Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.

Tropheryma whipplei is the bacterium associated with Whipple’s disease (WD), a chronic systemic infectious disease primarily involving the gastrointestinal tract. T. whipplei can also be detected in different body site of healthy individuals, including saliva and feces. Traditionally, Tropheryma whipplei has a higher prevalence in bronchoalveolar lavage fluid (BALF) of immunocompromised individuals. Few studies have explored the significance of the detection of T. whipplei in BALF. Herein, we retrospectively reviewed 1725 BALF samples which detected for metagenomic next-generation sequencing (mNGS) from March 2019 to April 2022 in Zhuhai, China. Seventy BALs (70/1725, 4.0%) from 70 patients were positive for T. whipplei. Forty-four patients were male with an average age of 50 years. The main symptoms included cough (23/70), expectoration (13/70), weight loss (9/70), and/or dyspnea (8/70), but gastrointestinal symptoms were rare. Chronic liver diseases were the most common comorbidity (n=15, 21.4%), followed by diabetes mellitus (n=13, 18.6%). Only nine patients (12.9%) were immunocompromised. Twenty-four patients (34.3%) were finally diagnosed with reactivation tuberculosis and 15 patients (21.4%) were diagnosed with lung tumors, including 13 primary lung adenocarcinoma and two lung metastases. Fifteen patients (21.4%) had pneumonia. Among the 20 samples, T. whipplei was the sole agent, and Mycobacterium tuberculosis complex was the most common detected other pathogens. Among the non-tuberculosis patients, 31 (31/46, 67.4%) had ground glass nodules or solid nodules on chest CT. Our study indicates that T. whipplei should be considered as a potential contributing factor in some lung diseases. For non-immunocompromised patients, the detection of T. whipplei also needs attention. The mNGS technology improves the detection and attention of rare pathogens. In the future, the infection, colonization, and prognosis of T. whipplei in lung still need to be studied.

The exploration of DNA-binding proteins (DBPs) is an important aspect of studying biological life activities. Research on life activities requires the support of scientific research results on DBPs. The decline in many life activities is closely related to DBPs. Generally, the detection method for identifying DBPs is achieved through biochemical experiments. This method is inefficient and requires considerable manpower, material resources and time. At present, several computational approaches have been developed to detect DBPs, among which machine learning (ML) algorithm-based computational techniques have shown excellent performance. In our experiments, our method uses fewer features and simpler recognition methods than other methods and simultaneously obtains satisfactory results. First, we use six feature extraction methods to extract sequence features from the same group of DBPs. Then, this feature information is spliced together, and the data are standardized. Finally, the extreme gradient boosting (XGBoost) model is used to construct an effective predictive model. Compared with other excellent methods, our proposed method has achieved better results. The accuracy achieved by our method is 78.26% for PDB2272 and 85.48% for PDB186. The accuracy of the experimental results achieved by our strategy is similar to that of previous detection methods.

Biliary tract cancer (BTC), encompassing diseases such as intrahepatic (ICC), extrahepatic cholangiocarcinoma (ECC), and gallbladder cancer, is not only increasing but also poses a significant and urgent health threat due to its high malignancy. Genomic differences point to the possibility that these subtypes represent distinct diseases. Elucidation of the specific distribution of T-cell subsets, critical to cancer immunity, across these diseases could provide better insights into the unique biology of BTC subtypes and help identify potential precision medicine strategies. To address this, we conducted single-cell RNA sequencing and T-cell receptor sequencing on CD3+ T cells from 36 samples from 16 patients with BTC across all subtypes and analyzed 355 pathologic slides to examine the spatial distribution of T cells and tertiary lymphoid structures. Compared with ICC and gallbladder cancer, ECC possessed a unique immune profile characterized by T-cell exhaustion, elevated CXCL13 expression in CD4+ T helper–like and CD8+CXCL13+ exhausted T cells, more mature tertiary lymphoid structures, and fewer desert immunophenotypes. Conversely, ICC displayed an inflamed immunophenotype with an enrichment of IFN-related pathways and high expression of LGALS1 in activated regulatory T cells, associated with immunosuppression. Inhibition of LGALS1 reduced tumor growth and regulatory T-cell prevalence in ICC mouse models. Overall, this study unveils T-cell diversity across BTC subtypes at the single-cell and spatial level that could open paths for tailored immunotherapies. Significance: Single-cell and spatial analyses detailed the T-cell characteristics specific to anatomic subtypes of biliary tract cancer, identifying unique immunologic features that could potentially be harnessed to improve patient outcomes.

Background: With an increase in life expectancy and improvement of surgical safety, more elderly patients with hepatocellular carcinoma (HCC), even with large tumors, are now considered for hepatectomy. This study aimed to clarify the impact of age on short- and long-term outcomes after major hepatectomy (≥ 3 segments) for large HCC (≥ 5 cm). Patients and Methods: Using a multicenter database, patients who underwent curative-intent major hepatectomy for large HCC between 2006 and 2016 were identified. Postoperative morbidity and mortality, overall survival (OS) and recurrence-free survival (RFS) were compared between the elderly (≥ 65 years) and younger (< 65 years) patients. Univariable and multivariable Cox-regression analyses were performed to identify the risk factors of OS and RFS in the entire and elderly cohorts, respectively. Results: Of 830 patients, 92 (11.1%) and 738 (88.9%) were elderly and younger patients, respectively. There were no significant differences in postoperative 30-day mortality and morbidity between the two groups (5.4% vs 2.6% and 43.5% vs 38.3%, both P> 0.05). The 5-year OS and RFS rates in elderly patients were also comparable to younger patients (35.0% vs 33.2% and 20.0% vs 20.8%, both P> 0.05). In the entire cohort, multivariable Cox-regression analyses identified that old age was not independently associated with OS and RFS. However, in the elderly cohort, preoperative alpha-fetoprotein level > 400 μg/L, multiple tumors, macrovascular invasion and microvascular invasion were independently associated with decreased OS and RFS. Conclusion: Carefully selected elderly patients benefited from major hepatectomy for large HCC as much as younger patients, and their long-term prognosis was determined by preoperative alpha-fetoprotein level, tumor number and presence of macro- or micro-vascular invasion.