Molecular characterizations of Cryptosporidium spp. in brown rat (Rattus norvegicus) from an animal feedlot in Xinjiang, China.

Cryptosporidium infection is a common occurrence in rodents worldwide. In this study, 435 wild brown rats were captured from an animal feedlot in Xinjiang, China, with a fecal sample obtained directly from the rectal contents of each rat. The DNA extracted from these fecal samples was analyzed for Cryptosporidium spp. using PCR targeting the SSU rRNA gene. The prevalence of Cryptosporidium infection in brown rats was found to be 5.5% (24 out of 435). Interestingly, the infection rates varied among different animal enclosures, with rates of 0% in the chicken coop (0/51), cowshed (0/3), and varying rates in other areas including the sheepfold (6.1%, 6/98), the pigsty (7.6%, 10/132), the dovecote (7.0%, 5/71), and outdoor environments (3.8%, 3/80). The study identified three species and one genotype of Cryptosporidium, namely C. occultus (n = 10), C. parvum (n = 4), C. ditrichi (n = 1), and Cryptosporidium rat genotype IV (n = 9). Additionally, two of the C. parvum isolates were successfully subtyped as IIdA19G1 (n = 2) at the gp60 gene. These results offer valuable insights into the prevalence and genetic diversity of Cryptosporidium in brown rats within the region.

Functional Analysis of Type III Effectors in Xanthomonas campestris pv. campestris Reveals Distinct Roles in Modulating Arabidopsis Innate Immunity.

Xanthomonas campestris pv. campestris (Xcc) is a significant phytopathogen causing black rot disease in crucifers. Its virulence relies heavily on the type III secretion system (T3SS), facilitating effector translocation into plant cells. The type III effectors (T3Es) disrupt cellular processes, promoting pathogen proliferation. However, only a few T3Es from Xcc have been thoroughly characterized. In this study, we further investigated two effectors using the T3Es-deficient mutant and the Arabidopsis protoplast system. XopE2Xcc triggers Arabidopsis immune responses via an unidentified activator of the salicylic acid (SA) signaling pathway, whereas XopLXcc suppresses the expression of genes associated with patterns-triggered immunity (PTI) and the SA signaling pathway. These two effectors exert opposing effects on Arabidopsis immune responses. Additionally, we examined the relationship between the specific domains and functions of these two effector proteins. Our findings demonstrate that the N-myristoylation motif and N-terminal domain are essential for the subcellular localization and virulence of XopE2Xcc and XopLXcc, respectively. These novel insights enhance our understanding of the pathogenic mechanisms of T3Es and contribute to developing effective strategies for controlling bacterial disease.

Exosomal miR-1228-5p down-regulates DUSP22 to promotes cell proliferation and migration in small cell lung cancer.

BACKGROUND: Exosomes play a crucial role in promoting tumor progression, dissemination, and resistance to treatment. These extracellular vesicles hold promise as valuable indicators for cancer detection. Our investigation focuses on exploring the significance and clinical relevance of exosomal miRNAs in small cell lung cancer (SCLC). METHODS: Serum exosomes were isolated from both SCLC patients and healthy controls, and subjected to exosomal miRNA sequencing analysis. Mimics and inhibitors were employed to investigate the function of exosomal miR-1128-5p in cell migration and proliferation, both in vitro and in vivo. Western blot and luciferase assay were utilized to identify the interaction between miR-1228-5p and dual specificity phosphatase 22 (DUSP22). RESULTS: Exosomal miRNA sequencing analysis revealed enrichment of specific miRNAs in SCLC compared to healthy controls. Circulating miR-1228-5p was upregulated in SCLC patients, associated with advanced stages, suggesting its potential oncogenic role. In vitro, miR-1228-5p expression was significantly higher in SCLC cells than in normal cells. SCLC cell-derived exosomes contained elevated levels of miR-1228-5p, facilitating its entry into co-cultured cells. Notably, migration and proliferation induced by SCLC exosomes were mainly mediated by miR-1228-5p. In vivo experiments confirmed these findings. Western blot analysis demonstrated miR-1228-5p's regulation of DUSP22 expression, and luciferase reporter assay validated DUSP22 as a direct target gene. Overexpressing DUSP22 counteracted miR-1228-5p's promotion of SCLC cell proliferation and migration. CONCLUSIONS: Collectively, our results suggest that exosomes play a role in facilitating cancer growth and metastasis by delivering miR-1228-5p. Moreover, circulating exosomal miR-1228-5p may serve as a potential marker for SCLC diagnosis and prognosis.

Whole-genome sequencing of Fusarium spp. causing sugarcane root rot on both chewing cane and sugar-making cane.

Previously we isolated three Fusarium strains (a F. sacchari strain namely GXUF-1, and another two F. commune strains namely GXUF-2 and GXUF-3), and we verified that GXUF-3 was able to cause sugarcane root rot to the chewing cane cultivar Badila. Considering that Fusarium spp. are a group of widely distributed fungal pathogens, we tested whether these three Fusarium isolates were able to cause root rot to Badila as well as sugar-making cane cultivar (Guitang42), using a suitable inoculation method established based on infection assays using Badila. We found that the three Fusarium strains were able to cause root rot symptoms to both Badila and Guitang42, to different extents. To better investigate the potential pathogenicity mechanisms, we performed Illumina high-throughput sequencing and analyzed the whole genomic sequence data of these three Fusarium strains. The results reveal that the assembly sizes of the three Fusarium strains were in a range of 44.7-48.2 Mb, with G + C contents of 48.0-48.5%, and 14,154-15,175 coding genes. The coding genes were annotated by multiple public databases, and potential pathogenic genes were predicted using proprietary databases (such as PHI, DFVF, CAZy, etc.). Furthermore, based on evolutionary analysis of the coding sequence, we found that contraction and expansion of gene families occurred in the three Fusarium strains. Overall, our results suggest a potential risk that the root rot disease may occur to the sugar-making canes although it was initially spotted from fruit cane, and provide clues to understand the pathogenic mechanisms of Fusarium spp. causing sugarcane root rot.

Molecular characterization of Enterocytozoon bieneusi genotypes in wild Altai marmot (Marmota baibacina) in Xinjiang, China: host specificity and adaptation.

Enterocytozoon bieneusi is responsible for opportunistic infections leading to gastrointestinal diseases in humans and animals worldwide. A total of 334 fresh fecal samples were collected from wild Altai marmots (Marmota baibacina) in Xinjiang, China, and E. bieneusi was screened via PCR amplification of the internal transcribed spacer (ITS) region of the small submit ribosomal RNA (SSU rRNA). The results indicated that 22.8% (76/334) of the wild Altai marmot fecal samples were positive for E. bieneusi, and the highest positive rate was detected in Akqi (51.9%, 27/52), with a significant difference from other sampling sites (p < 0.01). Four known genotypes (BEB6, CHG3, GX2, and YAK1) and three novel genotypes (XJHT2 to XJHT4) were identified in the present study. Genotype XJHT3 was dominant and detected in 48 fecal samples. In the phylogenetic analysis, the novel genotypes XJHT2 and XJHT3 were clustered in Group 1 together with the known genotype YAK1, while genotypes CHG3 and BEB6 were clustered in Group 2. The novel genotype XJHT4 was clustered together with other rodent-derived genotypes and generated a novel Group 14. These data confirmed the host specificity and adaptation of E. bieneusi in rodents. These findings enrich our understanding of the prevalence and genetic diversity of E. bieneusi in wild Altai marmots in Xinjiang, China.

Bioinformatics analysis of ferroptosis-related genes and immune cell infiltration in non-alcoholic fatty liver disease.

BACKGROUND: The morbidity and mortality rates of patients with non-alcoholic fatty liver disease (NAFLD) have been steadily increasing in recent years. Previous studies have confirmed the important role of ferroptosis in NAFLD development; however, the precise mechanism through which ferroptosis influences NAFLD occurrence remains unclear. The present study aimed to identify and validate ferroptosis-related genes involved in NAFLD pathogenesis and to investigate the underlying molecular mechanisms of NAFLD. METHODS: We downloaded microarray datasets GSE72756 and GSE24807 to identify differentially expressed genes (DEGs) between samples from healthy individuals and patients with NAFLD. From these DEGs, we extracted ferroptosis-related DEGs. GSE89632, another microarray dataset, was used to validate the expression of ferroptosis-related genes. A protein-protein interaction (PPI) network of ferroptosis-related genes was then constructed. The target genes were also subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Finally, competing endogenous RNA networks were constructed. We used the CIBERSORT package to evaluate the infiltration of immune cells infiltration in NAFLD. RESULTS: Five ferroptosis-related genes (SCP2, MUC1, DPP4, SLC1A4, and TF) were identified as promising diagnostic biomarkers for NAFLD. Enrichment analyses revealed that these genes are mainly involved in metabolic processes. NEAT1-miR-1224-5p-SCP2, NEAT1-miR-485-5p-MUC1, MALAT1-miR-485-5p-MUC1, and CNOT6-miR-145-5p-SLC1A4 are likely to be the potential RNA regulatory pathways that affect NAFLD development. Principal component analysis indicated significant differences in immune cell infiltration between the two groups. CONCLUSIONS: This study identified five ferroptosis-related genes as potential biomarkers for diagnosing NAFLD. The correlations between the expression of ferroptosis-related genes and immune cell infiltration might shed light on the study of the molecular mechanism underlying NAFLD development.

Improvement of resistance to rice blast and bacterial leaf streak by CRISPR/Cas9-mediated mutagenesis of Pi21 and OsSULTR3;6 in rice (Oryza sativa L.).

Rice (Oryza sativa L.) is a staple food in many countries around the world, particularly in China. The production of rice is seriously affected by the bacterial leaf streak and rice blast, which can reduce rice yield or even cause it to fail to be harvested. In this study, susceptible material 58B was edited by CRISPR/Cas9, targeting a target of the Pi21 gene and a target of the effector-binding element (EBE) of the OsSULTR3;6 gene, and the mutants 58b were obtained by Agrobacterium-mediated method. The editing efficiency of the two targets in the T0 generation was higher than 90.09%, the homozygous mutants were successfully selected in the T0 generation, and the homozygous mutation rate of each target was higher than 26.67%. The expression of the edited pi21 and EBE of Ossultr3;6 was significantly reduced, and the expression of defense responsive genes was significantly upregulated after infected with rice blast. The lesion areas of rice blast and bacterial leaf streak were significantly reduced in 58b, and the resistance of both was effectively improved. Furthermore, the gene editing events did not affect the agronomic traits of rice. In this study, the resistance of 58b to rice blast and bacterial leaf streak was improved simultaneously. This study provides a reference for using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) to accelerate the improvement of rice varieties and the development of new materials for rice breeding.

Characterization of a Bacillus velezensis strain isolated from Bolbostemmatis Rhizoma displaying strong antagonistic activities against a variety of rice pathogens.

Biological control is an effective measure in the green control of rice diseases. To search for biocontrol agents with broad-spectrum and high efficiency against rice diseases, in this study, a strain of antagonistic bacterium BR-01 with strong inhibitory effect against various rice diseases was isolated from Bolbostemmatis Rhizoma by plate confrontation method. The strain was identified as Bacillus velezensis by morphological observation, physiological and biochemical identification, and molecular characterization by 16S rDNA and gyrB gene sequencing analysis. The confrontation test (dual culture) and Oxford cup assays demonstrated that B. velezensis BR-01 had strong antagonistic effects on Magnaporthe oryzae, Ustilaginoidea virens, Fusarium fujikuroi, Xanthomonas oryzae pv. Oryzicola, and Xanthomonas oryzae pv. oryzae, the major rice pathogens. The genes encoding antimicrobial peptides (ituA, ituD, bmyB, bmyC, srfAA, fenB, fenD, bacA, and bacD) were found in B. velezensis BR-01 by PCR amplification with specific primers. B. velezensis BR-01 could produce protease, cellulase, ß-1,3-glucanase, chitinase, indoleacetic acid, siderophore, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and might produce three lipopeptide antibiotics, surfactin, iturin, and fengycin based on Liquid chromatography-mass spectrometry (LC-MS) results. Furthermore, the plant assays showed that B. velezensis BR-01 had significant control effects on rice bacterial blight and bacterial leaf streak by pot experiments in greenhouse. In conclusion, B. velezensis BR-01 is a broad-spectrum antagonistic bacterium and has the potential as the ideal biocontrol agent in controlling multiple rice diseases with high efficiency.

Comparative Genomic Analysis of Two Xanthomonas oryzae pv. oryzae Strains Isolated From Low Land and High Mountain Paddies in Guangxi, China.

Xanthomonas oryzae pv. textitoryzae (Xoo) is a causal agent of rice bacterial leaf blight (BLB), the major rice disease, which is seriously constraining rice production in Asia. The interaction between Xoo and rice is in a dynamic process, essentially the co-evolution. Tracking the occurrence of plant diseases and identifying the epidemic pathogens in time are critical to assessing the epidemic disease status and understanding the pathogen evolution. In 2020, the occurrences of rice BLB were spotted in many places of Guangxi, the major rice growing region in China. Two of the 2020-epidemic Xoo strains, namely, GXO20-01 and GXO20-06, were isolated from low land and high mountain paddies in Guangxi, respectively, and were demonstrated to be race R8 of Chinese Xoo strains, but with significantly different virulence on certain susceptible varieties of rice. The HiFi PacBio sequencing revealed that GXO20-01 and GXO20-06 share the highly syntenic genome structures and the major genome contents, but only differ in <10 genes, including one gene encoding for transcription activator-like effector (TALE). A phylogenomic analysis grouped GXO20-01 and GXO20-06 into the PX-A lineage, stood close to PXO563 and PXO71 strains, but stood away from the other Chinese Xoo strains; for example, the JL25 and YC11. A comparative genomic analysis revealed that the major pathogenicity/virulence genes are conserved in two, newly isolated Xoo strains and the other Xoo strains in PX-A lineage, including the majority genes for the TALomes. The genomic differences between the Xoo strains were pinpointed to a few tal genes, which were variable in both their numbers and sequences, even between GXO20-01 and GXO20-06, the two 2020-epidemic Xoo strains. The study further revealed the instability and variability of tal genes in Xoo and highlighted the utility of HiFi long-read sequencing in TALE analysis and pathogen tracking.

Engineering Resistance to Bacterial Blight and Bacterial Leaf Streak in Rice.

BACKGROUND: Xanthomonas oryzae (Xo) is one of the important pathogenic bacterial groups affecting rice production. Its pathovars Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) cause bacterial blight and bacterial leaf streak in rice, respectively. Xo infects host plants by relying mainly on its transcription activator-like effectors (TALEs) that bind to host DNA targets, named effector binding elements (EBEs), and induce the expression of downstream major susceptibility genes. Blocking TALE binding to EBE could increase rice resistance to the corresponding Xo. FINDINGS: We used CRISPR/Cas9 to edit the EBEs of three major susceptibility genes (OsSWEET11, OsSWEET14 and OsSULTR3;6) in the rice varieties Guihong 1 and Zhonghua 11. Both varieties have a natural one-base mutation in the EBE of another major susceptibility gene (OsSWEET13) which is not induced by the corresponding TALE. Two rice lines GT0105 (from Guihong 1) and ZT0918 (from Zhonghua 11) with target mutations and transgene-free were obtained and showed significantly enhanced resistance to the tested strains of Xoo and Xoc. Furthermore, under simulated field conditions, the morphology and other agronomic traits of GT0105 and ZT0918 were basically the same as those of the wild types. CONCLUSIONS: In this study, we first reported that the engineering rice lines obtained by editing the promoters of susceptibility genes are resistant to Xoo and Xoc, and their original agronomic traits are not affected.

