Molecular characteristics and phylogenetic analysis of pigeon paramyxovirus type 1 isolates from pigeon meat farms in Shanghai (2009-2012).

The majority of pigeon paramyxovirus type 1 (PPMV-1) strains are generally non-pathogenic to chickens; however, they can induce severe illness and high mortality rates in pigeons, leading to substantial economic repercussions. The genomes of 11 PPMV-1 isolates from deceased pigeons on meat pigeon farms during passive monitoring from 2009 to 2012 were sequenced and analyzed using polymerase chain reaction and phylogenetic analysis. The complete genome lengths of 11 isolates were approximately 15,192 nucleotides, displaying a consistent gene order of 3'-NP-P-M-F-HN-L-5'. ALL isolates exhibited the characteristic motif of 112RRQKRF117 at the fusion protein cleavage site, which is characteristic of velogenic Newcastle disease virus. Moreover, multiple mutations have been identified within the functional domains of the F and HN proteins, encompassing the fusion peptide, heptad repeat region, transmembrane domains, and neutralizing epitopes. Phylogenetic analysis based on sequences of the F gene unveiled that all isolates clustered within genotype VI in class II. Further classification identified at least two distinct sub-genotypes, with seven isolates classified as sub-genotype VI.2.1.1.2.2, whereas the others were classified as sub-genotype VI.2.1.1.2.1. This study suggests that both sub-genotypes were implicated in severe disease manifestation among meat pigeons, with sub-genotype VI.2.1.1.2.2 displaying an increasing prevalence among Shanghai's meat pigeon population since 2011. These results emphasize the value of developing pigeon-specific vaccines and molecular diagnostic tools for monitoring and proactively managing potential PPMV-1 outbreaks.

A multicenter, randomized, open-label, phase 2 clinical study of telitacicept in adult patients with generalized myasthenia gravis.

BACKGROUND AND PURPOSE: This study aimed to investigate the clinical efficacy and safety of telitacicept in patients with generalized myasthenia gravis (gMG) who tested positive for acetylcholine receptor antibodies or muscle-specific kinase antibodies and were receiving standard-of-care therapy. METHODS: Patients meeting the eligibility criteria were randomly assigned to receive telitacicept subcutaneously once a week for 24 weeks in addition to standard-of-care treatment. The primary efficacy endpoint was the mean change in the quantitative myasthenia gravis (QMG) score from baseline to week 24. Secondary efficacy endpoints included mean change in QMG score from baseline to week 12 and gMG clinical absolute score from baseline to week 24. Additionally, safety, tolerability and pharmacodynamics were assessed. RESULTS: Twenty-nine of the 41 patients screened were randomly selected and enrolled. The mean (± standard deviation [SD]) reduction in QMG score from baseline to week 24 was 7.7 (± 5.34) and 9.6 (± 4.29) in the 160 mg and 240 mg groups, respectively. At week 12, mean reductions in QMG scores for these two groups were 5.8 (± 5.85) and 9.5 (± 5.03), respectively, indicating rapid clinical improvement. Safety analysis revealed no adverse events leading to discontinuation or mortalities. All patients showed consistent reductions in serum immunoglobulin (Ig) A, IgG and IgM levels throughout the study. CONCLUSION: Telitacicept demonstrated safety, good tolerability and reduced clinical severity throughout the study period. Further validation of the clinical efficacy of telitacicept in gMG will be conducted in an upcoming phase 3 clinical trial.

Deciphering antifungal and antibiofilm mechanisms of isobavachalcone against Cryptococcus neoformans through RNA-seq and functional analyses.

