[Application and Prospect of Nanopore Sequencing Technology in Etiological Diagnosis of Blood Stream Infection].

Blood stream infection (BSI),a blood-borne disease caused by microorganisms such as bacteria,fungi,and viruses,can lead to bacteremia,sepsis,and infectious shock,posing a serious threat to human life and health.Identifying the pathogen is central to the precise treatment of BSI.Traditional blood culture is the gold standard for pathogen identification,while it has limitations in clinical practice due to the long time consumption,production of false negative results,etc.Nanopore sequencing,as a new generation of sequencing technology,can rapidly detect pathogens,drug resistance genes,and virulence genes for the optimization of clinical treatment.This paper reviews the current status of nanopore sequencing technology in the diagnosis of BSI.

Rapid and Accurate Detection of SARS Coronavirus 2 by Nanopore Amplicon Sequencing.

Objective: We aimed to evaluate the performance of nanopore amplicon sequencing detection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples. Method: We carried out a single-center, prospective cohort study in a Wuhan hospital and collected a total of 86 clinical samples, including 54 pharyngeal swabs, 31 sputum samples, and 1 fecal sample, from 86 patients with coronavirus disease 2019 (COVID-19) from Feb 20 to May 15, 2020. We performed parallel detection with nanopore-based genome amplification and sequencing (NAS) on the Oxford Nanopore Technologies (ONT) minION platform and routine reverse transcription quantitative polymerase chain reaction (RT-qPCR). In addition, 27 negative control samples were detected using the two methods. The sensitivity and specificity of NAS were evaluated and compared with those of RT-qPCR. Results: The viral read number and reference genome coverage were both significantly different between the two groups of samples, and the latter was a better indicator for SARS-CoV-2 detection. Based on the reference genome coverage, NAS revealed both high sensitivity (96.5%) and specificity (100%) compared with RT-qPCR (80.2 and 96.3%, respectively), although the samples had been stored for half a year before the detection. The total time cost was less than 15 h, which was acceptable compared with that of RT-qPCR (∼2.5 h). In addition, the reference genome coverage of the viral reads was in line with the cycle threshold value of RT-qPCR, indicating that this number could also be used as an indicator of the viral load in a sample. The viral load in sputum might be related to the severity of the infection, particularly in patients within 4 weeks after onset of clinical manifestations, which could be used to evaluate the infection. Conclusion: Our results showed the high sensitivity and specificity of the NAS method for SARS-CoV-2 detection compared with RT-qPCR. The sequencing results were also used as an indicator of the viral load to display the viral dynamics during infection. This study proved the wide application prospect of nanopore sequencing detection for SARS-CoV-2 and may more knowledge about the clinical characteristics of COVID-19.

GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma.

BACKGROUND: Aberrant TAK1 (transforming growth factor ß-activated kinase 1) activity is known to be involved in a variety of malignancies, but the regulatory mechanisms of TAK1 remain poorly understood. GRAMD4 (glucosyltransferase Rab-like GTPase activator and myotubularin domain containing 4) is a newly discovered p53-independent proapoptotic protein with an unclear role in HCC (hepatocellular carcinoma). RESULTS: In this research, we found that GRAMD4 expression was lower in HCC samples, and its downregulation predicted worse prognosis for patients after surgical resection. Functionally, GRAMD4 inhibited HCC migration, invasion and metastasis. Mechanistically, GRAMD4 interacted with TAK1 to promote its protein degradation, thus, resulting in the inactivation of MAPK (Mitogen-activated protein kinase) and NF-κB pathways. Furthermore, GRAMD4 was proved to recruit ITCH (itchy E3 ubiquitin protein ligase) to promote the ubiquitination of TAK1. Moreover, high expression of TAK1 was correlated with low expression of GRAMD4 in HCC patients. CONCLUSIONS: GRAMD4 inhibits the migration and metastasis of HCC, mainly by recruiting ITCH to promote the degradation of TAK1, which leads to the inactivation of MAPK and NF-κB signalling pathways.

Investigation of the effect of oxidation on the structure of ß-lactoglobulin by high resolution mass spectrometry.

In this work, high-resolution mass spectrometry was used to identify the oxidation sites and forms of ß-lactoglobulin (ß-Lg) induced by hydrogen peroxide with 1.5% concentration, and the influence of oxidation sites on the structure of ß-Lg was discussed from the molecular level. Twelve kinds of oxidation products and 36 oxidation sites were identified, including sulfoxidation in sulfur-containing amino acid residue, hydroxylation in aromatic group residue, deamination in amino-containing amino acid etc. The destruction of hydrogen bonds and disulfide bonds in ß-Lg caused by oxidation is the main factor causing its structural changes, which were manifested in the decrease of ß-sheet component and increase of ß-turns and random coil contents, intrinsic fluorescence intensity and surface hydrophobicity. In addition, several peptides as potential oxidative markers were found to be capable of monitoring the degree of oxidation of ß-Lg. In short, this work provided insights into structural changes of ß-Lg by oxidation.

Mechanism of Selenium Nanoparticles Inhibiting Advanced Glycation End Products.

Selenium nanoparticles (SeNPs) have been applied in fields of nanobiosensors, environment, nanomedicine, etc. as a result of their excellent characteristics. Early studies had shown that SeNPs have certain inhibition ability against glycation, but the inhibition mechanism, especially for the influence of SeNPs on the reaction activity of glycation sites, remains unclear. The aim of the presented research was to reveal the effects of SeNPs on the ß-lactoglobulin (ß-Lg)/d-ribose glycation system at the molecular level and explore the possible inhibitory mechanism of SeNPs on the formation of advanced glycation end products (AGEs) by analyzing the glycation sites via high-performance liquid chromatography (HPLC)-Orbitrap-tandem mass spectrometry (MS/MS). Changes in contents of AGE formation and free amino acid contents had indicated that SeNPs could significantly slow the glycation process, thus attenuating the formation of AGEs. HPLC-Orbitrap-MS/MS analysis revealed that, at 6, 12, and 24 h, the number of glycation sites of glycated ß-Lg decreased from 7, 7, and 9 to 5, 5, and 6 after the intervention of SeNPs, respectively. The glycation extent of each glycation site was controlled, and the dual-glycation ability of K8, K14, K47, K91, and K101 was changed. All of these results confirmed that SeNPs could indeed slow the process of protein glycation at the molecular level. This may be the reason for SeNPs reducing the formation of AGEs during glycation. Therefore, this study shed light on the insight of how SeNPs reduce the formation of AGEs.

Mechanism of the effect of 2, 2'-azobis (2-amidinopropane) dihydrochloride simulated lipid oxidation on the IgG/IgE binding ability of ovalbumin.

In this work, the mechanism of the effect of lipid oxidation on the IgG/IgE binding ability of ovalbumin (OVA) was investigated via the peroxyl radicals produced by 2, 2'-azobis (2-amidinopropane) dihydrochloride to simulate lipid oxidation. Results showed that the structure of OVA unfolded partially with an increase in oxidation degree, leading to the exposure of the allergenic epitopes and increasing the IgG/IgE binding ability of OVA. Nine oxidation sites were found on the α-helix, and these sites may unwind the α-helix and expose the allergenic epitopes on the OVA surface, leading to antibody recognition and combination. Consequently, the IgG/IgE binding ability of OVA was increased. In conclusion, the allergenic capacity of OVA can be promoted by modifying peroxyl radical oxidation in processing egg products.

Prevention and Control of Pathogens Based on Big-Data Mining and Visualization Analysis.