Target-induced activation of DNAzyme for sensitive detection of bleomycin by using a simple MOF-modified electrode.

In this work, a sensitive electrochemical method for bleomycin (BLM) determination was reported on the basis of BLM-mediated activation of Zn2+-dependent DNAzyme and the adsorption of signal probes by a metal-organic framework (MOF) modified electrode. Two hairpin DNAs were employed in this protocol, one (HP1) for BLM recognition and one (HP2) for amplified signal output. The presence of BLM and Fe2+ caused the formation of BLM-Fe (II) complex to cleave HP1, releasing DNAzyme fragments, which could further hybridize with substrate HP2 to form a partial double-stranded DNA duplex and enable the activation of Zn2+-dependent DNAzyme with the coexistence of Zn2+. The Zn2+-dependent DNAzyme catalyzed the cyclic cleavage of magnetic beads (MB)-immobilized HP2 to release massive DNA fragments with a Fc-labeled- terminal, which could be used for BLM quantification through electrochemical measurement after their adsorption on a MOF modified electrode. Attributing to the high catalytic efficiency of DNAzyme and excellent electrochemical performance of MOF modified electrode, our method revealed an impressive limit of detection as low as 4 pM BLM with a linear range of 5-2000 pM. Besides, the easy synthesis of MOF without further modification and the easy way of adsorption for signal achievement facilitated the operation process. In virtue of the high sensitivity, selectivity and the simple-to-implement features, this method is believed to hold a great promising application for BLM determination in biomedical and clinical study.

CuI/2-Aminopyridine 1-Oxide Catalyzed Amination of Aryl Chlorides with Aliphatic Amines.

A class of 2-aminopyridine 1-oxides are discovered to be effective ligands for the Cu-catalyzed amination of less reactive (hetero)aryl chlorides. A wide range of functionalized (hetero)aryl chlorides reacted with various aliphatic amines to afford the desired products in good to excellent yields under the catalyst of CuI/2-aminopyridine 1-oxides. Furthermore, the catalyst system worked well for the coupling of cyclic secondary amines and N-methyl benzylamine with (hetero)aryl chlorides.

Sensitive detection of streptomycin in milk using a hybrid signal enhancement strategy of MOF-based bio-bar code and target recycling.

A MOF-based bio-bar code material was synthesized and firstly applied to develop an electrochemical streptomycin (STR) aptasensor. By using MOF-based bio-bar code and enzyme-assisted target recycling for dual-signal amplification, highly sensitive detection of STR was achieved. The sensing surface was simply fabricated by immobilizing a mixed monolayer of thiolated cDNA/aptamer duplexes (dsDNA) and 6-mercapto-1-hexanol (MCH) on the gold nanoparticle modified screen printed carbon electrode (Au/SPCE). The presence of target STR caused highly efficient removal of the aptamers from dsDNA assisted by Exo I enzyme. Then MOF-based bio-bar codes were backfilled to achieve the adsorption of electroactive Ru(NH3)63+ (RuHex) on electrode surface. The electrochemical signal of the surface-confined RuHex was used for quantitation. The analytical performance for STR was satisfactory with a wide linear range of 0.005-150 ng mL-1, a low detection limit of 2.6 pg mL-1 and a good selectivity towards other three antibiotics. Moreover, the application of this aptasensor for determination of STR in real milk samples was also realized. With these merits, this dual-signal amplification assay might provide one of the effective ways for food safety monitoring.

Verification of Bacteroidales 16S rRNA markers as a complementary tool for detecting swine fecal pollution in the Yangtze Delta.

