The gut microbiota is essential for good health. It has also been demonstrated that the gut microbiota can regulate immune responses against respiratory tract infections. Since the outbreak of the COVID-19 pandemic, accumulating evidence suggests that there is a link between the severity of COVID-19 and the alteration of one’s gut microbiota. The composition of gut microbiota can be profoundly affected by COVID-19 and vice versa. Here, we summarize the observations of the mutual impact between SARS-CoV-2 infection and gut microbiota composition. We discuss the consequences and mechanisms of the bi-directional interaction. Moreover, we also discuss the immune cross-reactivity between SARS-CoV-2 and commensal bacteria, which represents a previously overlooked connection between COVID-19 and commensal gut bacteria. Finally, we summarize the progress in managing COVID-19 by utilizing microbial interventions.

Metagenomic next-generation sequencing (mNGS) has been a lack of method for pathogen quantitation. We explored the suitable concentration of T ₁ phage [internal control (IC) _phage ], Thermus thermophilus (IC _T.T ), and artificial DNA sequence (IC _DNA ) as mNGS ICs for pathogen quantitation and compared the quantitation efficiency among them. We prepared the simulated cerebrospinal fluid (CSF) samples composed of a pathogen cocktail, containing Staphylococcus aureus , Escherichia coli , Komagataella pastoris , and human cells to test the accuracy, linearity, and interference of IC quantitation. We also collected 15 clinical CSF conducting both mNGS and droplet digital PCR (ddPCR) to further verify the quantification efficacy of IC. In accuracy, the mNGS quantification of pathogen was more precise when the IC was 10 ³ and 10 ⁴ CFU/mL Thermus thermophilus or 10 ³ and 10 ⁴ PFU/mL T ₁ phage with the CV% of pathogens quantification most below 15%. The DNA sequence’ quantification was less accurate with all the CV% of pathogen quantitation above 15%. In linearity, compared to DNA sequence (all R ² 0.9). In interference, the mNGS quantification was affected obviously by human cell concentrations when the IC was DNA sequence ( P < 0.0001), and the quantification was not interfered when the IC was the 10 ³ CFU/mL Thermus thermophilus or 10 ⁴ PFU/mL T ₁ phage . Furthermore, we revealed the mNGS quantitation was highly consistent with ddPCR in clinical CSF according to the linear regression ( R ² = 0.9646, P < 0.0001, k = 0.9362) and Bland-Altman (the bias of average difference is 4.033, with the 95% confidence interval from − 24.61 to 32.68). Thermus thermophilus and T ₁ phages are comparable as mNGS IC in pathogen quantitation and are both superior than artificial DNA sequences. In total, 1,000-CFU/mL Thermus thermophilus as mNGS IC may allow us to reflect directly the variation of pathogens in central nervous system infection patients. IMPORTANCE: Metagenomic next-generation sequencing (mNGS) has been used broadly for pathogens detection of infectious diseases. However, there is a lack of method for the absolute quantitation of pathogens by mNGS. We compared the quantitative efficiency of three mNGS internal controls (ICs) Thermus thermophilus , T1 phages, and artificial DNA sequence and developed the most applicable strategies for pathogen quantitation via mNGS in central nervous system infection. The IC application strategy we developed will enable mNGS analysis to assess the pathogen load simultaneously with the detection of pathogens, which should provide critical information for quick decision-making of treatment as well as clinical prognosis.