Cryptococcus neoformans has been designated as critical fungal pathogens by the World Health Organization, mainly due to limited treatment options and the prevalence of antifungal resistance. Consequently, the utilization of novel antifungal agents is crucial for the effective treatment of C. neoformans infections. This study exposed that the minimum inhibitory concentration (MIC) of isobavachalcone (IBC) against C. neoformans H99 was 8 µg/mL, and IBC dispersed 48-h mature biofilms by affecting cell viability at 16 µg/mL. The antifungal efficacy of IBC was further validated through microscopic observations using specific dyes and in vitro assays, which confirmed the disruption of cell wall/membrane integrity. RNA-Seq analysis was employed to decipher the effect of IBC on the C. neoformans H99 transcriptomic profiles. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis was performed to validate the transcriptomic data and identify the differentially expressed genes. The results showed that IBC exhibited various mechanisms to impede the growth, biofilm formation, and virulence of C. neoformans H99 by modulating multiple dysregulated pathways related to cell wall/membrane, drug resistance, apoptosis, and mitochondrial homeostasis. The transcriptomic findings were corroborated by the antioxidant analyses, antifungal drug sensitivity, molecular docking, capsule, and melanin assays. In vivo antifungal activity analysis demonstrated that IBC extended the lifespan of C. neoformans-infected Caenorhabditis elegans. Overall, the current study unveiled that IBC targeted multiple pathways simultaneously to inhibit growth significantly, biofilm formation, and virulence, as well as to disperse mature biofilms of C. neoformans H99 and induce cell death.

Establishment of RPA-Cas12a-Based Fluorescence Assay for Rapid Detection of Feline Parvovirus.

Feline parvovirus (FPV) is highly infectious for cats and other Felidae and often causes severe damage to young kittens. In this study, we incorporated recombinase polymerase amplification (RPA) and Cas12a-mediated detection and developed an RPA-Cas12a-based real-time or end-point fluorescence detection method to identify the NS1 gene of FPV. The total time of RPA-Cas12a-based fluorescence assay is approximately 25 min. The assay presented a limit of detection (LOD) of 1 copies/µl (25 copies/per reaction), with no cross-reactivity with several feline pathogens. The clinical performance of the assay was examined using total genomic DNA purified from 60 clinical specimens and then compared to results obtained with qPCR detection of FPV with 93.3% positive predictive agreement and 100% negative predictive agreement. Together, the rapid reaction, cost-effectiveness, and high sensitivity make the RPA-Cas12a-based fluorescence assay a fascinating diagnostic tool that will help minimize infection spread through instant detection of FPV.

Isobavachalcone exhibits antifungal and antibiofilm effects against C. albicans by disrupting cell wall/membrane integrity and inducing apoptosis and autophagy.

Isobavachalcone (IBC) is a natural flavonoid with multiple pharmacological properties. This study aimed to evaluate the efficacy of IBC against planktonic growth and biofilms of Candida albicans (C. albicans) and the mechanisms underlying its antifungal action. The cell membrane integrity, cell metabolic viability, and cell morphology of C. albicans treated with IBC were evaluated using CLSM and FESEM analyses. Crystal violet staining, CLSM, and FESEM were used to assess the inhibition of biofilm formation, as well as dispersal and killing effects of IBC on mature biofilms. RNA-seq combined with apoptosis and autophagy assays was used to examine the mechanisms underlying the antifungal action of IBC. IBC exhibited excellent antifungal activity with 8 µg/mL of MIC for C. albicans. IBC disrupted the cell membrane integrity, and inhibited biofilm formation. IBC dispersed mature biofilms and damaged biofilm cells of C. albicans at 32 µg/mL. Moreover, IBC induced apoptosis and autophagy-associated cell death of C. albicans. The RNA-seq analysis revealed upregulation or downregulation of key genes involved in cell wall synthesis (Wsc1 and Fks1), ergosterol biosynthesis (Erg3, and Erg11), apoptisis (Hsp90 and Aif1), as well as autophagy pathways (Atg8, Atg13, and Atg17), and so forth, in response to IBC, as evidenced by the experiment-based phenotypic analysis. These results suggest that IBC inhibits C. albicans growth by disrupting the cell wall/membrane, caused by the altered expression of genes associated with ß-1,3-glucan and ergosterol biosynthesis. IBC induces apoptosis and autophagy-associated cell death by upregulating the expression of Hsp90, and altering autophagy-related genes involved in the formation of the Atg1 complex and the pre-autophagosomal structure. Together, our findings provide important insights into the potential multifunctional mechanism of action of IBC.

Analysis of the correlations between the extracranial internal carotid artery and extracranial vertebral artery and mild cognitive impairment.