To correctly assess and properly manage the public health risks associated with exposure to contaminated water, it is necessary to identify the source of fecal pollution in a watershed. In this study, we evaluated the efficacy of our two previously developed real time-quantitative PCR (qPCR) assays for the detection of swine-associated Bacteroidales genetic markers (gene 1-38, gene 3-53) in the Yangtze Delta watershed of southeastern China. The results indicated that the gene 1-38 and 3-53 markers exhibited high accuracy (92.5%, 91.7% conditional probability, respectively) in detecting Bacteroidales spp. in water samples. According to binary logistic regression (BLR), these two swine-associated markers were well correlated (P < 0.05) with fecal indicators (Escherichia coli and Enterococci spp.) and zoonotic pathogens (E. coli O157: H7, Salmonella spp. and Campylobacter spp.) in water samples. In contrast, concentrations of conventional fecal indicator bacteria (FIB) were not correlated with zoonotic pathogens, suggesting that they are noneffective at detecting fecal pollution events. Collectively, the results obtained in this study demonstrated that a swine-targeted qPCR assay based on two Bacteroidales genes markers (gene 1-38, gene 3-53) could be a useful tool in determining the swine-associated impacts of fecal contamination in a watershed.

Mechanistic insights into host adaptation, virulence and epidemiology of the phytopathogen Xanthomonas.

Xanthomonas is a well-studied genus of bacterial plant pathogens whose members cause a variety of diseases in economically important crops worldwide. Genomic and functional studies of these phytopathogens have provided significant understanding of microbial-host interactions, bacterial virulence and host adaptation mechanisms including microbial ecology and epidemiology. In addition, several strains of Xanthomonas are important as producers of the extracellular polysaccharide, xanthan, used in the food and pharmaceutical industries. This polymer has also been implicated in several phases of the bacterial disease cycle. In this review, we summarise the current knowledge on the infection strategies and regulatory networks controlling virulence and adaptation mechanisms from Xanthomonas species and discuss the novel opportunities that this body of work has provided for disease control and plant health.

Type VI secretion system is not required for virulence on rice but for inter-bacterial competition in Xanthomonas oryzae pv. oryzicola.

The type VI secretion system (T6SS), a multifunctional protein secretion device, plays very important roles in bacterial killing and/or virulence to eukaryotic cells. Although T6SS genes have been found in many Xanthomonas species, the biological function of T6SSs has not been elucidated in most xanthomonads. In this study, we identified two phylogenetically distinct T6SS clusters, T6SS1 and T6SS2, in a newly sequenced Chinese strain GX01 of Xanthomonas oryzea pv. oryzicola (Xoc) which causes bacterial leaf streak (BLS) of rice (Oryza sativa L.). Mutational assays demonstrated that T6SS1 and T6SS2 are not required for the virulence of Xoc GX01 on rice. Nevertheless, we found that T6SS2, but not T6SS1, played an important role in bacterial killing. Transcription and secretion analysis revealed that hcp2 gene is actively expressed and that Hcp2 protein is secreted via T6SS. Moreover, several candidate T6SS effectors were predicted by bioinformatics analysis that might play a role in the antibacterial activity of Xoc. This is the first report to investigate the type VI secretion system in Xanthomonas oryzae. We speculate that Xoc T6SS2 might play an important role in inter-bacterial competition, allowing this plant pathogen to gain niche advantage by killing other bacteria.

Type III effectors xopN and avrBS2 contribute to the virulence of Xanthomonas oryzae pv. oryzicola strain GX01.

Xanthomonas oryzae pv. oryzicola (Xoc) depends on its type III secretion system (T3SS) to translocate type III secreted effectors (T3SEs), including transcription activator-like effectors (TALEs) and non-transcription activator-like effectors (non-TALEs), into host cells. T3SEs can promote the colonization of Xoc and contribute to virulence by manipulating host cell physiology. We annotated 25 genes encoding non-TALEs in Xoc strain GX01, an isolate from Guangxi in the South China's rice growing region. Through systematic mutagenesis of non-TALEs, we found that xopN, the virulence contribution of which was previously unknown for Xoc, significantly contributes to the virulence of Xoc GX01, as does avrBs2.