Morbidity and mortality caused by infectious diseases rank first among all human illnesses. Many pathogenic mechanisms remain unclear, while misuse of antibiotics has led to the emergence of drug-resistant strains. Infectious diseases spread rapidly and pathogens mutate quickly, posing new threats to human health. However, with the increasing use of high-throughput screening of pathogen genomes, research based on big data mining and visualization analysis has gradually become a hot topic for studies of infectious disease prevention and control. In this paper, the framework was performed on four infectious pathogens (Fusobacterium, Streptococcus, Neisseria, and Streptococcus salivarius) through five functions: 1) genome annotation, 2) phylogeny analysis based on core genome, 3) analysis of structure differences between genomes, 4) prediction of virulence genes/factors with their pathogenic mechanisms, and 5) prediction of resistance genes/factors with their signaling pathways. The experiments were carried out from three angles: phylogeny (macro perspective), structure differences of genomes (micro perspective), and virulence and drug-resistance characteristics (prediction perspective). Therefore, the framework can not only provide evidence to support the rapid identification of new or unknown pathogens and thus plays a role in the prevention and control of infectious diseases, but also help to recommend the most appropriate strains for clinical and scientific research. This paper presented a new genome information visualization analysis process framework based on big data mining technology with the accommodation of the depth and breadth of pathogens in molecular level research.

Postoperative adjuvant transarterial chemoembolization for multinodular hepatocellular carcinoma within the Barcelona Clinic Liver Cancer early stage and microvascular invasion.

BACKGROUND: The survival benefit of postoperative adjuvant transcatheter arterial chemoembolization (PA-TACE) remained controversial. We aimed to investigate the prognosis effect of PA-TACE on the Barcelona Clinic Liver Cancer (BCLC) early stage multinodular hepatocellular carcinoma (MHCC) patients with/without microvascular invasion (MVI). METHODS: Two hundred and seventy-one patients from January 2010 to December 2014 undergoing curative hepatectomy were included in this study. Disease-free survival (DFS) rates and overall survival (OS) rates as well as prognostic factors were analyzed by the Kaplan-Meier method, the log-rank test and the Cox proportional hazard model. RESULTS: Thirty-four point four percent (44/128) MVI positive and 55.2% (79/143) MVI negative patients underwent PA-TACE. Multivariate analysis revealed that HBV DNA load >103 copy/mL, >three tumors, MVI, and without PA-TACE were independent risk factors for poor DFS. Higher alkaline phosphatase (ALP), three tumors, MVI, and without PA-TACE were independent risk factors for poor OS. Both DFS and OS were significantly improved in patients with MVI who received PA-TACE as compared to those who underwent hepatic resection alone (5-year DFS, 26.3% vs. 20.7%, P=0.038; 5-year OS, 73.6% vs. 47.7%, P=0.005). No differences were noted in DFS and OS among MVI negative patients with or without PA-TACE (5-year DFS, 33.7% vs. 33.0%, P=0.471; 5-year OS, 84.1% vs. 80.3%, P=0.523). Early recurrence was more likely to occur in patients without PA-TACE (P=0.001). CONCLUSIONS: PA-TACE was a safe intervention and could effectively prevent tumor recurrence and improve the survival of the BCLC early stage MHCC patients with MVI.

Genotyping of Klebsiella Pneumonia Strains Isolated from Eldly Inpatients by Multiple-locus Variable-number Tandem-repeat Analysis.

Objective To investigate the genotype of klebsiella pneumonia strains isolated from eldly inpatients by multiple-locus variable-number tandem-repeat analysis. Methods Totally 184 klebsiella pneumonia strains,isolated from eldly inpatients,were collected,and their genome DNA were extracted. The polymorphism of 7 variable-number tandem-repeat locus in the DNA samples was analyzed by multiple primers polymerase chain reaction and capillary electrophoresis. The clustering analysis of genotyping was carried out with the BioNumerics 5.1 software. Results A total of 139 genotypes were identified in 184 klebsiella pneumonia clinical strains,showing obvious genetic polymorphisms. With clustering analysis of genotypes,all the strains were categorized into three gene clusters (genogroups 1,2,and 3). The genogroup 1 was the biggest cluster,containing 93.06% of the isolated strains. Conclusion There was a predominant cluster in the klebsiella pneumonia strains isolated from eldly inpatients in our center,and the major source of klebsiella pneumonia infection remained the nosocomial infection.

