Fully functional monocytic MDSC generation from the murine HoxB8 cell line.

Myeloid-derived suppressor cells (MDSC) play a crucial role in controlling T-cell responses, but their development and suppressor mechanisms are not fully understood. To study the molecular functions of MDSC, a large number of standardized cells are required. Traditionally, bone marrow (BM) has been used to generate myeloid cell types, including MDSC. In this study, we demonstrate that a previously described protocol for generating monocytic MDSC (M-MDSC) from murine BM with GM-CSF can be fully transferred to BM cells that are conditionally transformed with HoxB8 gene (HoxB8 cells). HoxB8 cells have an extended lifespan and efficiently differentiate into MDSC that are quantitatively and qualitatively comparable to M-MDSC from BM cells. Flow cytometric analyses of LPS/IFN-γ activated cultures revealed the same iNOS+ and/or Arg1+ PD-L1high M-MDSC subsets in similar frequencies from BM or HoxB8 cells. In vitro suppression of CD4+ and CD8+ T-cell proliferations was also largely comparable in their efficacy and its iNOS- or Arg1-dependent suppressor mechanisms, which was confirmed by the similar amounts of nitric oxide (NO) secretion measured from the suppressor assay. Therefore, our data suggest that murine M-MDSC generation from HoxB8 cells with GM-CSF can be used to substitute BM cultures.

Guidelines for mouse and human DC generation.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34+ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34+ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

CRISPR/Cas9 editing in conditionally immortalized HoxB8 cells for studying gene regulation in mouse dendritic cells.

HoxB8 multipotent progenitors (MPP) are obtained by expression of the estrogen receptor hormone binding domain (ERHBD) HoxB8 fusion gene in mouse BM cells. HoxB8 MPP generate (i) the full complement of DC subsets (cDC1, cDC2, and pDC) in vitro and in vivo and (ii) allow CRISPR/Cas9-mediated gene editing, for example, generating homozygous deletions in cis-acting DNA elements at high precision, and (iii) efficient gene repression by dCas9-KRAB for studying gene regulation in DC differentiation.

Effect on 30-day mortality and duration of hospitalization of empirical antibiotic therapy in CRGNB-infected pneumonia.

BACKGROUND: The objective of this study was to investigate whether unreasonable empirical antibiotic treatment (UEAT) had an impact on 30-day mortality and duration of hospitalization in bacterial pneumonia caused by carbapenem-resistant gram-negative bacteria (CRGNB). METHODS: This was a retrospective cohort study involving CRGNB-infected pneumonia. All CRGNB-infected pneumonia patients received empirical and targeted antibiotic treatment (TAT), and they were divided into reasonable empirical antibiotic treatment (REAT) and UEAT according to whether the empirical antibiotic treatment (EAT) was reasonable. The data of the two groups were compared to analyze their influence on the 30-day mortality and hospitalization time in CRGNB-infected pneumonia patients. Moreover, we also considered other variables that might be relevant and conducted multivariable regression analysis of 30-day mortality and duration of hospitalization in CRGNB-infected pneumonia patients. RESULTS: The study collected 310 CRGNB-infected pneumonia patients, the most common bacterium is Acinetobacter baumannii (211/310 [68%]), the rest were Klebsiella pneumoniae (46/310 [15%]), Pseudomonas aeruginosa and others (53/310 [17%]). Among them, 76/310 (24.5%) patients received REAT. In the analysis of risk factors, dementia, consciousness were risk factors of 30-day mortality, pulmonary disease, hemodynamic support at culture taken day and recent surgery were risk factors for longer hospital stay. The analysis of 30-day mortality showed that UEAT was not associated with 30-day mortality for the 30-day mortality of REAT and UEAT were 9 of 76 (11.84%) and 36 of 234 (15.38%) (P = 0.447), respectively. Meanwhile, there was difference between REAT and UEAT (P = 0.023) in the analysis of EAT on hospitalization time in CRGNB-infected pneumonia patients. CONCLUSIONS: UEAT was not associated with 30-day mortality while was related to duration of hospitalization in CRGNB-infected pneumonia patients, in which Acinetobacter baumanniii accouned for the majority.

Characterization of Myostain (MSTN) and Myogenic Differentiation Antigen (MyoD) and the Effect of Dexamethasone on Their Expression in Large-Scale Loach Paramisgurnus dabryanus.

Myostatin (MSTN) and myogenic differentiation antigen (MyoD) play an essential role in specification and differentiation of skeletal muscle. However, the role of stress in the regulation of MyoD and MSTN has not been fully revealed and more evidence should be provided. Here, we reported the cloning and expressional analyses of MSTN and MyoD in Large-scale Loach Paramisgurnus dabryanus (hereafter PdMSTN and PdMyoD). Injecting individuals with 0, 60, 600, and 1,200 µg/kg dexamethasone (DXM) for five consecutive days resulted in a dose-dependent change of PdMSTN and PdMyoD expression. The expression of PdMSTN was upregulated with increasing DXM concentrations, while PdMyoD expression was downregulated. The changes in the expression of these genes at different time points for 10 consecutive days were studied after individuals were treated with 600 µg/kg DXM. Compared with the control group, PdMSTN expression decreased and PdMyoD expression increased before 12 h, and both PdMSTN and PdMyoD expression levels increased at 24 h, which was significantly higher than those in control group. At a prolonged treatment of 5-10 d, expression levels of PdMSTN and PdMyoD had significantly reduced. The results indicate that both PdMyoD and PdMSTN are involved in DXM-induced stress in Large-scale Loach.

miR-545 promoted enterovirus 71 replication via directly targeting phosphatase and tensin homolog and tumor necrosis factor receptor-associated factor 6.

Enterovirus 71 (EV71) is a small, nonenveloped icosahedral RNA virus and is the predominant causative pathogen of hand-foot-and-mouth disease. Recently, microRNAs (miRNAs) are reported to play important roles in the pathogenesis of EV71 replication. This study investigated the role of miR-545 in the EV71 replication and explored the underlying molecular mechanisms. We showed that miR-545 was upregulated in the EV71-infected human embryonic kidney (HEK) 293 cells and rhabdomyosarcoma (RD) cells. Overexpression of miR-545 promoted the viral replication of EV71 and attenuated the inhibitory effects of EV71 on cell viability in HEK293 and RD cells; while knockdown of miR-545 significantly suppressed the EV71 replication in these two cell lines. Bioinformatics analysis and luciferase reporter assay showed that miR-545 directly targeted the 3'untranslated region of phosphatase and tensin homolog (PTEN) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in HEK293 cells. Furthermore, miR-545 negatively regulated the messenger RNA (mRNA) and protein expression of PTEN and TRAF6. The mRNA and protein expression of PTEN and TRAF6 was also suppressed by EV71 infection, which was attenuated by miR-545 knockdown in HEK293 cells. Overexpression of PTEN and TRAF6 both suppressed the EV71 replication in HKE293 cells, and also attenuated the enhanced effects of miR-545 overexpression on the EV71 replication in HEK293 cells. Collectively, our study for the first time showed that miR-545 had an enhanced effect on the EV71 replication in HEK293 and RD cells. Further mechanistic results indicated that miR-545 promoted EV71 replication at least partly via targeting PTEN and TRAF6.

Expression of Matrix Metalloproteinase-2, Tissue Inhibitor of Matrix Metalloproteinase-2 and CD147 in the Traditional Chinese Medicine "Compound T11" for Treatment of Chronic Liver Injury.

OBJECTIVES: To measure the expression of matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinase inhibitor (TIMP)-2, and CD147 in mice with chronic liver injury induced by carbon tetrachloride after treatment with the traditional Chinese medicine (TCM) "Compound T11". METHOD: Sixty male ICR mice were divided randomly into 6 groups of 10: control (C), model (M), low-dose treatment (LT; 50 mg/mL of Compound T11), medium-dose treatment (MT, 100 mg/mL), high-dose treatment (HT, 150 mg/mL), and positive drug treatment (YT, 67.5 mg/mL). Each group was modeled for 7 weeks. Groups M, LT, MT, HT, and YT were injected (s.c.) with 20% carbon tetrachloride diluted with olive oil, and group C was given olive oil in the same way twice a week. After modeling, the treatment groups were administered Compound T11 at the concentrations shown above by oral gavage daily for 2 weeks, while group C was given 0.5% carboxymethyl cellulose sodium. After the final treatment, mice were killed and their liver tissues were excised. Immunohistochemical staining was performed to measure the protein expression of MMP-2, TIMP-2, and CD147, and western blotting was used to measure the protein expression of MMP-2, TIMP-2, CD147, and α-smooth muscle actin (SMA). MMP-2, TIMP-2, and CD147 mRNA expression was determined by quantitative fluorescence real-time PCR. RESULTS: Compound T11 increased the protein expression of MMP-2 and CD147 and decreased the protein expression of TIMP-2 and α-SMA. CONCLUSIONS: Treatment of chronic liver injury by TCM Compound T11 may be associated with changes to the expression of MMP-2 and CD147, and the inhibition of TIMP-2 expression.

Characterization of the Gut Microbiota in Six Geographical Populations of Chinese Rhesus Macaques (Macaca mulatta), Implying an Adaptation to High-Altitude Environment.

Knowledge about the impact of different geographical environments on rhesus macaque gut microbiota is limited. In this study, we compared the characteristics of gut microbiota in six different Chinese rhesus macaque populations, including Hainan, Nanning, Guizhou, Xichang, Jianchuan and Tibet. Through the composition analysis of operational taxonomic units (OTUs), we found that there were significant differences in the abundance of core overlapping OTUs in the six Chinese groups. Specifically, the Tibet population exhibited the highest gut microbial diversity and the most unique OTUs. Statistically significant differences in the composition of gut microbiota among the six groups at phylum and family level were evident. Specifically, Tibet had higher abundances of Firmicutes and lower abundances of Bacteroidetes than the other geographical groups, and the higher abundance of Firmicutes in the Tibetan group was mainly caused by a significant increase in the family Ruminococcaceae and Christensenellaceae. Phylogenetic investigation of communities by reconstruction of unobserved state analysis showed that the enrichment ratio for environmental information processing and organismal systems was the highest in the Tibet population. Additionally, our results suggested that in the adaptation process of rhesus macaques to different geographical environments, the abundance of the core common flora of the intestinal microbes had undergone varying degree of change and produced new and unique flora, both of which helped to reshape the gut microbiota of rhesus macaques. In particular, this change was more obvious for animals in the high-altitude environments.

Transcriptome-wide N 6 -methyladenosine methylome profiling of porcine muscle and adipose tissues reveals a potential mechanism for transcriptional regulation and differential methylation pattern.

BACKGROUND: N 6 -methyladenosine (m6A) is the most prevalent internal form of modification in messenger RNA in higher eukaryotes and potential regulatory functions of reversible m6A methylation on mRNA have been revealed by mapping of m6A methylomes in several species. m6A modification in active gene regulation manifests itself as altered methylation profiles in a tissue-specific manner or in response to changing cellular or species living environment. However, up to date, there has no data on m6A porcine transcriptome-wide map and its potential biological roles in adipose deposition and muscle growth. METHODS: In this work, we used methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq) technique to acquire the first ever m6A porcine transcriptome-wide map. Transcriptomes of muscle and adipose tissues from three different pig breeds, the wild boar, Landrace, and Rongchang pig, were used to generate these maps. RESULTS: Our findings show that there were 5,872 and 2,826 m6A peaks respectively, in the porcine muscle and adipose tissue transcriptomes. Stop codons, 3'-untranslated regions, and coding regions were found to be mainly enriched for m6A peaks. Gene ontology analysis revealed that common m6A peaks in nuclear genes are associated with transcriptional factors, suggestive of a relationship between m6A mRNA methylation and nuclear genome transcription. Some genes showed tissue- and breed-differential methylation, and have novel biological functions. We also found a relationship between the m6A methylation extent and the transcript level, suggesting a regulatory role for m6A in gene expression. CONCLUSION: This comprehensive map provides a solid basis for the determination of potential functional roles for RNA m6A modification in adipose deposition and muscle growth.

A Method for Detecting Five Carbapenemases in Bacteria Based on CRISPR-Cas12a Multiple RPA Rapid Detection Technology.

Introduction: As the last line of defense for clinical treatment, Carbapenem antibiotics are increasingly challenged by multi-drug resistant bacteria containing carbapenemases. The rapid spread of these multidrug-resistant bacteria is the greatest threat to severe global health problems. Methods: To solve the problem of rapid transmission of this multidrug-resistant bacteria, we have developed a rapid detection technology using CRPSPR-Cas12a gene editing based on multiple Recombinase polymerase amplification. This technical method can directly isolate the genes of carbapenemase-containing bacteria from samples, with a relatively short detection time of 30 minutes. The instrument used for the detection is relatively inexpensive. Only a water bath can complete the entire experiment of Recombinase polymerase amplification and trans cleavage. This reaction requires no lid during the entire process while reducing a large amount of aerosol pollution. Results: The detection sensitivity of this method is 1.5 CFU/mL, and the specificity is 100%. Discussion: This multi-scene detection method is suitable for screening populations in wild low-resource environments and large-scale indoor crowds. It can be widely used in hospital infection control and prevention and to provide theoretical insights for clinical diagnosis and treatment.

Molecular orbital breaking in photo-mediated organosilicon Schiff base ferroelectric crystals.

Ferroelectric materials, whose electrical polarization can be switched under external stimuli, have been widely used in sensors, data storage, and energy conversion. Molecular orbital breaking can result in switchable structural and physical bistability in ferroelectric materials as traditional spatial symmetry breaking does. Differently, molecular orbital breaking interprets the phase transition mechanism from the perspective of electronics and sheds new light on manipulating the physical properties of ferroelectrics. Here, we synthesize a pair of organosilicon Schiff base ferroelectric crystals, (R)- and (S)-N-(3,5-di-tert-butylbenzylidene)-1-((triphenylsilyl)oxy)ethanamine, which show optically controlled phase transition accompanying the molecular orbital breaking. The molecular orbital breaking is manifested as the breaking and reformation of covalent bonds during the phase transition process, that is, the conversion between C = N and C-O in the enol form and C-N and C = O in the keto form. This process brings about photo-mediated bistability with multiple physical channels such as dielectric, second-harmonic generation, and ferroelectric polarization. This work further explores this newly developed mechanism of ferroelectric phase transition and highlights the significance of photo-mediated ferroelectric materials for photo-controlled smart devices and bio-sensors.

Modulating the ferroelectric phases in cholesteryl-based organic compounds with perfluoroalkyl tail engineering.

Here, we synthesized a series of cholesteryl-based compounds, whose phases and their transformation can be modulated by temperature and the chain length of the fluoroalkyl moieties. To our knowledge, this is the first time that the phase transition could be modulated with perfluoroalkyl tail engineering in organic single-component ferroelectric crystals.

The Characteristics of Extended-Spectrum ß-Lactamases (ESBLs)-Producing Escherichia coli in Bloodstream Infection.

Background: Bloodstream infection (BSI) is a common type of infection frequently diagnosed in clinics. The emergence and spread of ESBLs-producing Escherichia coli (E. coli) has emerged as one of the biggest challenges in global community health. Methods: The production of ESBLs was determined by the composite disk diffusion method. The expression of the various resistance and virulence genes were detected by PCR and sequencing. Multi-locus sequence typing (MLST) and phylogenetic groups were used for the classification. The transfer of resistant plasmids was determined by conjugation assay. The statistical differences were analyzed using Statistical Product and Service Solutions (SPSS) version 23.0. Results: A total of 60 strains of ESBLs-producing E. coli were collected. The resistance genes that were identified included bla CTX-M, bla TEM, bla SHV, bla OXA-1 and mcr-1. The most common one was the bla CTX-M including bla CTX-M-27 (n = 16), bla CTX-M-14 (n = 15), bla CTX-M-15 (n = 11), bla CTX-M-55 (n = 14) and bla CTX-M-65 (n = 5). A total of 31 STs were detected, and the most abundant among which was ST131 (n = 16, 26.7%). Most of the E. coli (n = 46, 76.7%) belonged to the groups B2 and D. And some virulence genes were related to the classification of the E. coli. Among them, the detection rates of hek/hra, kpsMII and papGII-III in groups B2 and D were higher than those in groups A and B1. The detection rates of cnf1, iucC and papGII-III in ST131 were higher than those in non-ST131. And the distributions of hek/hra, iroN, iucC, kpsMII and papGII-III were related to the bla CTX-M subtypes. Finally, most bacterial (n = 32, 53.3%) resistance genes could be transferred between the bacteria by plasmids, especially IncFIB. Conclusion: ESBLs-producing E. coli in BSI exhibited had high resistance rates and carried a variety of virulence factors (VFs). This is necessary to strengthen the monitoring of ESBLs-producing isolates in the medical environment.

Combination of AS101 and Mefloquine Inhibits Carbapenem-Resistant Pseudomonas aeruginosa in vitro and in vivo.

Background: In recent years, carbapenem-resistant Pseudomonas aeruginosa (CRPA) has spread around the world, leading to a high mortality and close attention of medical community. In this study, we aim to find a new strategy of treatment for CRPA infections. Methods: Eight strains of CRPA were collected, and PCR detected the multi-locus sequence typing (MLST). The antimicrobial susceptibility test was conducted using the VITEK@2 compact system. The minimum inhibitory concentration (MIC) for AS101 and mefloquine was determined using the broth dilution method. Antibacterial activity was tested in vitro and in vivo through the chessboard assay, time killing assay, and a mouse model. The mechanism of AS101 combined with mefloquine against CRPA was assessed through the biofilm formation inhibition assay, electron microscopy, and detection of reactive oxygen species (ROS). Results: The results demonstrated that all tested CRPA strains exhibited multidrug resistance. Moreover, our investigation revealed a substantial synergistic antibacterial effect of AS101-mefloquine in vitro. The assay for inhibiting biofilm formation indicated that AS101-mefloquine effectively suppressed the biofilm formation of CRPA-5 and CRPA-6. Furthermore, AS101-mefloquine were observed to disrupt the bacterial cell wall and enhance the permeability of the cell membrane. This effect was achieved by stimulating the production of ROS, which in turn hindered the growth of CRPA-3. To evaluate the therapeutic potential, a murine model of CRPA-3 peritoneal infection was established. Notably, AS101-mefloquine administration resulted in a significant reduction in bacterial load within the liver, kidney, and spleen of mice after 72 hours of treatment. Conclusion: The present study showed that the combination of AS101 and mefloquine yielded a notable synergistic bacteriostatic effect both in vitro and in vivo, suggesting a potential clinical application of this combination in the treatment of CRPA.

A PCR-Reverse Dot Blot Hybridization Based Microfluidics Detection System for the Rapid Identification of 13 Fungal Pathogens Directly After Blood Cultures Over a Period of Time.

Introduction: It is time-consuming to identify fungal pathogens from positive blood cultures using the standard culture-based method. And delayed diagnosis of bloodstream infection leads to significantly increased mortality. Methods: We developed a PCR-reverse dot blot hybridization combined with microfluidic chip techniques to rapidly identify 13 fungal pathogens within 3-4 h using the sample of blood cultured over a period of time. Results: We performed clinical validation using 43 blood culture-positive samples with a sensitivity of 96.7%, a specificity of 100%, and a concordance rate of 97.7%. Samples with different culture durations were evaluated using our approach, showing a detection rate of 85.2% at 16 h and 96.3% at 24 h; the platform could reach a detection limit of 103cfu/mL for the Candida spp. and 103 copies/mL for Aspergillus spp. Discussion: The detection rate of the platform is much higher than the positive rates of concurrent blood cultures. This method bears substantial clinical application potential as it incorporates the microfluidic platform with low reagent consumption, automation, and low cost (about 10 dollars).

Comprehensive Study of Untargeted Metabolomics and 16S rRNA Reveals the Mechanism of Fecal Microbiota Transplantation in Improving a Mouse Model of T2D.

Background: Fecal microbiota transplantation (FMT) has emerged as a new therapy targeting gastrointestinal microbiota for the treatment of a growing number of diseases in recent years. Previous studies have suggested that FMT may be a potential therapy for type 2 diabetes (T2D), but the underlying mechanism remains unclear. Therefore, in the present study, we aimed to investigate the role of FMT in T2D and its underlying mechanisms. Methods: To induce T2D, mice were fed a high-fat diet and injected with low-dose streptozotocin (STZ) for four weeks. The mice were then randomly divided into four groups: control group (n = 7), T2D group (n = 7), metformin (MET)-treated group (n = 7), and FMT group (n = 7). The MET group was orally administered 0.2 g/kg MET, the FMT group was orally administered 0.3 mL of bacterial solution, and the other two groups were orally administered the same volume of saline for four weeks. Serum and fecal samples were collected for non-targeted metabolomics, biochemical indicators, and 16S rRNA sequencing, respectively. Results: Our results demonstrated that FMT had a curative effect on T2D by ameliorating hyperlipidemia and hyperglycemia. Using 16S rRNA sequencing and serum untargeted metabolomic analysis, we found that FMT could restore the disorders of gastrointestinal microbiota in T2D mice. Moreover, corticosterone, progesterone, L-urobilin, and other molecules were identified as biomarkers after FMT treatment. Our bioinformatics analysis suggested that steroid hormone biosynthesis, arginine, proline metabolism, and unsaturated fatty acid biosynthesis could be potential regulatory mechanisms of FMT. Conclusion: In summary, our study provides comprehensive evidence for the role of FMT in the treatment of T2D. FMT has the potential to become a promising strategy for the treatment of metabolic disorders, T2D, and diabetes-related complications.

A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation.

Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8) is a lineage determining transcription factor in hematopoiesis and master regulator of dendritic cells (DC), an important immune cell for immunity and tolerance. IRF8 is prominently upregulated in DC development by autoactivation and controls both DC differentiation and function. However, it is unclear how Irf8 autoactivation is controlled and eventually limited. Here, we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of Irf8 transcription start site and expressed specifically in mouse plasmacytoid DC (pDC), referred to as lncIrf8. The lncIrf8 locus interacts with the lrf8 promoter and shows differential epigenetic signatures in pDC versus classical DC type 1 (cDC1). Interestingly, a sequence element of the lncIrf8 promoter, but not lncIrf8 itself, is crucial for mouse pDC and cDC1 differentiation, and this sequence element confers feedback inhibition of Irf8 expression. Taken together, in DC development Irf8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIrf8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of Irf8 that orchestrates its own expression and thereby controls DC differentiation.

Establishment and Methodological Evaluation of a Method for Rapid Detection of Helicobacter pylori and Virulence Genes Based on CRISPR-Cas12a.

Introduction: More than half of the world's people are infected or have been infected with Helicobacter pylori. This infection is related to many diseases, with its pathogenicity related to virulence factors. Therefore, the rapid diagnosis of H. pylori and genotyping of virulence genes play an extremely important role in the clinical treatment and control of transmission. Methods: To this end, we developed a molecular detection method based on RPA- CRISPR-Cas12a technology for the specific genes 16S rDNA gene, cytotoxin associated gene A(cagA), and vacuolating cytotoxin A (vacA) of H. pylori. Results: The results of which were displayed by lateral flow strips. Macroscopic observation takes only about 25 minutes and the sensitivity is 2ng/microliter. Discussion: The method is simple, convenient to operate and has low costs, and can therefore be applied widely to the detection and typing of H. pylori in various environments such as primary hospitals, community clinics, outdoors, and large medical institutions.

Rapid visualization of Clostridioides difficile toxins A and B by multiplex RPA combined with CRISPR-Cas12a.

Purpose: Clostridioides difficile (C. difficile) infection is the most common cause of nosocomial infection, which is a severe challenge in modern medical care. Currently, many laboratory diagnostic methods for C. difficile are available, such as PCR, culture-based tests, and antigen-based tests. However, these methods are not suitable for rapid point-of-care testing (POCT). Therefore, it is of great significance to develop a rapid, sensitive, and cost-effective method to detect C. difficile toxin genes. Methods: Recently, the development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for rapid POCT. In this study, we developed a rapid and specific detection platform for dual C. difficile toxins by combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a. Results: The platform includes multiplex RPA-cas12a-fluorescence assay and multiplex RPA-cas12a-LFS (Lateral flow strip) assay, through which the detection limit for tcdA and tcdB was 10 copies/µL and 1 copy/µL, respectively. The results can be more clearly distinguished using a violet flashlight, which realized a portable visual readout. The platform can be tested within 50 min. Furthermore, our method did not cross-react with other pathogens that cause intestinal diarrhea. The results of 10 clinical samples using our method was 100% consistent with those from real-time PCR detection. Conclusion: In conclusion, the CRISPR-based double toxin gene detection platform for C. difficile is an effective, specific, and sensitive detection method, which can be used as a powerful on-site detection tool for POCT in the future.

Study on the Regulation of Exogenous Hormones on the Absorption of Elements and the Accumulation of Secondary Metabolites in the Medicinal Plant Artemisia argyi Leaves.

As an important medicinal plant, we still do not know the effect of exogenous hormones on absorption of elements and accumulation of secondary metabolites in Artemisia argyi leaves. In this work, we analyzed the difference in 21 elements absorbed by A. argyi leaves under three exogenous hormone (MeJA, SA and ABA) treatments, and also clarified the correlation between 21 elements and eight bioactive components. Different hormone treatments changed the absorption and enrichment of elements, and the composition also changed significantly. The contents of eight bioactive components changed significantly under different hormone treatments. When A. argyi was stimulated by exogenous hormones, the content of secondary metabolites was adjusted in the leaves through changes in the absorption and enrichment of elements. The widely untargeted metabolomic analysis further confirmed that ABA changes the metabolic direction of secondary metabolites in A. argyi leaves and stimulates the biosynthesis of multiple secondary metabolites including phenylpropanoids, flavonoids, terpenoids, alkaloids and others. These results provide a new perspective for the changes in element absorption and the mechanism of secondary metabolic components in A. argyi leaves under exogenous hormone treatments, and also deepen people's understanding of the interaction mechanism between medicinal plants and hormones.