BACKGROUND: Vascular tortuosity is a prevalent morphological change that frequently occurs in arteries across different parts of the body. OBJECTIVE: To analyze the relationship between the tortuosities of the extracranial internal carotid artery (EICA) and extracranial vertebral artery (EVA) with mild cognitive impairment. METHODS: The tortuosity index (TI), vascular deviation degree, tortuosity degree, and angle number of the EICA and EVA were retrospectively analyzed and calculated in 160 patients who underwent computed tomography angiography (CTA) in this study's department, and the Montreal cognitive assessment was adopted to evaluate the cognitive function of the patients. RESULTS: The differences in age, gender, arterial hypertension (AH), and diabetes mellitus (DM) between the normal group and the mild cognitive impairment group were statistically significant (p< 0.01). The TI was negatively correlated with the score of cognitive function. The tortuosities of the EICA and EVA were correlated with mild cognitive impairment (p< 0.05). The reduction in visual-spatial ability was correlated with the right EICA tortuosity, and the reduction in memory was correlated with the EVA tortuosity. Age, gender, HP, DM, and coronary heart disease (CHD) were potential risk factors for carotid tortuosity (p< 0.05). CONCLUSION: There was a significant correlation observed between the TIs of both the EICA and EVA and the presence of mild cognitive impairment. Advanced age, female, HP, DM, and CHD were independent risk factors for EICA and EVA tortuosities.

Establishment of RT-RPA-Cas12a assay for rapid and sensitive detection of human rhinovirus B.

Human rhinovirus B (HRV-B) is a major human viral pathogen that can be responsible for various kinds of infections. Due to the health risks associated with HRV-B, it is therefore crucial to explore a rapid, specific, and sensitive method for surveillance. Herein, we exploited a novel detection method for HRV-B by combining reverse-transcription recombinase polymerase amplification (RT-RPA) of nucleic acids isothermal amplification and the trans-cleavage activity of Cas12a. Our RT-RPA-Cas12a-based fluorescent assay can be completed within 35-45 min and obtain a lower detection threshold to 0.5 copies/µL of target RNA. Meanwhile, crRNA sequences without a specific protospacer adjacent motif can effectively activate the trans-cleavage activity of Cas12a. Moreover, our RT-RPA-Cas12a-based fluorescent method was examined using 30 clinical samples, and exhibited high accuracy with positive and negative predictive agreement of 90% and 100%, respectively. Taken together, a novel promising, rapid and effective RT-RPA-Cas12a-based detection method was explored and shows promising potential for on-site HRV-B infection in resource-limited settings.

A Recombinase-Aided Amplification Assay for the Detection of Chlamydia felis.

Chlamydia felis is an important zoonotic agent for humans and various animals. A recombinase-aided amplification (RAA) assay was developed for detecting C. felis. RAA can be performed in a closed tube at 39°C within 30 min. The detection limit was 10.6 copies of the C. felis plasmid DNA per reaction. No positive signals for other pathogens were detected. The coincidence rate of RAA and conventional PCR was 95.24% (20/21) and 100% (96/96) for positive and negative samples, respectively. The established RAA assay is a simple, rapid, highly sensitive, and specific method for detecting C. felis.

Development of RPA-Cas12a-fluorescence assay for rapid and reliable detection of human bocavirus 1.

Human bocavirus (HBoV) 1 is considered an important pathogen that mainly affects infants aged 6-24 months, but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging. Herein, we present a novel faster, lower cost, reliable method for the detection of HBoV1, which integrates a recombinase polymerase amplification (RPA) assay with the CRISPR/Cas12a system, designated the RPA-Cas12a-fluorescence assay. The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37°C without the need for sophisticated instruments. The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens. Furthermore, the method was appraised using 28 clinical samples, and displayed high accuracy with positive and negative predictive agreement of 90.9% and 100%, respectively. Therefore, our proposed rapid and sensitive HBoV1 detection method, the RPA-Cas12a-fluorescence assay, shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care. The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection. The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/µl.

Sensitive and rapid RT-RPA-Cas12a-mediated detection method capable of human rhinovirus A and/or C species by targeting VP4.

Human rhinovirus (HRV), the main etiologic agent of the common cold, is responsible for significant morbidity, medical costs, and the loss of productivity in the workplace and school. To prevent the spread of HRV, accurate, low-cost and rapid diagnostics of HRV is crucial for identifying those at-risk for the illness associated with HRV, with the most frequently detected species, including HRV species A (HRV-A) and C (HRV-C). Here, a novel HRV-A and/or HRV-C molecular diagnostic assay that integrates reverse-transcription recombinase polymerase amplification assay (RT-RPA) amplification with CRISPR/Cas12a detection, with the result readout using a fluorescence detector or lateral flow strip (LFS). The established assay could be completed within 50 min without complex instruments and skilled technicians. The limit of detection of the RT-RPA-Cas12a-mediated real-time fluorescence or LFS assay could reach 0.1 copy/µl, and 0.5 copy/µl for the end-point fluorescence assay with a UV light illuminator readout, respectively. Meanwhile, the assay demonstrates excellent specificity without cross-reactivity to non-target viruses. Furthermore, they were appraised using 80 clinical samples, and RT-RPA-Cas12a-mediated fluorescence or LFS assay displayed high-accuracy with positive and negative predictive agreement of 96.7%, 95% and 100%, respectively. Taken together, the RT-RPA-Cas12a-mediated assay is a rapid, sensitive, and specific detection tool for routine and on-site detection method for HRV-A and/or HRV-C infections, and shows great promise for use in resource-poor or constrained settings.

Ultrasensitive and visual detection of human norovirus genotype GII.4 or GII.17 using CRISPR-Cas12a assay.

BACKGROUND: Integrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens. METHODS: RT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored. RESULTS: The results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/µL within approximately 30-40 min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement. CONCLUSIONS: Together, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings.

Antifungal and Antibiofilm In Vitro Activities of Ursolic Acid on Cryptococcus neoformans.

Ursolic acid (UA) exists in a variety of medicinal plants. UA exhibits antimicrobial activity against several microorganisms; however, little is known regarding the potential antifungal effect of UA on Cryptococcus neoformans (C. neoformans). The antifungal and antibiofilm activities of UA on C. neoformans H99 were evaluated in this study. Minimum inhibitory concentration (MIC) of UA against C. neoformans H99 was determined by microdilution technique, and its action mode was elucidated by clarifying the variations in cell membrane integrity, capsule, and melanin production. Moreover, the inhibition and dispersal effects of UA on biofilm formation and mature biofilms by C. neoformans H99 were evaluated using crystal violet (CV) assay, optical microscopy, field emission scanning electron microscopy and confocal laser scanning microscopy. The results indicated that the MIC value of UA against C. neoformans H99 was 0.25 mg/mL. UA disrupted the cell membrane integrity, inhibited the capsule and melanin production of C. neoformans H99 in a concentration-dependent manner. Further, UA presented the inhibitory effect on biofilm formation and dispersed mature biofilms, as well as compromised the cell membrane integrity of C. neoformans H99 cells within biofilms. Together, these results indicate that UA might be a potential therapeutic option for the treatment of C. neoformans-related infections.

Exploitation of the antifungal and antibiofilm activities of plumbagin against Cryptococcus neoformans.

Cryptococcus neoformans is an important opportunistic fungal pathogen that causes various infections. Here, the antifungal and antibiofilm activities of plumbagin against C. neoformans and the underlying mechanisms were evaluated. The minimum inhibitory concentration (MIC) of plumbagin against C. neoformans H99 was 8 µg ml-1. Plumbagin disrupted the cell membrane integrity and reduced the metabolic activities of C. neoformans H99. C. neoformans H99 biofilm cells were damaged by plumbagin at a concentration of 64 µg ml-1, whereas 48-h mature biofilms were dispersed at a plumbagin concentration of 128 µg ml-1. Whole-transcriptome analysis of plumbagin-treated C. neoformans H99 in the biofilm and planktonic states identified differentially expressed genes enriched in several important cellular processes (cell membrane, ribosome biogenesis, fatty acid synthesis, melanin and capsule production). Notably, plumbagin damaged biofilm cells by downregulating FAS1 and FAS2 expression. Thus, plumbagin can be exploited as an antifungal agent to combat C. neoformans-related infections.

Relationship Between Antibiotic Resistance, Biofilm Formation, and Biofilm-Specific Resistance in Escherichia coli Isolates from Ningbo, China.

Purpose: Several Escherichia coli pathotypes still constitute an important public health concern owing to its pathogenicity and antimicrobial resistance. Moreover, biofilm formation of E. coli can allow the strains to interfere with host and antimicrobial eradication, thus conferring additional resistance. The association between the formation of biofilm and antimicrobial resistance determinants has been extensively exploited; nevertheless, there is still no definite conclusion. The purpose of this study was to provide additional data to augment the present knowledge about the subject. Methods: Antibiotic resistance/susceptibility profiles of 81 isolates from pediatric individuals in China between 2011 and 2014 against 20 antibiotics were assessed using the VITEK 2 system. Biofilm-forming capacities were evaluated using the crystal violet staining method, confocal laser scanning microscopy (CLSM), and field emission scanning electron microscopy. Biofilm compositions inside the biofilm formed by representative strains were assessed using CLSM. The effects of antibiotics on biofilms generated by E. coli strains of different biofilm-forming ability were examined using CLSM in combination with gatifloxacin. The relationships between antibiotic resistance, biofilm formation, and biofilm-specific resistance in E. coli isolates were investigated. Results: The results showed that 23 isolates were classified as multidrug-resistant, and 57 isolates were classified as extensively drug-resistant (XDR). Among the 69 isolates with the ability to form biofilms, 46 isolates were stronger biofilm formers. Correlation analysis demonstrated that strain populations exhibiting more robust biofilm formation likely contained larger proportions of XDR isolates. Conclusion: Together, our study implies that there was an association between biofilm-formation and resistance to several antibiotics for XDR-E. coli isolates, and would provide novel insights regarding the prevention and treatment against E. coli-related infections.

Antifungal and Antibiofilm Efficacy of Paeonol Treatment Against Biofilms Comprising Candida albicans and/or Cryptococcus neoformans.

Fungal populations are commonly found in natural environments and present enormous health care challenges, due to increased resistance to antifungal agents. Paeonol exhibits antifungal activities; nevertheless, the antifungal and antibiofilm activities of paeonol against Candida albicans and Cryptococcus neoformans remain largely unexplored. Here, we aimed to evaluate the antifungal and antibiofilm activities of paeonol against C. albicans and/or C. neoformans (i.e., against mono- or dual-species). The minimum inhibitory concentrations (MICs) of paeonol for mono-species comprising C. albicans or C. neoformans were 250 µg ml-1, whereas the MIC values of paeonol for dual-species were 500 µg ml-1. Paeonol disrupted cell membrane integrity and increased the influx of gatifloxacin into cells of mono- and dual-species cells, indicating an antifungal mode of action. Moreover, paeonol at 8 times the MIC damaged mono- and dual-species cells within C. albicans and C. neoformans biofilms, as it did planktonic cells. In particular, at 4 and 8 mg ml-1, paeonol efficiently dispersed preformed 48-h biofilms formed by mono- and dual-species cells, respectively. Paeonol inhibited effectively the yeast-to-hyphal-form transition of C. albicans and impaired capsule and melanin production of C. neoformans. The addition of 10 MIC paeonol to the medium did not shorten the lifespan of C. elegans, and 2 MIC paeonol could effectively protect the growth of C. albicans and C. neoformans-infected C. elegans. Furthermore, RNA sequencing was employed to examine the transcript profiling of C. albicans and C. neoformans biofilm cells in response to 1/2 MIC paeonol. RNA sequencing data revealed that paeonol treatment impaired biofilm formation of C. albicans by presumably downregulating the expression level of initial filamentation, adhesion, and growth-related genes, as well as biofilm biosynthesis genes, whereas paeonol inhibited biofilm formation of C. neoformans by presumably upregulating the expression level of ergosterol biosynthesis-related genes. Together, the findings of this study indicate that paeonol can be explored as a candidate antifungal agent for combating serious single and mixed infections caused by C. albicans and C. neoformans.

Effect of paeonol against bacterial growth, biofilm formation and dispersal of Staphylococcus aureus and Listeria monocytogenes in vitro.

Previous studies have demonstrated the antibacterial activity of paeonol against bacterial pathogens, but its anti-biofilm activities against Staphylococcus aureus and Listeria monocytogenes remain largely unexplored. Here, the antibacterial and anti-biofilm activities of paeonol against S. aureus and L. monocytogenes were examined using the crystal violet staining assay (CVSA), field emission scanning electron microscopy (FESEM), and confocal laser scanning microscopy (CLSM) analysis. Paeonol effectively inhibited the growth of S. aureus and L. monocytogenes with a minimum inhibitory concentration (MIC) of 500 and 125 µg ml-1, respectively, and disrupted the integrity of cell membranes. Moreover, sub-MIC paeonol exhibited an inhibitory effect on the attachment of S. aureus and L. monocytogenes to the abiotic surface and biofilm formation. Further, paeonol effectively destroyed cell membranes within biofilms, and dispersed mature biofilms of both strains. The results indicate that paeonol might be a promising antibacterial and anti-biofilm agent for combating infections caused by S. aureus and L. monocytogenes.

Cantharidin induces senescence via inhibition of AMP-activated protein kinase and activation of NLRP3 inflammasome in H9c2 cardiomyocytes.

Cantharidin is a natural compound with cardiotoxicity. Cellular senescence and senescence-associated secretory phenotype (SASP) are implicated in chemotherapy-associated cardiotoxicity. We here investigated how cantharidin induced cardiomyocyte senescence. H9c2 cells were treated with cantharidin. Senescence, mitochondrial functions, SASP, NOD-like receptor thermal protein domain associated protein 3 (NLRP3) signaling and AMP-activated protein kinase (AMPK) phosphorylation were examined. Cantharidin inhibited viability and increased expression of senescence-associated ß--galactosidase (SA-ß-Gal), p16 and p21 in H9c2 cells, suggesting occurrence of senescence. Cantharidin impaired mitochondrial functions evidenced by reduction in basal respiration, ATP levels and spare respiratory capacity. Cantharidin also decreased mitochondrial DNA copy number and down-regulated mRNA levels of cytochrome c oxidase-I, -II and -III. Moreover, cantharidin suppressed activity of mitochondria complex-I and -II. Examinations of SASP showed that cantharidin promoted expression and secretion of SASP cytokines interleukin-1ß-, -6 and -8 and tumor necrosis factor-α, associated with activation of NLRP3/caspase-1 pathway. Finally, cantharidin suppressed AMPK phosphorylation. AMPK activator GSK621 abrogated the up-regulation of SA-ß--Gal, p16 and p21 and counteracted the activation of NLRP3 and caspase-1 in cantharidin-challenged H9c2 cells. In conclusion, cantharidin stimulated senescence and SASP in cardiomyocytes through activation of NLRP3 inflammasome and inhibition of AMPK, providing novel molecular insights into cantharidin-induced cardiotoxicity.

Plasma Proteomics Characteristics of Subclinical Vitamin E Deficiency of Dairy Cows During Early Lactation.

Vitamin E (VE) is an essential fat-soluble nutrient for dairy cows. Vitamin E deficiency leads to immune suppression and oxidative stress and increases the susceptibility of cows to reproductive disorders in the early post-partum period. However, studies on plasma proteomics of VE deficiency have not been reported so far. Therefore, the purpose of this study was to understand the changes of blood protein profile in cows with subclinical VE deficiency in the early post-partum period. In this study, plasma protein levels of 14 healthy cows (>4 µg/ml α-tocopherol) and 13 subclinical VE-deficient cows (2-3 µg/ml α-tocopherol) were analyzed by tandem mass tag (TMT). The results showed that there were 26 differentially expressed proteins (DEPs) in the plasma of cows with subclinical VE deficiency compared with healthy controls. