influence of Trim59 deficiency on experimental stroke outcomes and the cerebral proteomic profile using myeloid cell Trim59 conditional knockout (Trim59-cKO) mice and a label-free quantitative proteomic profiling technique. The possible mechanisms by which TRIM59 affected stroke onset were elucidated by in vivo and in vitro experiments. Results: Immunofluorescence staining results showed that TRIM59 expression was up-regulated after cerebral ischemia and co-localized with macrophages. Myeloid cell Trim59 deficiency exacerbated ischemic injury on day 3 after experimental stroke. In proteomic analysis, 23 differentially expressed proteins were identified in ischemic brain of Trim59-cKO mice as compared to Trim59flox/flox mice. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the differentially expressed proteins were enriched in complement and coagulation cascades. Protein-protein interaction analysis suggested the central role of clusterin in the interaction network. ELISA and Western blot assays confirmed the reduced levels of clusterin protein in the ischemic brains of Trim59-cKO mice. Further experimental results showed that clusterin was expressed in neurons. Conditional co-culture experiments of primary neurons and bone marrow-derived macrophages demonstrated that LPS stimulated macrophages to secrete complement C3. In addition, TRIM59 may affect the changes in clusterin expression in an indirect manner by influencing the secretion of complement C3 in macrophages. In vivo experiments also proved a significant increase in C3 levels in the brains of Trim59-cKO mice after ischemia. Conclusion: Myeloid cell Trim59 deficiency aggravated ischemic stroke outcomes in conjunction with a distinct cerebral proteomic profile, and the underlying mechanism may be related to the regulation of macrophage C3 expression by TRIM59.

R-ketorolac ameliorates cancer-associated cachexia and prolongs survival of tumour-bearing mice.

BACKGROUND: Cancer-associated cachexia (CAC) is a debilitating syndrome associated with poor quality of life and reduced life expectancy of cancer patients. CAC is characterized by unintended body weight reduction due to muscle and adipose tissue loss. A major hallmark of CAC is systemic inflammation. Several non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested for CAC treatment, yet no single medication has proven reliable. R-ketorolac (RK) is the R-enantiomer of a commonly used NSAID. The effect of RK on CAC has not yet been evaluated. METHODS: Ten- to 11-week-old mice were inoculated with C26 or CHX207 cancer cells or vehicle control (phosphate-buffered saline [PBS]). After cachexia onset, 2 mg/kg RK or PBS was administered daily by oral gavage. Body weight, food intake and tumour size were continuously measured. At study endpoints, blood was drawn, mice were sacrificed and tissues were excised. Immune cell abundance was analysed using a Cytek® Aurora spectral flow cytometer. Cyclooxygenase (COX) activity was determined in lung homogenates using a fluorometric kit. Muscle tissues were analysed for mRNA and protein expression by quantitative real-time PCR and western blotting analysis, respectively. Muscle fibre size was determined on histological slides after haematoxylin/eosin staining. RESULTS: Ten-day survival rate of C26-bearing animals was 10% while RK treatment resulted in a 100% survival rate (P = 0.0009). Chemotherapy resulted in a 10% survival rate 14 days after treatment initiation, but all mice survived upon co-medication with RK and cyclophosphamide (P = 0.0001). Increased survival was associated with a protection from body weight loss in C26 (-0.61 ± 1.82 vs. -4.48 ± 2.0 g, P = 0.0004) and CHX207 (-0.49 ± 0.33 vs. -2.49 ± 0.93 g, P = 0.0003) tumour-bearing mice treated with RK, compared with untreated mice. RK ameliorated musculus quadriceps (-1.7 ± 7.1% vs. -27.8 ± 8.3%, P = 0.0007) and gonadal white adipose tissue (-18.8 ± 49% vs. -69 ± 15.6%, P = 0.094) loss in tumour-bearing mice, compared with untreated mice. Mechanistically, RK reduced circulating interleukin-6 (IL-6) concentrations from 334 ± 151 to 164 ± 123 pg/mL (P = 0.047) in C26 and from 93 ± 39 to 35 ± 6 pg/mL (P = 0.0053) in CHX207 tumour-bearing mice. Moreover, RK protected mice from cancer-induced T-lymphopenia (+1.8 ± 42% vs. -49.2 ± 12.1% in treated vs. untreated mice, respectively). RK was ineffective in ameliorating CAC in thymus-deficient nude mice, indicating that the beneficial effect of RK depends on T-cells. CONCLUSIONS: RK improved T-lymphopenia and decreased systemic IL-6 concentrations, resulting in alleviation of cachexia and increased survival of cachexigenic tumour-bearing mice, even under chemotherapy and independent of COX inhibition. Considering its potential, we propose that the use of RK should be investigated in patients suffering from CAC.

