Objective To explore the presence and potential interactions of microbes and bacteriophages in the blood of healthy individuals by employing in-depth bioinformatics mining to analyze the structure and function of the blood microbi-ome ecosystem.Methods Blood plasma samples from 1 600 voluntary blood donors collected at Mianyang Central Blood Station from 2012 to 2018 were subjected to DNA extraction and library construction.High-throughput sequencing was con-ducted using the Illumina HiSeq 4500 platform,followed by extensive bioinformatics analysis.Microbial abundance in blood samples was analyzed using metagenomic analysis software such as Bowtie2,Trimmomatic and Kraken.Subsequent phage-ome analysis included sequence quality control,assembly,identification,clustering and functional annotation using software such as Megahit,geNomad,CheckV and eggNOG-mapper.Phylogenetic trees,species annotation and host analysis and pre-diction for the identified blood bacteriophages were constructed using iTOL,BLAST and PhaBOX software.Results Met-agenomic sequencing identified microbes across 36 phyla,151 orders,338 families,338 genera and 3 757 species in the plasma samples.At the species level,the most abundant species included Bacillus cereus,Lactobacillus murinus,L.johnso-nii,Faecalibacterium prausnitzii,B.thuringiensis,L.reuteri,Cutibacterium acnes,Dietzia sp.JS16-p6b,Mycoplasma hyo-rhinis,M.hyopneumoniae and Staphylococcus aureus.Through phageome analysis,202 viral Operational Taxonomic Units(vOTUs)were identified,revealing 24 types of bacteriophages.Host analysis using the viral host database completed mat-ches for 15 potential bacteriophage hosts,including Stenotrophomonas maltophilia,Rhodoferax lacus,Pseudoalteromonas marina,Thalassotalea loyana,Vibrio alginolyticus,V.tasmaniensis,V.vulnificus,Pseudomonas sp.,Agrobacterium sp.ST15.13/040,Enterococcus gallinarum,Flavobacterium sp.,Thermotoga naphthophila,Chryseobacterium sp.RU33C,L.acidipiscis and Neisseria mucosa.Conclusion The study of the healthy human blood microbiome and phageome reveals the presence of microbes and phages in the blood,which may have profound impacts on human health.

OBJECTIVE: To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology. METHODS: We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated. RESULTS: This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively. CONCLUSION By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.
Humans ; COVID-19 ; CRISPR-Cas Systems ; Genotype ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/genetics* ; RNA ; COVID-19 Testing

Cancer stands as one of the predominant causes of mortality globally, necessitating ongoing efforts to develop innovative therapeutics. Historically, natural products have been foundational in the quest for anticancer agents. Bulbocodin D (BD) and Bulbocodin C (BC), two bibenzyls derived from Pleione bulbocodioides (Franch.) Rolfe, have demonstrated notable in vitro anticancer activity. In human lung cancer A549 cells, the IC_50s for BD and BC were 11.63 and 11.71 μmol·L^-1, respectively. BD triggered apoptosis, as evidenced by an upsurge in Annexin V-positive cells and elevated protein expression of cleaved-PARP in cancer cells. Furthermore, BD and BC markedly inhibited the migratory and invasive potentials of A549 cells. The altered genes identified through RNA-sequencing analysis were integrated into the CMap dataset, suggesting BD's role as a potential signal transducer and activator of transcription 3 (STAT3) inhibitor. SwissDock and MOE analyses further revealed that both BD and BC exhibited a commendable binding affinity with STAT3. Additionally, a surface plasmon resonance assay confirmed the direct binding affinity between these compounds and STAT3. Notably, treatment with either BD or BC led to a significant reduction in p-STAT3 (Tyr 705) protein levels, regardless of interleukin-6 stimulation in A549 cells. In addition, the extracellular signal-regulated kinase (ERK) was activated after BD or BC treatment. An enhancement in cancer cell mortality was observed upon combined treatment of BD and U0126, the MEK1/2 inhibitor. In conclusion, BD and BC emerge as promising novel STAT3 inhibitors with potential implications in cancer therapy.
Humans ; Lung Neoplasms/metabolism* ; STAT3 Transcription Factor/metabolism* ; Antineoplastic Agents/chemistry* ; A549 Cells ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation

Objective:To learn about the characteristics of natural foci and the spatial distribution of mosquitoes and ticks in Yadong County, Tibet Autonomous Region (Tibet for short).Methods:The eco-geographical characteristics, demographic information, agricultural and animal husbandry data of the natural foci in Yadong County, Tibet were collected from the Yadong County 2020 statistical yearbook, vector data were collected from the Yadong County Center for Disease Control and Prevention, and the data of the meteorological station in Yadong County from 2010 to 2021 were collected from the World Weather Network. In July 2021, a total of 20 mosquito and tick sampling points were selected for field investigation according to the distribution characteristics of vegetation and animal husbandry of Yadong County. The light trap method was used to trap mosquitoes, and the manual inspection and flag dragging method were used to catch ticks. The spatial distribution characteristics of mosquitoes and ticks were analyzed.Results:The natural epidemic foci in Yadong County, Tibet, were high in the north and low in the south, with an elevation difference of about 5 000 m. It was rich in water resources, and the average runoff of Yadong River was 20.1 m 3/s. The climate in the north was cold and dry, and the south was mild and humid. From 2010 to 2021, the annual average station air pressure in Yadong County was 452.8 mmHg (1 mmHg = 0.133 kPa), the maximum monthly average temperature, precipitation and relative humidity of air were 8.7 ℃, 134.5 mm and 81.3%, respectively, all in July. A total of 6 897 diptera insects were attracted by the light trap method, all of which were non-blood-sucking mosquitoes. The total density was 163.77 pieces/(lamp·h), the dominant population was Muscaridae, accounting for 89.69% (6 186/6 897). In different sampling areas, Xiayadong Township collected the most mosquitoes, accounting for 67.17% (4 633/6 897); the others were Yadong County and Shangyadong Township, accounting for 27.36% (1 887/6 897) and 5.47% (377/6 897), respectively. A total 2 014 host animals were examined, and 23 parasitic ticks were caught, of which 20 were of the genus Ixodes and 3 were of the genus Haemophilus. No free ticks were caught in all ticks sampling points. Conclusions:The climate and ecological environment of the natural foci in Yadong County, Tibet have obvious vertical gradient. Mosquitoes and ticks are active, but the density is not high, the density of mosquitoes is generally high in the south and low in the north.

Objective:To understand the pathogen infection and epidemiological characteristics of children with diarrhea in Tianjin.Methods:Stool samples from 1 466 children with diarrhea in Tianjin Children's Hospital from August 2017 to July 2018 were collected, and all samples were tested for five intestinal-related pathogens (norovirus, rotavirus, Clostridium difficile toxin, adenovirus, and astrovirus). Results:Among the 1 466 samples, 627 samples were positive for nucleic acid detection of intestinal pathogens, with a positive rate of 42.8%. The detection rate of norovirus was the highest (26.3%), followed by rotavirus (15.3%), Clostridium difficile toxin (4.6%), adenovirus (4.1%), and astrovirus (1.84%). The infection has obvious seasonality. The positive detection rates of the five pathogens were similar among children of different sexes, and only the positive detection rates of norovirus and rotavirus were statistically significant among different ages ( P<0.05). There were 110 cases of mixed infection, and the mixed infection of norovirus and rotavirus was the most common, with a total of 37 cases. Conclusions:The pathogen spectrum of infant infectious diarrhea in Tianjin is complex and diverse, and the main pathogens are norovirus and rotavirus.

Objective:To investigate the molecular epidemiologic and clinical characteristics of norovirus (NoV) in hospitalized children with acute gastroenteritis (AGE) in Tianjin to provide theoretical basis for clinical diagnosis and treatment.Methods:Fecal specimens were collected from children with AGE in Tianjin Children′s Hospital from August 2017 to July 2020. NoV was detected by real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR). Partial sequence of the capsid protein VP1 of positive samples was amplified by conventional RT-PCR. The products were sent for sequencing and genotyping based on the sequence. As the same time, the clinical symptoms were compared among children infected with different NoV genotypes.Results:Among the 6 432 specimens, 1 703 (26.5%) were positive for NoV. Sequence analysis showed that 761 identified NoV strains could be divided into 7 genotypes. Genotypes GII.4, GII.3, GII.2, GII.17, GII.1, GII.13 and GII.6 accounted for 55.5% (422/761), 36.7% (279/761), 4.9% (37/761), 0.9% (7/761), 0.8% (6/761), 0.8% (6/761) and 0.5% (4/761), respectively. All of the GII.4 belonged to GII.4 Sydney 2012. There was also a significant difference in the seasonal distribution of children infected with GII.4 and other genotypes ( χ2=103.53, P<0.001). Children infected with GII.4 NoV were more likely to have diarrhea, vomiting and dehydration ( χ2=8.42, P=0.004; χ2=20.39, P<0.001; χ2=4.99, P=0.025). Conclusions:GII.4 and GII.3 were mainly genotypes of NoV infection in Tianjin from 2017 to 2020 and genotypes were diverse. In addition, children with GII.4 NoV were more likely to suffer from diarrhea, vomiting and dehydration.

