One-Step Reverse-Transcription Recombinase-Aided Amplification CRISPR/Cas12a-Based Lateral Flow Assay for Fast Field Screening and Accurate Differentiation of Four Major Tobamoviruses Infecting Tomato and Pepper.

Several tobamoviruses cause substantial economic losses to tomato and pepper crops globally, especially the pepper mild mosaic virus (PMMoV), tomato brown rugose fruit virus (ToBRFV), tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV). A fast and accurate detection method is essential for virus identification. An all-in-one reaction method combining a one-step reverse-transcription recombinase-aided amplification (RT-RAA) and CRISPR/Cas12a-based lateral flow assay in one mixture was developed to rapidly screen and accurately differentiate among these four tobamoviruses for field detection in tomato and pepper plants. With a generic RT-RAA primer set and a mix of four specific crRNAs, along with a portable metal incubator and the use of a crude extraction method, this method screened for PMMoV, ToBRFV, ToMV, and ToMMV concurrently in less than 1 h, enabling field workers to take action immediately. The accurate differentiation of these four viruses could be achieved by later adding a single specific crRNA.

First report of bean common mosaic virus naturally infecting rice bean (Vigna umbellata) in China.

Rice bean [Vigna umbellata (Thunb.) Ohwi et Ohashi] is a traditional grain legume in Asia. From 2020 and 2021, approximately 15% plants exhibiting virus-like symptoms of mosaic, crumpling, and stunting were observed in two rice bean fields in Changping district, Beijing, China. To unveil the viral agent(s) leading to the disease symptoms, leaf samples from nine symptomatic plants were collected, and used for total RNA extraction with RNAprep pure plant kit (DP432, Tiangen Biotech, Beijing, China). The RNA samples were then subjected to reverse transcription (RT)-PCR testing with degenerate primers targeting the partial NIb gene of potyvirus (Marie-Jeanne et al. 2000) as well as the respective specific primer pairs for 18 viruses that are known to infect Fabaceae crops (Suppl Table S1). While none of the 18 target viruses was detected from any of the samples, a fragment of 327 bp was obtained from each of the diseased leaf samples, but not from healthy plants, with the degenerate potyvirus primer pair. The suspected potyvirus-amplicons were Sanger sequenced at SinoGenMax (SinoGenoMax Co., Ltd. Beijing, China). BLASTn analysis of the sequences showed that they shared 100% nt identity with themselves and 99.1% with the partial NIb gene of bean common mosaic virus (BCMV; Potyvirus; Potyviridae) isolate BCMV-Az (accession no. KP903372). To confirm the BCMV identity of the virus as well as its molecular characteristics, a fragment around 3,388 bp corresponding to NIa, NIb, CP and 3'UTR of BCMV genome was amplified from 4 diseased samples with the primer pair BCMV-F (5'-AGCAAGTCAATTTACAAGGGACTTC-3') and BCMV-R (5'-GGAACAACAAACATTGCCGTAGCTAC-3') (Li et al., 2014). Five independent clones from each diseased sample (20 clones in total) were Sanger sequenced, and all showed 100% identity with each other. One representative sequence has been deposited in GenBank under the accession number OP422522. BLASTn analysis showed that the sequence shares >98% identity with a number of BCMV sequences in GenBank (e.g., 98.9% with KP903372, the isolate BCMV-Az), thus confirming the BCMV identity of the virus. Further analysis of plants (n = 88) resulting from seeds of the 9 abovementioned diseased plants revealed an infection rate of 41% based on symptoms and RT-PCR assay with the degenerate primers, demonstrating the seed transmissibility of the virus in rice bean. The virus isolate is also mechanical transmissible as mechanical inoculation of 10 healthy rice bean plants led to the successful infection of four plants, which showed mosaic symptoms and tested positive for BCMV with RT-PCR assay with BCMV-specific primers reported in Manjunatha et al. (2014). BCMV can infect many leguminous plants, such as azuki bean (Vigna angularis) (Li et al., 2014), yam bean (Pachyrhizus erosus) (Han et al., 2023), and mungbean (Vigna radiata) (Cui et al. 2014). However, to the best of our knowledge, this is the first report that BCMV naturally infects rice bean in China. Further attention should be paid to this emerging viral disease, and proper measures need to be developed and implemented to minimize the spread of the virus in bean crops in the region.

Hyperglycemia Aggravates Periodontitis via Autophagy Impairment and ROS-Inflammasome-Mediated Macrophage Pyroptosis.

Macrophage pyroptosis drives the secretion of IL-1ß, which has been recently reported to be a featured salivary biomarker for discriminating periodontitis in the presence of diabetes. This study aimed to explore whether macrophage pyroptosis plays a role in the development of diabetes mellitus-periodontitis, as well as potential therapeutic strategies. By establishing a model of experimental diabetes mellitus-periodontitis in rats, we found that IL-1ß and gasdermin D were highly expressed, leading to aggravated destruction of periodontal tissue. MCC950, a potent and selective molecule inhibitor of the NLRP3 inflammasome, effectively inhibited macrophage pyroptosis and attenuated alveolar bone losses in diabetes mellitus-periodontitis. Consistently, in vitro, high glucose could induce macrophage pyroptosis and thus promoted IL-1ß production in macrophages stimulated by lipopolysaccharide. In addition, autophagy blockade by high glucose via the mTOR-ULK1 pathway led to severe oxidative stress response in macrophages stimulated by lipopolysaccharide. Activation of autophagy by rapamycin, clearance of mitochondrial ROS by mitoTEMPO, and inhibition of inflammasome by MCC950 could significantly reduce macrophage pyroptosis and IL-1ß secretion. Our study demonstrates that hyperglycemia promotes IL-1ß production and pyroptosis in macrophages suffered by periodontal microbial stimuli. Modulation of autophagy activity and specific targeting of the ROS-inflammasome pathway may offer promising therapeutic strategies to alleviate diabetes mellitus-periodontitis.

Portable rapid detection of maize chlorotic mottle virus using RT-RAA/CRISPR-Cas12a based lateral flow assay.

Introduction: Maize lethal necrosis seriously threatens maize production worldwide, which was caused by coinfection by maize chlorotic mottle virus (MCMV) and a potyvirid. To effectively control maize lethal necrosis, it is vital to develop a rapid, sensitive, and specific detection method for the early diagnosis of MCMV in host plant tissues. Methods: We established a rapid detection procedure by combining the one-step reverse-transcription recombinase-aided amplification (one-step RT-RAA) and CRISPR/Cas12a-based lateral flow assay in one tube (one-tube one-step RT-RAA/CRISPR-Cas12a), which can be implemented on a portable metal incubator at 37~42°C. Furthermore, the crude extract of total RNA from plant materials using alkaline-PEG buffer can be directly used as the template for one-step RT-RAA. Results: The developed one-tube one-step RT-RAA/CRISPR-Cas12a lateral flow assay can detect as low as 2.5 copies of the coat protein (CP) gene of MCMV and 0.96 pg of the total RNA extracted from MCMV infected maize leaves. Furthermore, the MCMV infected maize leaves at 5 dpi having no obvious symptoms was detected as weak positive. Discussion: The crude extraction method of total RNA from plant materials required no complicated device, and all the procedures could be implemented at room temperature and on a portable metal incubator, costing a total time of about 1h. The one-step RT-RAA reagents and CRISPR/Cas12a reagents can be lyophilized for easy storage and transportation of reagents, which makes this method more feasible for the filed detection. This method presents rapidness, robustness and on-site features in detecting viral RNA, and is a promising tool for the field application in minimally equipped laboratories.

Development of one-step multiplex RT-PCR assay for rapid simultaneous detection of five RNA viruses and Acidovorax citrulli in major cucurbitaceous crops in China.

Cucurbitaceous fruits and vegetables are important crops. Viral and bacterial diseases cause substantial economic losses to cucurbit crops globally. For rapid detection of these pathogens and improved disease control, a one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) system was created. This method allowed for the concurrent detection of Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), and Acidovorax citrulli. Five pairs of specific primers were created according to the conserved regions around the coat protein (CP) genes of each virus, and one pair was based on the A. citrulli internal transcribed spacer (ITS). To limit false negatives, one pair of primers, created based on the Transcriptional elongation factor 1-α (EF1-α) from the major cucurbitaceous crop species, was put into the mRT-PCR reaction system. Primer concentrations, annealing temperature, extension time, and amplification cycles were optimized. Anticipated fragments of 152 bp (TMV), 205 bp (ZYMV), 318 bp (WMV), 419 bp (CGMMV), 529 bp (CMV), 662 bp (A. citrulli), and 821 bp (EF1-α) were amplified by the multiplex RT-PCR system, and their origin was established via DNA sequencing. This method was successfully used to examine field-collected seed samples of cucurbitaceous crops from China. The results demonstrated that the one-step mRT-PCR technique is a quick, efficient, and sensitive assay for the concurrent detection of six pathogens of cucurbits. It provides a method for monitoring and preventing these diseases.

Cost and effectiveness of microwave ablation versus video-assisted thoracoscopic surgical resection for ground-glass nodule lung adenocarcinoma.

Purpose: To retrospectively evaluate the cost and effectiveness in consecutive patients with ground-glass nodules (GGNs) treated with video-assisted thoracoscopic surgery (VATS; i.e., wedge resection or segmentectomy) or microwave ablation (MWA). Materials and methods: From May 2017 to April 2019, 204 patients who met our study inclusion criteria were treated with VATS (n = 103) and MWA (n = 101). We calculated the rate of 3-year overall survival (OS), local progression-free survival (LPFS), and cancer-specific survival (CSS), as well as the cost during hospitalization and the length of hospital stay. Results: The rates of 3-year OS, LPFS, and CSS were 100%, 98.9%, and 100%, respectively, in the VATS group and 100%, 100% (p = 0.423), and 100%, respectively, in the MWA group. The median cost of VATS vs. MWA was RMB 54,314.36 vs. RMB 21,464.98 (p < 0.001). The length of hospital stay in the VATS vs. MWA group was 10.0 vs. 6.0 d (p < 0.001). Conclusions: MWA had similar rates of 3-year OS, LPFS, and CSS for patients with GGNs and a dramatically lower cost and shorter hospital stay compared with VATS. Based on efficacy and cost, MWA provides an alternative treatment option for patients with GGNs.

Antisense Oligonucleotide-Based Therapy on miR-181a-5p Alleviates Cartilage Degradation of Temporomandibular Joint Osteoarthritis via Promoting SIRT1.

Temporomandibular joint osteoarthritis (TMJOA) condylar cartilage degeneration and abnormal subchondral bone pathological remodeling induce pain and joint dysfunction, and cartilage degeneration is considered irreversible. Very few therapeutic approaches are administrated in practice. Nucleotides have demonstrated considerable potential as a next-generation medication, and they have been applied in several models of osteoarthritis. There is a need to establish an effective protocol for TMJOA gene therapy. In the current study unilateral anterior crossbite (UAC) surgery was used to simulate mechanical stress-induced TMJOA in mice. Degeneration of condylar cartilage and destruction of subchondral bone were observed in damaged joints, and miR-181a-5p was elevated in chondrocytes. Intra-articular injection of miR-181a-5p antisense oligonucleotide (ASO) could reduce the cartilage damage and alleviate UAC-induced TMJOA progression, but it did not restore injured subchondral bone. Mechanically, miR-181a-5p evidently targeted the 3' untranslated region of Sirt1 directly, resulting in inhibition of silent information regulator 1 expression and promoting apoptosis by elevating p53-dependent signaling, indicating that miR181a-5p ASO promoted chondrocyte survival. The present study suggests that ASO-based gene therapy may be an effective TMJOA treatment.

Zhurong reveals recent aqueous activities in Utopia Planitia, Mars.