Thirty Years of Experience of Acute Flaccid Paralysis Surveillance for Polio - China, 1993-2022.

Introduction: Detecting poliovirus infections proves to be highly challenging due to their asymptomatic nature and infectious potential, highlighting the crucial importance of effective detection methods in the context of polio eradication efforts. In many countries, including China, the primary approach for identifying polio outbreaks has been through acute flaccid paralysis (AFP) surveillance. In this study, we conducted an evaluation spanning three decades (1993-2022) to assess the effectiveness of AFP surveillance in China. Methods: Data on all AFP cases identified since 1993 and national-level AFP surveillance system quality indicators aligned with the World Health Organization (WHO) standards were collected for analysis. The quality indicators assess surveillance sensitivity, completeness, timeliness of detection notification, case investigation, and laboratory workup. Surveillance sensitivity is determined by the non-polio AFP (NPAFP) detection rate among children under 15 years of age. Results: Between 1993 and 2022, a total of 150,779 AFP cases were identified and reported. Within this pool, surveillance identified 95 cases of wild poliovirus (WPV) and 24 cases due to vaccine-derived poliovirus. From 1995 onwards, the detection rate of NPAFP cases consistently adhered to the WHO and national standards of ≥1 case per 100,000, falling between 1.38 and 2.76. Starting in 1997, all timeliness indicators consistently achieved the criteria of 80%, apart from the consistency in meeting standards set for the rate of positive specimens sent to the national laboratory. Conclusions: AFP surveillance has been instrumental in China's accomplishment of maintaining a polio-free status. The ongoing adherence to key performance indicators, ensuring sensitivity and prompt specimen collection, demonstrates that AFP surveillance is proficient in detecting poliovirus in China. As we move into the post-eradication phase, AFP surveillance remains crucial for the sustained absence of polioviruses in the long term.

Evolutionary Diversity of Coxsackievirus A6 Causing Severe Hand, Foot, and Mouth Disease - China, 2012-2023.

Introduction: Coxsackievirus A6 (CVA6) has emerged as a significant pathogen responsible for severe cases of hand, foot, and mouth disease (HFMD). This study aims to delineate the demographic characteristics and analyze the viral evolution of severe HFMD associated with CVA6, thereby assisting in its surveillance and management. Methods: In this investigation, 74 strains of CVA6 were isolated from samples collected from severe HFMD cases between 2012 and 2023. The VP1 gene sequences of CVA6 were amplified and analyzed to assess population historical dynamics and evolutionary characteristics using BEAST, DnaSP6, and PopART. Results: A significant portion (94.4%) of severe CVA6-associated HFMD cases (51 out of 54, with 20 lacking age information) were children under 5 years old. Among the 74 CVA6 strains analyzed, 72 belonged to the D3a sub-genotype, while only two strains were D2 sub-genotype. The average genetic distance between VP1 sequences prior to 2015 was 0.027, which increased to 0.051 when compared to sequences post-2015. Historical population dynamics analysis indicated three significant population expansions of severe CVA6-associated HFMD during 2012-2013, 2013-2014, and 2019-2020, resulting in the formation of 65 distinct haplotypes. Consistent with the MCC tree findings, transitioning between regional haplotypes required multiple base substitutions, showcasing an increase in population diversity during the evolutionary process (from 14 haplotypes in 2013 to 55 haplotypes over the subsequent decade). Conclusions: CVA6, associated with severe HFMD, is evolving and presents a risk of outbreak occurrence. Thus, enhanced surveillance of severe HFMD is imperative.

Genotype F of Echovirus 25 with multiple recombination pattern have been persistently and extensively circulating in Chinese mainland.

Echovirus 25 (E25), a member of the Enterovirus B (EV-B) species, can cause aseptic meningitis (AM), viral meningitis (VM), and acute flaccid paralysis (AFP). However, systematic studies on the molecular epidemiology of E25, especially those concerning its evolution and recombination, are lacking. In this study, 18 strains of E25, isolated from seven provinces of China between 2009 and 2018, were collected based on the Chinese hand, foot, and mouth disease (HFMD) surveillance network, and 95 sequences downloaded from GenBank were also screened. Based on the phylogenetic analysis of 113 full-length VP1 sequences worldwide, globally occurring E25 strains were classified into 9 genotypes (A-I), and genotype F was the dominant genotype in the Chinese mainland. The average nucleotide substitution rate of E25 was 6.08 × 10-3 substitutions/site/year, and six important transmission routes were identified worldwide. Seventeen recombination patterns were determined, of which genotype F can be divided into 9 recombination patterns. A positive selector site was found in the capsid protein region of genotype F. Recombination analysis and pressure selection analysis for genotype F showed multiple recombination patterns and evolution characteristics, which may be responsible for it being the dominant genotype in the Chinese mainland. This study provides a theoretical basis for the subsequent prevention and control of E25.

Evolution, recombination and geographic spreading of global Coxsackievirus A6.

BACKGROUND: The increasing incidence of hand, foot, and mouth disease (HFMD) associated with Coxsackievirus A6 (CVA6) has become a very significant public health problem. The aim of this study is to investigate the recombination, geographic transmission, and evolutionary characteristics of the global CVA6. METHODS: From 2019 to 2022, 73 full-length CVA6 sequences were obtained from HFMD patients in China and analyzed in combination with 1032 published whole genome sequences. Based on this dataset, the phylogenetic features, recombinant diversity, Bayesian phylodynamic characteristics, and key amino acid variations in CVA6 were analyzed. RESULTS: The four genotypes of CVA6, A, D, E, and F, are divided into 24 recombinant forms (RFs, RF-A - RF-X) based on differences in the P3 coding region. The eastern China region plays a key role in the dissemination of CVA6 in China. VP1-137 and VP1-138 are located in the DE loop on the surface of the CVA6 VP1 protein, with the former being a highly variable site and the latter having more non-synonymous substitutions. CONCLUSIONS: Based on whole genome sequences, this study contributes to the CVA6 monitoring, early warning, and the pathogenic mechanism by studying recombination diversity, geographical transmission characteristics, and the variation of important amino acid sites.

Anti-BCMA/GPRC5D bispecific CAR T cells in patients with relapsed or refractory multiple myeloma: a single-arm, single-centre, phase 1 trial.

BACKGROUND: Some challenges still exist with single-target B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell therapies due to variable or negative BCMA expression, although they have yielded remarkable efficacy in relapsed or refractory multiple myeloma. We developed anti-BCMA/GPRC5D bispecific CARs to mitigate the limitations and potentiate the functions of CAR T cells. METHODS: This single-arm, phase 1 trial was conducted at the Affiliated Hospital of Xuzhou Medical University (Xuzhou, China). The trial enrolled patients aged 18-75 years with relapsed or refractory multiple myeloma and an Eastern Cooperative Oncology Group performance status of 0-3. Anti-BCMA/GPRC5D bispecific CAR T cells were administered at 0·5 × 106, 1·0 × 106, 2·0 × 106, and 4·0 × 106 CAR T cells per kg in the dose-escalation phase, with additional patients included at the dose selected for the dose-expansion phase. The primary endpoint was safety, which included dose-limiting toxicity and maximum tolerated dose. Activity was also evaluated as a secondary endpoint. The maximum tolerated dose was chosen for the dose-expansion phase. Safety and activity analyses were done in all patients who received anti-BCMA/GPRC5D bispecific CAR T cells as defined in the protocol. This trial is registered with ClinicalTrials.gov (NCT05509530) and is complete. FINDINGS: Between Sept 1, 2022, and Nov 3, 2023, 24 patients were enrolled and underwent apheresis. Three patients were excluded after apheresis (two patients discontinued due to rapid disease progression and one patient was withdrawn because of failed manufacture of CAR T cells), so 21 patients were infused with anti-BCMA/GPRC5D bispecific CAR T cells. Median follow-up was 5·8 months (IQR 5·2-6·7). Median age was 62 years (IQR 56-67). Eight (38%) patients were male, and 13 (62%) female. All patients were Chinese. At the 4·0 × 106 CAR T cells per kg dose, two patients had dose-limiting toxicities, of whom one died of subarachnoid haemorrhage (which was not considered to be related to the study treatment). The maximum tolerated dose was identified as 2·0 × 106 CAR T cells per kg. The most common grade 3 or worse adverse events were haematological toxicities in 19 (90%) patients (except lymphopenia). 15 (71%) patients had cytokine release syndrome, of which all cases were grade 1 or 2. One case of grade 1 immune effector cell-associated neurotoxicity syndrome (ICANS) was observed in a patient who received 4·0 × 106 CAR T cells per kg. No ICANS or grade 3 or worse organ toxicities were observed in patients who received 0·5-2·0 × 106 CAR T cells per kg. The overall response rate was 86% (18 of 21 patients), with 13 (62%) patients having a complete response or better, and 17 (81%) patients having measurable residual disease negativity. Of the 12 patients who received 2·0 × 106 CAR T cells per kg (three in the dose-escalation phase and an addition nine in the dose-expansion phase), the overall response rate was 92% (11 of 12 patients) with nine (75%) patients having a complete response or better. INTERPRETATION: Anti-BCMA/GPRC5D bispecific CAR T cells show a good safety profile and encouraging activity in patients with relapsed or refractory multiple myeloma. FUNDING: National Natural Science Foundation of China. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Molecular Epidemiology and Evolution of Coxsackievirus A14.

As the proportion of non-enterovirus 71 and non-coxsackievirus A16 which proportion of composition in the hand, foot, and mouth pathogenic spectrum gradually increases worldwide, the attention paid to other enteroviruses has increased. As a member of the species enterovirus A, coxsackievirus A14 (CVA14) has been epidemic around the world until now since it has been isolated. However, studies on CVA14 are poor and the effective population size, evolutionary dynamics, and recombination patterns of CVA14 are not well understood. In this study, 15 CVA14 strains were isolated from HFMD patients in mainland China from 2009 to 2019, and the complete sequences of CVA14 in GenBank as research objects were analyzed. CVA14 was divided into seven genotypes A-G based on an average nucleotide difference of the full-length VP1 coding region of more than 15%. Compared with the CVA14 prototype strain, the 15 CVA14 strains showed 84.0-84.7% nucleotide identity in the complete genome and 96.9-97.6% amino acid identity in the encoding region. Phylodynamic analysis based on 15 CVA14 strains and 22 full-length VP1 sequences in GenBank showed a mean substitution rate of 5.35 × 10-3 substitutions/site/year (95% HPD: 4.03-6.89 × 10-3) and the most recent common ancestor (tMRCA) of CVA14 dates back to 1942 (95% HPD: 1930-1950). The Bayesian skyline showed that the effective population size had experienced a decrease-increase-decrease fluctuation since 2004. The phylogeographic analysis indicated two and three possible migration paths in the world and mainland China, respectively. Four recombination patterns with others of species enterovirus A were observed in 15 CVA14 strains, among which coxsackievirus A2 (CVA2), coxsackievirus A4 (CVA4), coxsackievirus A6 (CVA6), coxsackievirus A8 (CVA8), and coxsackievirus A12 (CVA12) may act as recombinant donors in multiple regions. This study has filled the gap in the molecular epidemiological characteristics of CVA14, enriched the global CVA14 sequence database, and laid the epidemiological foundation for the future study of CVA14 worldwide.

Uricase sensitizes hepatocellular carcinoma cells to 5-fluorouracil through uricase-uric acid-UMP synthase axis

Conventional chemotherapy plays a key role in hepatocellular carcinoma (HCC) treatment, however, with intrinsic or acquired chemoresistance being a major constraint. Here, we aimed to identify potential target to reverse such chemoresistance. In the present study, we found significant difference in uridine monophosphate synthetase (UMPS) expression between 5-FU resistant and sensitive HCC cell lines and the overexpression or downregulation of UMPS impacted 5-FU response in HCC cells. We further found that inhibition of UMPS activity with uric acid at concentration present in human plasma decreased the 5-FU sensitivity of HCC cells, while reduction of uric acid levels with uricase improved the 5-FU sensitivity of HCC cells as well as colorectal cancer cells. In vivo studies also suggested that modulation of uric acid levels did affect 5-FU sensitivity of tumors. These data indicated that UMPS was correlated with the 5-FU resistance in HCC cells and uricase sensitized cancer cells to 5-FU through uricase-uric acid-UMP synthase axis, which provided a potential strategy to improve the efficacy of 5-FU-based chemotherapy for human cancers. (AU)