Effect and potential mechanism of nitrite reductase B on nitrite degradation by Limosilactobacillus fermentum RC4.

Nitrite has the potential risk of hypoxic poisoning or cancer in pickled food. In our previous study, Limosilactobacillus fermentum (L. fermentum) RC4 is effective in nitrite degradation by producing nitrite reductase B (NirB). To investigate the detailed mechanism from the genome, response, and regulation of NirB, the whole-genome sequence of L. fermentum RC4 was analyzed, the L. fermentum-EGFP-nirB with enhanced green fluorescent protein (EGFP) labeled the nitrite reductase large subunit nirB, and the recombined L. fermentum-NirB with overexpression NirB strain was conducted. The key genes within the dominant metabolism pathways may be involved in stress tolerance to regulate the degrading process. The green fluorescence density of EGFP indicated that NirB activity has a threshold and peaked under 300 mg/L nitrite concentration. NirB overexpressed in L. fermentum RC4 boosted the enzyme activity by 39.6% and the degradation rate by 10.5%, when fermented in 300 mg/L for 40 h, compared to the control group. RNA-seq detected 248 differential genes mainly enriched in carbohydrate, amino acid, and energy metabolism. The ackA gene for pyruvate metabolism and the mtnN gene for cysteine metabolism were up-regulated. NirB regulates these genes to produce acid and improve stress resistance for L. fermentum RC4 to accelerate nitrite degradation.

Application of a Multiplex Ligation-Dependent Probe Amplification-Based Next-Generation Sequencing Approach for the Detection of Pathogenesis of Duchenne Muscular Dystrophy and Spinal Muscular Atrophy Caused by Copy Number Aberrations.

Both Duchenne muscular dystrophy (DMD; OMIM no. 310200) and spinal muscular atrophy (SMA; OMIM no. 253300/253550/253400/271150) are genetic disorders characterized by progressive muscle degeneration and weakness. Genetic copy number aberrations in the pathogenetic genes DMD and SMN1 lead to alterations in functional proteins, resulting in DMD and SMA, respectively. Multiplex ligation-dependent probe amplification (MLPA) has become a standard method for the detection of common copy number aberrations (CNAs), including DMD and SMN1 deletions, both of which are associated with poor clinical outcomes. However, traditional MLPA assays only accommodate a maximum of 60 MLPA probes per test. To increase the number of targeted sequences in one assay, an MLPA-based next-generation sequencing (NGS) assay has been developed that is based on the standard MLPA procedure, allows high-throughput screening for a large number of fragments and samples by integrating additional indices for detection, and can be analyzed on all Illumina NGS platforms.

Molecular pathology and clinical treatment of independent HPV primary serous carcinoma of the uterine cervix (USCC): A case report.

On October 23, 2020, a 69-year-old Chinese female patient was admitted to Yuncheng Hospital due to a history of postmenopausal bleeding and lower abdominal pain for 5 months. The HPV test and pathology results indicated the presence of independent HPV in primary serous carcinoma of the uterine cervix. The genetic testing identified variants of uncertain significance (PAX8 p.Tyr 410 Ter and TP53 p.Asn 247 Ile), microsatellite instability stable (MSI-S), tumor mutational burden (TMB) 7.33Muts/Mb, and an elevated tumor neoantigen burden. Before undergoing radical hysterectomy treatment, the patient exhibited a positive response to three cycles of intravenous docetaxel (100 mg/3 h) and carboplatin (450 mg/1 h). Following the surgery, she received an additional three cycles of docetaxel (100 mg/3 h) and carboplatin (500 mg/1 h), accompanied by 25 cycles of radiation therapy (DT 46Gy/2Gy/23f). Concurrently, cisplatin (450 mg/1 h) was administered. As of now, the patient has achieved 20 months of disease-free survival.

Targeted treatments after chemoradiotherapy failure in a patient with relapsed, advanced non­small cell lung cancer with on­therapy circulating tumor biomarker monitoring: A case report.