The Mars' climate is cold and dry in the most recent epoch, and liquid water activities are considered extremely limited. Previous orbital data only show sporadic hydrous minerals in the northern lowlands of Mars excavated by large impacts. Using the short-wave infrared spectral data obtained by the Zhurong rover of China's Tianwen-1 mission, which landed in southern Utopia Planitia on Mars, we identify hydrated sulfate/silica materials on the Amazonian terrain at the landing site. These hydrated minerals are associated with bright-toned rocks, interpreted to be duricrust developed locally. The lithified duricrusts suggest that formation with substantial liquid water originates by either groundwater rising or subsurface ice melting. In situ evidence for aqueous activities identified at Zhurong's landing site indicates a more active Amazonian hydrosphere for Mars than previously thought.

Recombinase polymerase amplification assay for sensitive and rapid detection of southern rice black-streaked dwarf virus in plants.

Southern rice black-streaked dwarf virus (SRBSDV) naturally infects rice and maize plants through white-backed planthopper (Sogatella furcifera) causing significant crop losses in China and Vietnam. Thus, rapid and accurate detection methods for SRBSDV are urgently needed. Recombinase polymerase amplification (RPA) is a novel technique for rapid and sensitive detection of nucleic acids. In this research, a reverse transcription (RT)-RPA-based method was developed for the detection of SRBSDV. A pair of RPA primers targeting the conserved sequences within the SP10 (major coat protein) gene on genomic RNA S10 of SRBSDV were designed. The assay was performed in a single tube at 39 °C for 20 min and demonstrated that the RPA assay is an efficient alternative for rapid detection of SRBSDV.

A novel pathogenicity determinant hijacks maize catalase 1 to enhance viral multiplication and infection.

Pathogens have evolved various strategies to overcome host immunity for successful infection. Maize chlorotic mottle virus (MCMV) can cause lethal necrosis in maize (Zea mays) when it coinfects with a virus in the Potyviridae family. However, the MCMV pathogenicity determinant remains largely unknown. Here we show that the P31 protein of MCMV is important for viral accumulation and essential for symptom development. Ectopic expression of P31 using foxtail mosaic virus or potato virus X induced necrosis in systemically infected maize or Nicotiana benthamiana leaves. Maize catalases (CATs) were shown to interact with P31 in yeast and in planta. P31 accumulation was elevated through its interaction with ZmCAT1. P31 attenuated the expression of salicylic acid (SA)-responsive pathogenesis-related (PR) genes by inhibiting catalase activity during MCMV infection. In addition, silencing of ZmCATs using a brome mosaic virus-based gene silencing vector facilitated MCMV RNA and coat protein accumulation. This study reveals an important role for MCMV P31 in counteracting host defence and inducing systemic chlorosis and necrosis. Our results have implications for understanding the mechanisms in defence and counter-defence during infection of plants by various pathogens.

AIM2 Inflammasome's First Decade of Discovery: Focus on Oral Diseases.

A common feature of many acute and chronic oral diseases is microbial-induced inflammation. Innate immune responses are the first line of defense against pathogenic microorganisms and are initiated by pattern recognition receptors (PRRs) that specifically recognize pathogen-associated molecular patterns and danger-associated molecular patterns. The activation of certain PRRs can lead to the assembly of macromolecular oligomers termed inflammasomes, which are responsible for pro-inflammatory cytokine maturation and secretion and thus activate host inflammatory responses. About 10 years ago, the absent in melanoma 2 (AIM2) was independently discovered by four research groups, and among the "canonical" inflammasomes [including AIM2, NLR family pyrin domain (NLRP)1, NLRP3, NLR family apoptosis inhibitory protein (NAIP)/NLR family, caspase activation and recruitment domain (CARD) containing (NLRC)4, and pyrin], AIM2 so far is the only one that simultaneously acts as a cytosolic DNA sensor due to its DNA-binding ability. Undoubtedly, such a double-faceted role gives AIM2 greater mission and more potential in the mediation of innate immune responses. Therefore, AIM2 has garnered much attention from the broad scientific community during its first 10 years of discovery (2009-2019). How the AIM2 inflammasome is related to oral diseases has aroused debate over the past few years and is under active investigation. AIM2 inflammasome may potentially be a key link between oral diseases and innate immunity. In this review, we highlight the current knowledge of the AIM2 inflammasome and its critical role in the pathogenesis of various oral diseases, which might offer future possibilities for disease prevention and targeted therapy utilizing this continued understanding.

Novel Method to Reliably Determine the Photon Helicity in B→K_{1}γ.

A sizable right-handed photon polarization in b→sγ is a clear signal for new physics. In this Letter, we point out that the photon helicity in b→sγ can be unambiguously extracted by combining the measurements in B→K_{1}γ and the Cabibbo-favored D→K_{1}e^{+}ν decay. We propose a ratio of up-down asymmetries in D→K_{1}e^{+}ν to quantify the hadronic effects. A method for measuring, in experiment, the involved partial decay widths in the ratio is discussed, and experimental facilities like BESIII, Belle-II and LHCb are likely to measure this ratio. We also give the angular distribution that is useful for extracting the photon polarization in the presence of different kaon resonances.

Mechanotransduction pathways in the regulation of cartilage chondrocyte homoeostasis.

Mechanical stress plays a critical role in cartilage development and homoeostasis. Chondrocytes are surrounded by a narrow pericellular matrix (PCM), which absorbs dynamic and static forces and transmits them to the chondrocyte surface. Recent studies have demonstrated that molecular components, including perlecan, collagen and hyaluronan, provide distinct physical properties for the PCM and maintain the essential microenvironment of chondrocytes. These physical signals are sensed by receptors and molecules located in the cell membrane, such as Ca2+ channels, the primary cilium and integrins, and a series of downstream molecular pathways are involved in mechanotransduction in cartilage. All mechanoreceptors convert outside signals into chemical and biological signals, which then regulate transcription in chondrocytes in response to mechanical stresses. This review highlights recent progress and focuses on the function of the PCM and cell surface molecules in chondrocyte mechanotransduction. Emerging understanding of the cellular and molecular mechanisms that regulate mechanotransduction will provide new insights into osteoarthritis pathogenesis and precision strategies that could be used in its treatment.

Mitochondrial DNA haplogroups participate in osteoarthritis: current evidence based on a meta-analysis.

Mitochondrial genes' variants encoded in both the nuclear and mitochondrial genomes can disrupt mitochondrial function, resulting in losing of cartilage and generating osteoarthritis (OA). However, the association between mtDNA haplogroups and OA still lacks strength evidence supporting. The aim of this meta-analysis is to assess the role of mtDNA haplogroups in speculating the pathogenesis and progression of OA. PubMed, Embase, the Cochrane Central Register of Controlled Trials, and World Health Organization clinical trials' registry center were searched to identify relevant studies up to the end of March 2019. Inclusion citations required a case-control or cohort study to demonstrate the association between mtDNA haplogroups and OA's prevalence or progression. Title, abstract, and full-text screening were sequentially assessed by three reviewers. Data were analyzed using STATA. Besides, publication bias and meta-regression analysis were conducted to explore potential heterogeneities. We collected results from 7 articles. The cluster TJ cases showed a lower proportion in OA cases (RR = 0.83, 95% CI 0.72, 0.96). However, there is no evidence that revealed this kind of impact originated from neither type J nor type T individually. Besides, the type B and G analyses among Asian populations also elucidated a negative association. Moreover, the cluster TJ of mtDNA haplogroups revealed a lower cumulative probability of radiographic OA progression (ES = 0.77, 95% CI 0.63, 0.94), which was contributed by type T (ES = 0.61, 95% CI 0.45, 0.82).The mtDNA haplogroups do have impacts on the prevalence and progression of OA. Cluster TJ could help reduce the prevalence and slow down the radiographic changes; however, the impacts came from type J and type T, respectively.

Natural variation in the Arabidopsis AGO2 gene is associated with susceptibility to potato virus X.