Objective:To investigate the molecular epidemiological characteristics of norovirus (NoV) in hospitalized children with sporadic acute gastroenteritis in Tianjin in 2019.Methods:Fecal specimens and clinical data were collected from 3 116 hospitalized children with sporadic acute gastroenteritis possibly caused by viral infection in Tianjin Children′ Hospital between January and December, 2019. Real-time quantitative PCR was used to detect NoV. Partial sequences of RNA-dependent RNA polymerase (RdRp) and capsid genes of NoV were amplified by RT-PCR. Sequence alignment and phylogenetic analysis were performed for further analysis.Results:Among the 3 116 specimens, 809 (26.0%) were positive for NoV. There were significant differences in NoV detection rate between different age groups ( P=0.000), and the highest NoV detection rate (31.6%) was observed in the age group of 7-12 months. Moreover, the detection rate of NoV varied with seasons ( P=0.000), and the NoV detection rate was highest in winter (39.0%). Based on the sequence analysis of RdRp and capsid genes, 286 identified NoV strains belonged to six genotypes, which were GⅡ.P12-GⅡ.3, GⅡ.P16-GⅡ.2, GⅡ.P17-GⅡ.17, GⅡ.Pe-GⅡ.2, GⅡ.Pe-GⅡ.3 and GⅡ.Pe-GⅡ.4. The predominant genotype was GⅡ.Pe-GⅡ.4 Sydney 2012 (61.2%), followed by GⅡ.P12-GⅡ.3 (33.6%, 96/286), GⅡ.Pe-GⅡ.3 (2.4%, 7/286), GⅡ.P16-GⅡ.2 (2.1%, 6/286), GⅡ.Pe-GⅡ.2 (0.3%, 1/286) and GⅡ.P17-GⅡ.17 (0.3%, 1/286). Patients carrying the NoV of GⅡ.Pe-GⅡ.4 Sydney 2012 genotype were likely to suffer from vomiting than those positive for NoV of GⅡ.P12-GⅡ.3 genotype. Conclusions:NoV was an important pathogen causing acute gastroenteritis in children. GⅡ.Pe-GⅡ.4 Sydney 2012 and GⅡ.P12-GⅡ.3 were the major genotypes of NoV in hospitalized children with sporadic acute gastroenteritis in Tianjin in 2019.

Objective:To investigate the prevalence and clinical characteristics of Mycoplasma pneumoniae( Mp) genotypes and subtypes in children in Tianjin. Methods:Children with pneumonia admitted to Tianjin Children′s Hospital from December 2017 to December 2019 were selected as the research objects. Bronchoalveolar lavage fluid was collected by fiberoptic bronchoscopy. The positive samples were detected by real-time fluorescent quantitative PCR and Mp culture. PCR-restriction fragment length polymorphism(RFLP) and multiple variable number tandem repeats were used for genotyping. Detailed clinical and laboratory data were collected for all cases. Results:The results of RFLP showed that there were 138 cases (78.9%) of typeⅠand 37 cases (21.1%) of type Ⅱ; 37 cases of type M3-5-6-2, including six subtypes B, G, M, S, V and Y; 138 cases of M4-5-7-2 were detected, including seven subtypes of E, J, P, U, X, Z and a. In M3-5-6-2 type, there were 1 case of P1-Ⅰtype (2.7%), 36 cases of P1-Ⅱtype (97.3%), 137 cases of P1-Ⅰ type (99.2%) and 1 case of P1-Ⅱ type (0.7%) in M4-5-7-2 type. There was no significant difference in genotype distribution among different age groups. There were statistical differences in the distribution of four seasons among the 13 genotypes of B, G, M, S, V, Y and E, J, P, U, X, Z, a. All Mp infected children had symptoms of fever and cough. The hospitalization time, fever duration, high fever (>39℃), cough duration, skin changes, digestive system symptoms and liver function injury rate of P1-Ⅰ/M4-5-7-2 pneumonia children were higher than those of P1-Ⅱ/M3-5-6-2 pneumonia children, but the difference was not statistically significant. The WBC count of P1-Ⅱ/M3-5-6-2 types was higher than that of typeⅠand M4-5-7-2; the LDH of P1-Ⅰ/M4-5-7-2 was higher than that of Ⅱ and M3-5-6-2, with statistical difference. There was no significant difference in the incidence of inflammatory consolidation, atelectasis, pleural thickening and pleural effusion among different genotypes. Conclusions:Mp infection in children with pneumonia in Tianjin is mainly P1-Ⅰ/ M4-5-7-2, and P1-Ⅱ is on the rise. P1-Ⅰ and M4-5-7-2 were associated with fever and severe symptoms.