Ongoing investigations of targeted therapeutic agents and their increased clinical applications, together with research in genomics and proteomics, have explored a variety of novel approaches for treatment of lung cancer, and 'molecular subtypes' have been defined based on specific actionable genetic aberrations. Liquid biopsies, including circulating tumor DNA (ctDNA) testing, are of value for cancer diagnosis and comprehensive genomic profiling, such as the identification of cancer subtypes and major genetic alterations in cancer cells. The case of a 66-year-old male patient with newly-diagnosed driver mutation-negative advanced non-small cell lung cancer (NSCLC) who underwent conventional therapy is described in the present report. The patient underwent regular monitoring, including continuous ctDNA analysis, imaging and assessment of tumor marker levels such as carcinoembryonic antigen (CEA). The patient initially presented with deep vein thrombosis which affected both lower extremities and without any symptoms in the lung, with a positron emission tomography scan identifying irregular pulmonary nodules in the right lower lobe and enlarged right supraclavicular lymph nodes. Subsequent ultrasound-guided fine-needle aspiration with nodule biopsy indicated advanced unresectable disease at stage IIIB based on the Tumor-Node-Metastasis staging system by the American Joint Committee on Cancer. Next-generation sequencing of tumor tissue and peripheral blood confirmed driver mutation-negative genes, including epidermal growth factor receptor, rat sarcoma, ALK receptor tyrosine kinase, ROS1 proto-oncogene receptor tyrosine kinase and rearrangement during transfection (RET). After 5 years of chemoradiotherapy and surveillance of ctDNA and CEA levels, detectable kinesin family member 5B (KIF5B)-RET fusion in ctDNA and rising CEA levels prompted early scans, which identified disease progression. The patient subsequently received the oral RET inhibitor pralsetinib, with treatment being currently ongoing for ≥17 months without detectable KIF5B-RET ctDNA or elevated CEA levels, with an ongoing minor response and stable disease based on Response Evaluation Criteria in Solid Tumors v1.1 on imaging. The present case illustrates the potential role of on-therapy circulating tumor biomarker monitoring as a non-traumatic method to evaluate therapy response and detect early disease progression in patients with advanced NSCLC. Integration of circulating tumor biomarker testing into the management of patients with advanced NSCLC requires additional prospective studies to actively assess and elucidate optimal treatment strategies.

Improved diagnosis of colorectal cancer using combined biomarkers including Fusobacterium nucleatum, fecal occult blood, transferrin, CEA, CA19-9, gender, and age.

BACKGROUND: Conventional blood and stool tests are normally used for early screening of colorectal cancer (CRC) but the accuracy and efficiency remain to be improved. Recent findings suggest Fusobacterium nucleatum to be a biomarker for CRC. This study evaluated the role of F. nucleatum and developed CRC diagnostic models by combining F. nucleatum with fecal occult blood (FOB), transferrin (TRF), carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), gender, and age. MATERIALS AND METHODS: Candidates including 71 healthy individuals and 59 CRC patients were recruited. Abundance of F. nucleatum in stool or tissue samples was measured by quantitative real-time PCR. CEA, CA19-9, TRF, and FOB were measured in parallel. These biomarkers together with genders and ages were the seven parameters used to develop CRC diagnostic models. Ten different machine learning algorithms were tested to achieve the best performance. RESULTS: Fecal F. nucleatum abundance was found significantly higher in CRC group compared to healthy group (p = 0.0005). Among the CRC patients, F. nucleatum abundance in tumor tissue was significantly higher than that in paracancerous tissue (p = 0.0087). CRC diagnostic models using different parameters were generated based on Logistic Regression algorithm, which showed good performance. The area under the curve (AUC) score of fecal F. nucleatum as the single diagnostic biomarker was 0.68 while the accuracy was 0.56. The diagnostic performance was obviously improved with the highest AUC (0.93) and accuracy (0.87) achieved when using all the 7 clinical parameters. The combination F. nucleatum + FOB + gender + age had the second highest AUC (0.92) and accuracy (0.85). A more utilitarian model using F. nucleatum + FOB showed relatively high AUC at 0.86 and accuracy at 0.81. CONCLUSIONS: F. nucleatum is valuable for CRC diagnosis. Combination of different clinical parameters could significantly improve CRC diagnostic performance. The combination F. nucleatum + FOB + gender + age may be an effective and noninvasive method for clinical application.

Early detection of gram-negative bacteria using metagenomic next-generation sequencing in acute respiratory distress syndrome: A case report.

Metagenomic next-generation sequencing (mNGS) is an effective method that can be used for the identification of early pathogens in patients with suspected severe pneumonia. However, the potential of mNGS for evaluating the prognosis of acute respiratory distress syndrome (ARDS) in patients with severe pneumonia remains unclear. In the present report, hospital-acquired gram-negative bacteria infections were detected in a case using metagenomic next-generation sequencing (mNGS) in a sample of bronchoalveolar fluid. This was obtained from a 58-year-old male patient with traumatic wet lung after a neurosurgery. According to the results, of which the profiles of the resistance genes were detected by mNGS, drugs designed to control infection were adjusted, namely to polymyxin B (500,000 U/12 h), azithromycin (0.5 g/24 h) and ganciclovir (0.25 g/12 h). Following adjusting treatment for 8 days, the symptoms of lung infection and hypoxemia were markedly improved, resulting in the patient being transferred out of the intensive care unit 15 days after treatment. To conclude, observations from the present report suggest that mNGS is a useful method for the early identification of pathogens in patients with pneumonia caused by ARDS. However, further studies are required to identify the complementary role of mNGS in supporting conventional microbiological methods in routine clinical practice.

Sources, Processing-Related Transformation, and Gut Axis Regulation of Conventional and Potential Prebiotics.

One strategy to achieve a balanced intestinal microbiota is to introduce prebiotics. Some substances present in the diet, such as soybean extracts, koji glycosylceramides, grape extracts, tea polyphenols, and seaweed extracts, can be considered as potential prebiotics, because they can selectively stimulate the proliferation of beneficial bacteria in the intestine. However, the discovery of novel prebiotics also involves advances in screening methods and the use of thermal and non-thermal processing techniques to modify and enhance the properties of beneficial organisms. The health benefits of prebiotics are also reflected by their participation in regulating the microbiota in different gut axes. In the present review, we introduced the field of prebiotics, focusing on potential prebiotic substances, the process of screening potential prebiotics, the transformation of prebiotics by food-processing technologies, and the roles of prebiotics on gut axis regulation, which, it is hoped, will promote the discovery and utilization of novel prebiotics.

Metabolomics Analysis for Nitrite Degradation by the Metabolites of Limosilactobacillus fermentum RC4.

Nitrite (NIT), a commonly used food additive, especially in pickled and cured vegetables and meat products, might cause acute and chronic diseases. Fermentation with lactic acid bacteria (LAB) is an effective method for degrading NIT and improving the flavor of pickled and cured foods. In this study, Limosilactobacillus fermentum (L. fermentum) RC4 with a high NIT degradation ability was found to degrade NIT in a new manner when compared with reported enzymatic and acid degradation, namely, metabolite degradation during fermentation in MRS broth, which shows a synergistic effect with acid to increase NIT degradation. Liquid chromatography-mass spectrometry analysis identified 134 significantly different metabolites, of which 11 metabolites of L. fermentum RC4, namely, γ-aminobutyric acid (GABA), isocitric acid, D-glucose, 3-methylthiopropionic acid (MTP), N-formyl-L-methionine, dimethyl sulfone (MSM), D-ribose, mesaconate, trans-aconitic acid, L-lysine, and carnosine, showed significant NIT degradation effects compared with the control group (MRS broth). Verification experiments showed that adding the above 11 metabolites to 100 mg/L NIT and incubating for 24 h resulted in NIT degradation rates of 5.07%, 4.41%, 6.08%, 16.93%, 5.28%, 2.41%, 0.93%, 18.93%, 12.25%, 6.42%, and 3.21%, respectively. Among these, three metabolites, namely, mesaconate, MTP, and trans-aconitic acid, showed efficient NIT degradation abilities that might be related to the degradation mechanism involving decarboxylation reactions. This is the first systematic study of NIT degradation by LAB, resulting in the identification of a new metabolite degradation pathway and three efficient NIT degradation metabolites.

A multiplex ligation­dependent probe amplification­based next­generation sequencing approach for the detection of copy number variations in the human genome.

The aim of the present study was to describe a multiplex ligation­dependent probe amplification (MLPA)­based next­generation sequencing (NGS) assay that exhibited a significantly higher efficiency in detecting copy number variations (CNVs) and known single­nucleotide variants, compared with traditional MLPA. MLPA polymerase chain reaction products were used to construct a library with indexed adapters, which was subsequently tested on an NGS platform, and the resulting data were analyzed by a series of analytical software. The reads from each probe reflected genetic variations in the target regions, and fragment differentiation was based on the specific base composition of the sequences, rather than fragment length, which was determined by capillary electrophoresis. The results of this approach were not only consistent with the MLPA results following capillary electrophoresis, but also coincided with the CNV results from the single­nucleotide polymorphism array chip. This method allowed high­throughput screening for the number of fragments and samples by integrating additional indices for detection. Furthermore, this technology precisely and accurately performed large­scale detection and quantification of DNA variations, thereby serving as an effective and sensitive method for diagnosing genetic disorders caused by CNVs and known single­nucleotide variations. Notably, MLPA­NGS circumvents the problems associated with the inaccuracies of NGS in CNV detection due to the use of target sequence capture.