RNA silencing functions as an anti-viral defence in plants through the action of DICER-like (DCL) and ARGONAUTE (AGO) proteins. Despite the importance of this mechanism, little is known about the functional consequences of variation in genes encoding RNA silencing components. The AGO2 protein has been shown to be important for defense against multiple viruses, and we investigated how naturally occurring differences in AGO2 between and within species affects its antiviral activities. We find that the AGO2 protein from Arabidopsis thaliana, but not Nicotiana benthamiana, effectively limits potato virus X (PVX). Consistent with this, we find that the A. thaliana AGO2 gene shows a high incidence of polymorphisms between accessions, with evidence of selective pressure. Using functional analyses, we identify polymorphisms that specifically affect AGO2 antiviral activity, without interfering with other AGO2-associated functions such as anti-bacterial resistance or DNA methylation. Our results suggest that viruses adapt to overcome RNA silencing in their hosts. Furthermore, they indicate that plant-virus interactions have influenced natural variation in RNA-silencing components and that the latter may be a source of genetically encoded virus resistance.

The roles of circRFWD2 and circINO80 during NELL-1-induced osteogenesis.

Bone defects caused heavy social and economic burdens worldwide. Nel-like molecule, type 1 (NELL-1) could enhance the osteogenesis and the repairment of bone defects, while the specific mechanism remains to be elucidated. Circular RNAs (circRNAs) have been found to play critical roles in the tissue development and serve as biomarkers for various diseases. However, it remains unclear that the expression patterns of circRNAs and the roles of them played in recombinant NELL-1-induced osteogenesis of human adipose-derived stem cells (hASCs). In this study, we performed RNA-sequencing to investigate the expression profiles of circRNAs in recombinant NELL-1-induced osteogenic differentiation and identified two key circRNAs, namely circRFWD2 and circINO80. These two circRNAs were confirmed to be up-regulated during recombinant NELL-1-induced osteogenesis, and knockdown of them affected the positive effect of NELL-1 on osteogenesis. CircRFWD2 and circINO80 could interact with hsa-miR-6817-5p, which could inhibit the osteogenesis. Silencing hsa-miR-6817-5p could partially reverse the negative effect of si-circRFWD2 and si-circINO80 on the osteogenesis. Therefore, circRFWD2 and circINO80 could regulate the expression of hsa-miR-6817-5p and influence the recombinant NELL-1-induced osteogenic differentiation of hASCs. It opens a new window to better understanding the effects of NELL-1 on the osteogenic differentiation of hASCs and provides potential molecular targets and novel methods for bone regeneration efficiently and safely.

Characterization of Maize miRNAs in Response to Synergistic Infection of Maize Chlorotic Mottle Virus and Sugarcane Mosaic Virus.

The synergistic infection of maize chlorotic mottle virus (MCMV) and sugarcane mosaic virus (SCMV) causes maize lethal necrosis, with considerable losses to global maize production. microRNAs (miRNAs) are conserved non-coding small RNAs that play essential regulatory roles in plant development and environmental stress responses, including virus infection. However, the characterization of maize miRNAs in response to synergistic infection of MCMV and SCMV remains largely unknown. In this study, the profiles of small RNAs from MCMV and SCMV single- and co-infected (S + M) maize plants were obtained by high-throughput sequencing. A total of 173 known miRNAs, belonging to 26 miRNA families, and 49 novel miRNAs were profiled. The expression patterns of most miRNAs in S + M-infected maize plants were similar to that in SCMV-infected maize plants, probably due to the existence of RNA silencing suppressor HC-Pro. Northern blotting and quantitative real-time PCR were performed to validate the accumulation of miRNAs and their targets in different experimental treatments, respectively. The down-regulation of miR159, miR393, and miR394 might be involved in antiviral defense to synergistic infection. These results provide novel insights into the regulatory networks of miRNAs in maize plants in response to the synergistic infection of MCMV and SCMV.