Objective:To analyze the epidemiological characteristics and molecular classification of Human adenovirus (HAdV) and Human bocavirus (HBoV) infection in hospitalized children with acute respiratory infection in Tianjin Children′s Hospital.Methods:A total of 1 171 nasopharyngeal aspirates were collected from children with acute respiratory infection in Tianjin Children′s Hospital from March 2019 to February 2020. The specific primers designed by gene sequence were amplified by polymerase chain reation (PCR), and the positive amplification products were determined by sequencing. The sequences of HAdV and HBoV were compared in GenBank, molecular typed and phylogenetic tree analyzed of HAdV by MEGA7.0.26. The positive rate of HAdV and HBoV in different age groups(<6 months, 6-11 months, 12-23 months, 24-35 months, 36-47 months, ≥48 months) and seasons were compared by SPSS20.0.Results:Thirty HAdV were detected in 1 171 specimens, with a positive rate of 2.56% (30/1 171) and 84 cases with HBoV, with a positive rate of 7.17% (84/1 171).The positive detection rates of HAdV and HBoV in different age groups were 1.02% (4/392)-6.61% (8/121) and 4.09% (7/171)-11.45% (26/227), respectively. There was a significant difference in the positive detection rate of HAdV and HBoV in each age group (χ2=12.862, P=0.025; χ2=14.178, P=0.015).Winter is the peak period of HAdV infection, with a positive rate of 5.54% (15/271). The peak of HBoV infection is autumn and winter with a positive rate of 12.00% (36/300) and 12.5% (34/271), respectively, higher than that of the other two seasons (χ2=43.753, P<0.05). There was a significant difference in different season groups (χ2=13.287, P=0.004; χ2=43.753, P<0.05). The sequences of 29 adenoviruses were HAdV-3, 7 serotypes of HAdV-B subgroup and HAdV-1, 2, 5 serotypes of HAdV-C subgroup. Conclusion:HAdV and HBoV play important roles in children′s respiratory tract infections, and are closely related to factors such as the season and the age of the child. They should attract clinical attention.

Objective:To evaluate the prevalence of human rhinovirus (HRV) infection in hospitalized children in Tianjin and investigate the clinical impact of HRV infections.Methods:From July 2017 to December 2019, 2 945 nasopharyngeal secretion specimens were screened for HRV using polymerase chain reaction (PCR). VP4/VP2 sequences of HRV were further characterized. The clinical characteristics of the HRV infection were analyzed. The detection results of HRV for different groups and different months were compared using SPSS 19.0.Results:HRV-positive specimens accounted for 8.15% (240/2 945), of which 74.78% (86/115) were diagnosed with pneumonia, 40.83%(98/240) had co-infections with other common pathogens. HRV infections could be detected throughout the year with peaks in spring (11.00%, 66/660) and autumn (9.29%, 81/872). The positive rate of HRV was 4.14%(29/700) in winter. By VP4/VP2 sequence analysis, HRV-A was the most frequently detected strain(50.00%, 78/156), followed by HRV-C (41.67%, 65/156).46.15% (30/65) of HRV-C infections occurred in October and November. There were several different HRV-A types and HRV-C types. The most commonly detected HRV-A types were A12(11.54%, 9/78), A49(6.41%, 5/78), A22, A101, and A66(5.13%, 4/78), etc. The most common HRV-C types were C2(20.00%, 13/65), C22(9.23%, 6/65), C26, C43, C54 and C53(4.62%,3/65). Patients with HRV-A infections are more likely to show fever symptoms than HRV-C (χ2=5.411, P<0.05). No significant difference in other symptoms were found between the two types. Conclusions:HRV was a commonly detected virus among infants and had a clear seasonal distribution. It′s also possible for the HRV patients to have co-infections with other pathogens.HRV showed high genetic diversity.