Background: Clear cell renal cell carcinoma (ccRCC), the most prevalent kidney cancer subtype, has a high mortality rate. Crystallin lambda 1 (CRYL1) encodes an enzyme that catalyzes the dehydrogenation of L-gulonate into dehydro-L-gulonate in uronate cycle. CRYL1 dysregulation has been linked to the progression of several cancers. This research aimed to evaluate the prognostic significance of CRYL1 expression in ccRCC prognosis. Methods: Clinical data and gene expression profiles on ccRCC were retrieved from the University of California Santa Cruz Xena platform. Differences (variations) in the expression profiles of CRYL1 in ccRCC and healthy tissues were found using RNA-sequencing data, and these findings were validated using qPCR with real-world samples. CRYL1 expression levels were also linked to clinicopathological characteristics, survival, and immune microenvironments. The potential pathway via which CRYL1 expression levels impact the prognosis of patients with ccRCC was investigated using gene set enrichment analysis (GSEA). Results: In ccRCC tissues, CRYL1 expression levels were lower compared to healthy renal tissues in TCGA cohort (n = 535, P < 0.001), which was validated in another real-world cohort (n = 14, P < 0.001). Lower CRYL1 expression levels were linked to unfavorable clinicopathological characteristics and prognoses (P < 0.001). According to multivariate Cox regression analysis (P < 0.001), CRYL1 expression levels in patients with ccRCC could serve as an independent prognostic indicator. Furthermore, a strong link between CRYL1 expression levels and immune microenvironment was observed (P < 0.001). Finally, GSEA revealed that CRYL1 expression levels (P < 0.001) were associated with fatty acid metabolism, G2M checkpoint delays, and epithelial-mesenchymal transitions in ccRCC. Conclusion: Our study found that lower levels of CRYL1 expression were linked to unfavorable clinicopathological characteristics and worse prognoses, and CRYL1 could serve as a new target for the treatment of ccRCC, which is useful for personalized medicine.

Background: pGX9501 is a prophylactic DNA vaccine encoding the spike protein of SARS-CoV-2 and can induce immune response in the human body so as to prevent COVID-19. With respect to non-clinical studies, pGX9501 has been demonstrated to induce both cellular and humoral immune responses in various animal models. It was found that the level of antibody titers following a two-dose regimen was higher than that following a single-dose regimen in nonhuman primate challenge model. Methods: In China, a phase I, randomized, double-blind, placebo-controlled clinical trial has been conducted in Huashan Hospital, Shanghai, China to evaluate the safety, tolerability, and immunogenicity of DNA vaccine pGX9501 administered intradermally (ID) followed by electroporation (EP) in 45 Chinese healthy volunteers aged 18 to 59 years old. Results: No adverse events of special interest (AESIs), death, or treatment-related SAEs occurred in this study. All the treatment-related (vaccine or EP) adverse events (TRAEs) were of grade 1 and 2 in severity. The solicited AEs were reported in thirty-two (32/36, 88.9%) and nine (9/9, 100.0%) subjects, respectively, in the DNA vaccine and placebo group. The frequency of solicited AEs did not increase with vaccine dose level and frequency. The DNA vaccine pGX9501 effectively enhanced both humoral and cellular immune responses in a dose-dependent manner, with increased antibody GMTs and peak seroconversion rates observed on day 42. The significant rise in IFN-γ levels confirmed the vaccine’s ability to induce cellular immune responses. Variations in the microbiome structure suggested a tangible impact of the gut microbiota on vaccine immunogenicity. Conclusions: The findings from this study confirm the immunogenicity and safety of the DNA vaccine pGX9501 and point to the potential role of the gut microbiota in vaccine immune responses. These insights provide practical references for the future design and development of DNA vaccines.

Background: Wastewater-based epidemiology (WBE) is a promising strategy for detecting pathogens and may provide efficient and convenient early-warning signals for infectious disease outbreaks. Throughout the COVID-19 pandemic, monitoring SARS-CoV-2 in wastewater has proven a cost-efficient method for understanding infection spread in communities. Methods: Continuous wastewater surveillance from December 2022 to December 2025 was conducted at two terminal wastewater treatment plants (WWTPs) serving approximately 16 million residents in urban Shanghai, and wastewater surveillance data was compared with clinical data. We developed the Shanghai WBE Warning System (SHWWS), and compared with four existing methods. Findings: Wastewater surveillance tracks five distinct infection waves with high temporal concordance with clinical surveillance, although the wastewater signal generally synchronized with or lagged clinical indicators by days rather than leading them. SHWWS demonstrated favorable early-warning performance with fewer false alarms compared with existing methods (PC, WVAL, MK and RSI). WBE from the terminal WWTPs sequentially identified the major circulating variants and detected the emergence and accumulation of variant-defining mutations before the official designation. Interpretation: Our study demonstrates the potential of WBE at terminal WWTPs to effectively reflect community infection conditions and track viral variant diversity in a megacity, providing a locally calibrated template that may aid public health efforts in monitoring and managing disease outbreaks. Funding: The study was supported by the National Key Research and Development Program of China (2023YFC3043501 and 2024YFC2309201), by Guangzhou National Laboratory (GZNL2024A01007), and by Shanghai Municipal Ecology and Environment Bureau (Grant No.2024-04).