Comparison of splenocyte microRNA expression profiles of pigs during acute and chronic toxoplasmosis.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that infects humans and other warm-blooded animals. Previous quantitative proteomic analyses of infected host cells revealed that the expression of many host proteins is modulated by T. gondii infection. However, at present limited data are available on the differentially expressed miRNAs (DEMs) associated with the pathology and host immune responses induced by acute and chronic infection with T. gondii in pigs in vivo. In this study, high-throughput sequencing was used to investigate expression profiles of spleen miRNAs at 10, 25 and 50 days post-infection (DPI) in pigs infected with Chinese I genotype strain T. gondii isolated from a dead pig. RESULTS: When compared to the control group, 34, 6 and 86 DEMs were found in spleens of infected pigs at 10, 25 and 50 DPI, respectively. Gene Ontology (GO) enrichment analysis of the target genes of DEMs showed that no GO terms were enriched at 25 DPI, whereas 28 and 241 GO terms, of which two and 215 were sample-specific, were significantly enriched at 10 and 50 DPI, respectively. The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the target genes of DEMs included signal transduction, immune system, metabolism and diseases. miRNA-gene network analysis revealed that the DEMs played important roles in the host immune response to T. gondii infection by modulating expression levels of cellular immunity-related cytokines and immune-related C-type lectins. CONCLUSION: Our results not only showed that host miRNA expression is altered by T. gondii but also revealed differences in the regulation of key biological processes and pathways involved in host responses to acute versus chronic T. gondii infection. This will aid future research into miRNA-target interactions during T. gondii infection in pigs and the development of novel therapies against T. gondii.

Virus-Derived Small Interfering RNAs Affect the Accumulations of Viral and Host Transcripts in Maize.

RNA silencing is a conserved surveillance mechanism against invading viruses in plants, which involves the production of virus-derived small interfering RNAs (vsiRNAs) that play essential roles in the silencing of viral RNAs and/or specific host transcripts. However, how vsiRNAs function to target viral and/or host transcripts is poorly studied, especially in maize (Zea mays L.). In this study, a degradome library constructed from Sugarcane mosaic virus (SCMV)-inoculated maize plants was analyzed to identify the cleavage sites in viral and host transcripts mainly produced by vsiRNAs. The results showed that 42 maize transcripts were possibly cleaved by vsiRNAs, among which several were involved in chloroplast functions and in biotic and abiotic stresses. In addition, more than 3000 cleavage sites possibly produced by vsiRNAs were identified in positive-strand RNAs of SCMV, while there were only four cleavage sites in the negative-strand RNAs. To determine the roles of vsiRNAs in targeting viral RNAs, six vsiRNAs were expressed in maize protoplast based on artificial microRNAs (amiRNAs), of which four could efficiently inhibit the accumulations of SCMV RNAs. These results provide new insights into the genetic manipulation of maize with resistance against virus infection by using amiRNA as a more predictable and useful approach.

Genotyping and virulence analysis of Toxoplasma gondii isolates from a dead human fetus and dead pigs in Jiangsu province, Eastern China.

Toxoplasma gondii is an obligate intracellular parasite with worldwide distribution. Virulence of T. gondii is a multigenic trait. Genetic and virulence data for T. gondii isolates from humans and animals in China have been reported. However, almost all biological materials used for genotyping of T. gondii from humans and pigs were DNA samples prepared from tissues, and T. gondii strains used for virulence analysis were isolated mainly from cats. In this study, one isolate from a dead human fetus was identified as type I (ToxoDB #10) while the two isolates from dead pigs were type Chinese I (ToxoDB #9) with PCR-restriction fragment length polymorphism using 10 markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). Three isolates were comfirmed as virulent strains in mice. By cloning and sequences analysis, all isolates contained a Pvu II restriction site (572-577 bp) in the KHB fragment and five tandem repeats in the 5' UTR region of SAG1, which were associated with T. gondii virulence. The type Chinese I isolates contained two deletions of 15 and 3 bp at positions 635 to 649 and 658 to 660 in the GRA6, which were correlated with genotype, but not with virulence. To our knowledge, this is the first report on the systematic analysis of murine virulence of type Chinese I strain from pigs, and the associations of sequences of the KHB fragment and SAG1 with virulence of type Chinese I strain. The Chinese I genotype was more closely related to type II strains.