Rapid Identification of Bacterial Species Associated with Bronchiectasis via Metagenomic Approach.

Bronchiectasis is a chronic lung disorder and a number of bacterial pathogens are involved. However, 30%-40% of sputum and purulent samples in good quality failed to grow any pathogenic bacteria, making it difficult to confirm the pathogen. In this study, we collected bronchoalveolar lavage fluid from a bronchiectasis patient undergoing acute exacerbation, and sent for 16S rDNA pyrosequencing by a 454 GS Junior machine. Metagenomic analysis showed the composition of bacterial community in sample was complex. More than a half of reads (51.3%) were from Pseudomonas aeruginosa. This result was corresponding with the culture result but came out 2 d earlier, which is meaningful for early diagnosis and treatment. The detection with 16S rDNA pyrosequencing technology is more sensitive and rapid than routine culture, and can detect the co-infection or symbiosis in airway, giving us a novel and convenient approach to perform rapid diagnosis.

Identification and molecular analysis of pathogenic yeasts in droppings of domestic pigeons in Beijing, China.

Feral pigeons are known as reservoirs of pathogenic yeasts that cause opportunistic infections in human. In the outskirts of Beijing, China, pigeons are more frequently raised at homes than are encountered in public areas. Many studies have focused on the presence of pathogenic yeasts in the excreta (fresh or withered) of a variety kinds of birds, pigeon crop and cloacae. One hundred and forty-three samples of fresh droppings were collected from three suburban pigeon-raising homes in an area of northern Beijing, China. The internal transcribed sequences (ITS) of all strains (except for 8 strains of Rhodotorula sp. ) were sequenced and compared with those of the databases of the National Center for Biotechnology Information website ( http://www.ncbi.nlm.nih.gov ) using the Basic Local Alignment Search Tool (BLAST). Yeasts representing 8 genera, Cryptococcus, Filobasidium, Rhodotorula, Candida, Debaryomyces, Saccaromyces, Trichosporon and Sporidiobolus, were identified from 120 isolates. Cryptococcus was the most prolific genera represented by eight species. The populations of yeast species isolated from fresh pigeon droppings were different among homes. Although it is well established that Cryptococcus neoformans exists mainly in old pigeon guano, several C. neoformans strains were still isolated from fresh pigeon excreta, providing a clue that live cryptococcal cells could move through the gastrointestinal tract of the pigeons. Eight genera identified from fresh droppings of domestic pigeons further confirm that pigeons serve as reservoirs, carriers and even spreaders of Cryptococcus species and other medically significant yeasts. The proportion of pathogenic yeasts in all isolates is more than 90 %.

[Multilocus sequence typing and pulsed-field gel electrophoresis analysis of Salmonella Paratyphi A isolates from 2000 to 2008, China].

OBJECTIVE: To analyze molecular and evolution characteristics of Salmonella Paratyphi A isolates from 2000 to 2008, China. METHODS: Using pulsed-field gel electrophoresis (PFGE) method with SpeI restriction enzyme, and multilocus sequence typing (MLST) method based on housekeeping genes (aroC, thrA, hisD, purE, sucA, dnaN, hemD, adk, and purA), the genomic variations of 118 Salmonella Paratyphi A isolates from 10 regions during 2000 to 2008 were analyzed. RESULTS: Using PFGE method, 118 Salmonella Paratyphi A isolates were clustered into 32 PFGE patterns, and 5 patterns were predominant (5 isolates or above). However, only 2 MLST types were identified for all isolates with MLST method. Among all Salmonella Paratyphi A isolates, the sequences of housekeeping genes were highly conservative and showed a high degree of cloning. CONCLUSION: For Chinese epidemic Salmonella Paratyphi A isolates during 2000 - 2008, MLST method showed low discrimination power and the MLST method should not be applied to outbreak and epidemiological surveillance of Salmonella Paratyphi A. Currently, nationwide paratyphoid fever epidemics is caused by highly clonal isolates in China. As the time changes, these isolates also accumulate sporadic mutations.