Genetic diversity, population structure, and DNA fingerprinting of Ailanthus altissima var. erythrocarpa based on EST-SSR markers.

Ailanthus altissima var. erythrocarpa is an A. altissima variety with high economic, ecological and ornamental value, but there have been no reports on the development of SSR primers for it. According to the SSR primer information provided by the transcriptome of A. altissima var. erythrocarpa, 120 individuals with different redness levels were used to screen polymorphic primers. Transcriptomic analysis revealed 10,681 SSR loci, of which mononucleotide repeats were dominant (58.3%), followed by dinucleotide and trinucleotide repeats (16.6%, 15.1%) and pentanucleotide repeats (0.2%). Among 140 pairs of randomly selected primers, nineteen pairs of core primers with high polymorphism were obtained. The average number of alleles (Na), average number of effective alleles (Ne), average Shannon's diversity index (I), average observed heterozygosity (Ho), average expected heterozygosity (He), fixation index (F) and polymorphic information content (PIC) were 11.623, 4.098, 1.626, 0.516, 0.696, 0.232 and 0.671, respectively. Nineteen EST-SSR markers were used to study the genetic diversity and population structure of A. altissima var. erythrocarpa. The phylogenetic tree, PCoA, and structure analysis all divided the tested resources into two categories, clearly showing the genetic variation between individuals. The population showed high genetic diversity, mainly derived from intraspecific variation. Among nineteen pairs of primers, 4 pairs (p33, p15, p46, p92) could effectively distinguish and be used for fingerprinting of the tested materials. This study is of great significance for genetic diversity analysis and molecular-assisted breeding of A. altissima var. erythrocarpa.

Effects of metronidazole on colorectal cancer occurrence and colorectal cancer liver metastases by regulating Fusobacterium nucleatum in mice.

OBJECTIVE: Colorectal cancer (CRC) represents a leading cause of cancer-related deaths. Metronidazole (MNZ) is exceedingly implicated in CRC. This study explored the roles of MNZ in mouse CRC occurrence and liver metastasis (CRLM). METHODS: Male BALB/c nude mice were subjected to CRC and CRLM modeling, orally administration with MNZ (1 g/L) 1 week before modeling, and disease activity index (DAI) evaluation. Fresh stool and anal swab samples were collected on the morning of the 28th day after modeling. The relative expression of Fusobacterium nucleatum (F. nucleatum) DNA was assessed by quantitative polymerase chain reaction. After euthanasia, tumor tissues and liver tissues were separated and the tumor volume and weight change were measured. The liver tissues were stained with hematoxylin-eosin to quantitatively analyze the metastatic liver nodules. Malignant tumor biomarker Ki67 protein levels in liver tissues/DNA from stool samples were detected by immunohistochemistry/high-throughput 16S rRNA gene sequencing. Bioinformatics analysis was performed on the raw sequence data to analyze microbial community richness (Chao1 index, ACE index) and microbial community diversity (Shannon index). RESULTS: The DAI and F. nucleatum DNA relative expression in feces and anal swabs of the CRC and CRLM groups were raised and repressed after MNZ intervention. MNZ repressed tumor occurrence and growth in mice to a certain extent, alleviated CRLM malignant degree (reduced liver metastases and Ki67-positive cell density/number), and suppressed CRC liver metastasis by regulating intestinal flora structure, which affected the intestinal characteristic flora of CRC and CRLM mice. CONCLUSION: MNZ suppressed CRC occurrence and CRLM in mice by regulating intestinal F. nucleatum.

The basis of complications in the context of SARS-CoV-2 infection: Pathological activation of ADAM17.

The virulence of SARS-CoV-2 decreases with increasing infectivity, the primary approaches for antiviral treatments will be preventing or minimizing the complications resulting from virus infection. ADAM metallopeptidase domain 17 (ADAM17) activation by SARS-CoV-2 infection has a dual effect on the development of the disease: increased release of inflammatory cytokines and dysregulation of Angiotensin converting enzyme II (ACE2) on cell surfaces, inflammatory cytokine infiltration and loss of ACE2 protective function lead to a significant increase in the incidence of related complications. Importantly, pathologically activated ADAM17 showed superior features than S protein in regulating ACE2 expression and participating in the intra cellular replication of SARS-CoV-2. In short, SARS-CoV-2 elicits only a limited immune response when it promotes its own replication and pathogenicity through ADAM17. Therefore, the pathological activation of ADAM17 may also represent a diminished innate antiviral defense and an altered strategy of SARS-CoV-2 infection. In this review, we summarized recent advances in our understanding of the pathophysiology of ADAM17, with a focus on the new findings that SARS-CoV-2 affects ADAM17 expression through Furin protein converting enzyme and Mitogen-activated protein kinase (MAPK) pathway, and raises the hypothesis that SARS-CoV-2 may mediates the pathological activation of ADAM17 by hijacking the actin regulatory pathway, and discussed the underlying biological principles.

Three specific gut bacteria in the occurrence and development of colorectal cancer: a concerted effort.

Colorectal cancer (CRC), which develops from the gradual evolution of tubular adenomas and serrated polyps in the colon and rectum, has a poor prognosis and a high mortality rate. In addition to genetics, lifestyle, and chronic diseases, intestinal integrity and microbiota (which facilitate digestion, metabolism, and immune regulation) could promote CRC development. For example, enterotoxigenic Bacteroides fragilis, genotoxic Escherichia coli (pks+ E. coli), and Fusobacterium nucleatum, members of the intestinal microbiota, are highly correlated in CRC. This review describes the roles and mechanisms of these three bacteria in CRC development. Their interaction during CRC initiation and progression has also been proposed. Our view is that in the precancerous stage of colorectal cancer, ETBF causes inflammation, leading to potential changes in intestinal ecology that may provide the basic conditions for pks+ E. coli colonization and induction of oncogenic mutations, when cancerous intestinal epithelial cells can further recruit F. nucleatum to colonise the lesion site and F. nucleatum may contribute to CRC advancement by primarily the development of cancer cells, stemization, and proliferation, which could create new and tailored preventive, screening and therapeutic interventions. However, there is the most dominant microbiota in each stage of CRC development, not neglecting the possibility that two or even all three bacteria could be engaged at any stage of the disease. The relationship between the associated gut microbiota and CRC development may provide important information for therapeutic strategies to assess the potential use of the associated gut microbiota in CRC studies, antibiotic therapy, and prevention strategies.

Recombination and Mutation in a New HP-PRRSV Strain (SD2020) from China.

A new HP-PRRSV strain (SD2020) was isolated from pigs with suspected highly pathogenic porcine reproductive and respiratory syndrome disease in a pig farm in Shandong Province, China, and its genome was sequenced. This pig farm has been using the VR-2332 vaccine strain to immunize pigs for a long time. The phylogenic and single nucleotide polymorphisms (SNPs) analysis of the viruses isolated from dead pigs showed that SD2020 was a natural recombinant virus of the VR-2332 vaccine strain and the JXA1 similar strain, and that two splicing fragments highly homologous to JXA1 in the virus genome were probably derived from the JXA1 wild strain and JXA1-R vaccine strain, respectively. Therefore, the possible recombination events of SD2020 and its mutation site might be related to high pathogenicity.

Regulatory role of ncRNAs in pulmonary epithelial and endothelial barriers: Molecular therapy clues of influenza-induced acute lung injury.

Influenza A virus is globally widespread, causing a large number of infections and deaths due to acute lung injury (ALI) every year. The destruction and impairment of alveolar epithelial and microvascular endothelial cell barrier functions are the key inducers of ALI and acute respiratory distress syndrome caused by influenza virus infection. Although noncoding ribonucleic acids (ncRNAs) do not encode proteins in host cells, they possess the ability of protein regulation and signal transduction. Moreover, studies have shown that ncRNAs are significantly and differentially expressed following influenza virus infection, and these ncRNAs play vital roles in the pathogenesis of influenza virus infection. By analyzing the recently published literature, we found that ncRNA could regulate alveolar epithelial and microvascular endothelial cell barrier functions in different ways, which include influencing the innate and acquired immune responses of host cells, affecting apoptosis and autophagy, regulating tight and adherent junctions, etc. In the present paper, we reviewed the roles and regulatory mechanisms of these ncRNAs and discussed the effects of these ncRNAs on pulmonary epithelial and endothelial cell barriers. Further, by sorting and analyzing available research data, we proposed the possibility of applying these ncRNAs for treating ALI in influenza cases, thereby alleviating the permeability of pulmonary epithelial and endothelial cell barriers. Moreover, we discussed future research and development prospects. Our review suggests that targeted therapy and drug research based on ncRNAs would provide an important direction for the molecular therapy of influenza-induced ALI.

Dominant subtype switch in avian influenza viruses during 2016-2019 in China.

We have surveyed avian influenza virus (AIV) genomes from live poultry markets within China since 2014. Here we present a total of 16,091 samples that were collected from May 2016 to February 2019 in 23 provinces and municipalities in China. We identify 2048 AIV-positive samples and perform next generation sequencing. AIV-positive rates (12.73%) from samples had decreased substantially since 2016, compared to that during 2014-2016 (26.90%). Additionally, H9N2 has replaced H5N6 and H7N9 as the dominant AIV subtype in both chickens and ducks. Notably, novel reassortants and variants continually emerged and disseminated in avian populations, including H7N3, H9N9, H9N6 and H5N6 variants. Importantly, almost all of the H9 AIVs and many H7N9 and H6N2 strains prefer human-type receptors, posing an increased risk for human infections. In summary, our nation-wide surveillance highlights substantial changes in the circulation of AIVs since 2016, which greatly impacts the prevention and control of AIVs in China and worldwide.

Genetic diversities and phylogenetic analyses of three Chinese main ethnic groups in southwest China: A Y-Chromosomal STR study.

Short tandem repeats (STRs) located on the Y chromosome with the properties of male-specific inheritance and haploidy are widely used in forensics to analyze paternal genealogies and match male trace donors to evidence. Besides, Y-chromosomal haplotypes play an important role in providing breathtaking insights into population genetic history. However, the genetic diversity and forensic characteristics of Y-STRs in Guizhou main ethnic groups (Hans, Miaos and Bouyeis) remain uncharacterized. Here, we obtained Y-chromosomal 23-marker haplotypes in three Guizhou populations and submitted the first batch of Y-STR haplotype data to the YHRD. The HD in the aforementioned three populations are 0.99990, 0.99983, and 0.99979, respectively, and DC values are 0.9902, 0.9908, and 0.97959, respectively. Subsequently, genetic differentiation between our newly studied populations and reference groups along ethnic/administrative divisions, as well as national/continental boundaries were investigated via AMOVA, MDS, and phylogenetic relationship reconstruction. Significant genetic differentiations from our subjects and other groups are identified in ethnically, linguistically and geographically diverse populations, including most prominently Tibetans and Uyghurs among 30 mainland Chinese populations, Taiwanese groups and others among 58 Asian populations, as well as African groups and others among 89 worldwide populations. Qiannan Bouyei has a close genetic relationship with Guangxi Zhuang, and Zunyi Han and Qiandongnan Miao have close genetic affinity with Hunan Han and Guizhou Shui, respectively. Collectively, this new-generation Y-STR amplification system can be used as a supplementary tool in forensic identification and male parentage testing and even pedigree search.

Forensic characterization and genetic polymorphisms of 19 X-chromosomal STRs in 1344 Han Chinese individuals and comprehensive population relationship analyses among 20 Chinese groups.

X-chromosomal short tandem repeats (X-STRs) may assist resolution of complex forensic kinship cases and complement autosomal and Y-chromosomal STRs in routine forensic practice and population genetics. In the present study, we investigated the allele/haplotype diversity and forensic genetic characteristics of 19 X- STRs in 206 Guizhou Han and 1344 Meta-Han Chinese individuals using AGCU X19 PCR amplification system. Population relationships within five Han Chinese population groups (1344 individuals), between Guizhou Han and other 19 Chinese reference populations belonging to four language families (5074 individuals), as well as between Meta-Han Chinese and other 15 minorities (3730 individuals) were performed using Reynolds's, Nei's and Fst genetic distances, principal component analysis (PCA), multidimensional scaling (MDS), Structure and Neighbor-Joining tree. Mean paternity exclusion chance (MEC) in Duos > 0.99999999453588 and in trios > 0.99999999999781, as well as power of discrimination (PD) > 0.99999999999980 in Guizhou Han on the basis of allele frequencies. Consistent high MECs and PDs can be observed in Meta-Han Chinese population based on both allele diversities of 19 markers and haplotype diversities of seven linkage groups (LG). DXS10135 and LG1 are the most informative and polymorphic in Han Chinese group. The comprehensive population comparisons reveal that Han Chinese is a homogenous population and has the genetically closer relationship with Hmong-Mien-speaking groups than Tibetan-Burman-speaking and Turkic-speaking populations. In summary, AGCU X19 PCR amplification system is highly polymorphic and informative in Guizhou Han and Han Chinese populations. The comprehensive population data from 20 Chinese populations analyzed in this study may be used as a reference Chinese frequency database of X-STRs for forensic casework applications.

Recyclable ferromagnetic chitosan nanozyme for decomposing phenol.

Decomposing phenol and phenolic compounds to purify the environment is a focus of social attention. The use of ferromagnetic nanoparticles (MNP) to degrade phenol and phenolic compounds possesses many advantages and has received extensive attention. However, the unsatisfied catalyst activity and stability of MNP hamper its industrial applications. To improve MNP's properties, a ferromagnetic chitosan nanozyme (MNP@CTS) was synthesized via an improved hydrothermal method and molecular self-assembly technology. Its particle size was 11.76 nm, polydispersity index (PDI) was 0.073, surface zeta potential was 40.34 mV, saturation magnetization value was 35.28 emu g-1 and coercivity value was 17.56 Oe. The catalytic condition was extensively optimized among a range of pH and temperature, as well as initial concentrations of the substrate and H2O2, and MNP@CTS removed over 95% phenol from an aqueous solution within 5 h under the optimum conditions. Moreover, MNP@CTS was stable and could be regenerated for reuse for at least ten rounds. Thus, our findings open up a wide spectrum and lay a foundation of environmental friendly applications of MNP@CTS, showing several attractive features, such as easy preparation, low cost, excellent catalytic activity, good stability and reusability.

Cyclophilin B facilitates the replication of Orf virus.

BACKGROUND: Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored. METHODS: Suppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up-regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID50) assay and qRT-PCR detection. RESULTS: In the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome. CONCLUSIONS: Cellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV.

Three new 1-(p-hydroxybenzyl)phenanthrenes from Bletilla striata.

Three new benzylphenanthrenes, named 1-(p-hydroxybenzyl)-4,7-dimethoxyphenanthrene-2-ol (1), 1-(p-hydroxybenzyl)-4,7-dimethoxyphenanthrene-2,8-diol (2), and 1-(p-hydroxybenzyl)-4,7-dimethoxyphenanthrene-2,6-diol (3), along with a known analog were isolated from tubers of Bletilla striata. The structures of these new compounds were established by means of HR-ESI-MS, 1D, and 2D NMR.

In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.

Orf, which is caused by orf virus (ORFV), is distributed worldwide and is endemic in most sheep- and/or goat-raising countries. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, the specific effect of RNAi on the replication of ORFV was explored. The application of RNA interference (RNAi) inhibited the replication of ORFV in cell culture by targeting the ORF025 gene of ORFV, which encodes the viral polymerase. Three small interfering RNA (siRNA) (named siRNA704, siRNA1017 and siRNA1388) were prepared by in vitro transcription. The siRNAs were evaluated for antiviral activity against the ORFV Jilin isolate by the observation of cytopathic effects (CPE), virus titration, and real-time PCR. After 48 h of infection, siRNA704, siRNA1017 and siRNA1388 reduced virus titers by 59- to 199-fold and reduced the level of viral replication by 73-89 %. These results suggest that these three siRNAs can efficiently inhibit ORFV genome replication and infectious virus production. RNAi targeting of the DNA polymerase gene is therefore potentially useful for studying the replication of ORFV and may have potential therapeutic applications.

Rapid detection of orf virus by loop-mediated isothermal amplification based on the DNA polymerase gene.

At present, there are no effective antiviral treatments available for contagious ecthyma, and rapid diagnosis is therefore critical for effective control of the disease. Recently, the invention of a novel LAMP technique that can rapidly amplify nucleic acids with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic acid-based diagnostic tests and has made on-site diagnosis possible. To establish a flexible loop-mediated isothermal amplification (LAMP) assay for the rapid detection of orf virus, two pairs of primers, including outer primers F3/B3 and inner primers FIP/BIP, were designed to amplify the DNA polymerase gene. Optimal time and temperature conditions for LAMP were found to be 45 min and 62 °C, respectively. The LAMP assay was shown to be specific, with no cross-reactivity with sheeppox virus, goatpox virus, avian molluscum roup virus or vesicular stomatitis virus. Additionally, the sensitivity of the LAMP method was similar to that of real-time PCR and demonstrated greater sensitivity than a conventional polymerase chain reaction (PCR) assay. To assess the utility of LAMP in the detection of orf virus in clinical samples, a total of 35 samples collected from orf virus-infected sheep and goats were tested using the optimized LAMP assay, real-time PCR, and conventional PCR. Of the samples, 26 were found to be positive by LAMP, and 25 (74.3 %) were positive by real-time PCR, whereas only 18 (51.4 %) were positive by conventional PCR. Our results have shown that the LAMP assay developed in this study can be used for the rapid detection of orf virus.

Sequence analysis of the NSP2, ORF5, and ORF7 genes of 11 PRRS virus isolates from China.

During a tracing investigation of blue ear disease in China conducted from January to November 2008, 11 porcine reproductive and respiratory syndrome virus (PRRSV) isolates were collected from eight provinces including Liaoning, Jilin, Hebei, Shandong, Henan, Shanghai, Zhejiang, and Guangxi. The complete gene sequences of the NSP2, ORF5, and ORF7 genes from these 11 PRRSV isolates were amplified, cloned and detected by RT-PCR and then compared with the published sequences of other strains. The results showed that all of the isolates genotypically belonged to an American strain, but shared high homology with JXA1, the highly pathogenic strain endemic to China. All of the 11 PRRSV isolates demonstrated a 90-nucleotide deletion in the NSP2 gene, suggesting that the main epidemic of PRRSV in 2008 was due to this gene deletion isolate. More consistent mutations were found in specific regions of the ORF5 and ORF7 genes of the 11 PRRSV isolates, such as the signal peptide and transmembrane regions of GP5 and the Pat 7 motif of the N protein. Whether these mutations influence nuclear localization of PRRSV requires confirmation by future studies.

Development of reverse-transcription loop-mediated isothermal amplification for the detection of infectious bursal disease virus.

To establish a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of infectious bursal disease virus (IBDV), four primers specific to six regions of the VP3 gene were designed; the VP3 region was selected because it is a conserved part of the IBDV genome. After amplification in an isothermal water bath for 70 min, samples containing IBDV generated the expected ladder-like products while other viruses generated no product. The sensitivity and specificity of the RT-LAMP assay were evaluated by comparison with reverse-transcription polymerase chain reaction (RT-PCR) and virus isolation. The assay was significantly more sensitive than normal gel-based RT-PCR. Because it is specific and simple, the RT-LAMP assay can be widely applied in clinical laboratories for rapid detection of IBVD.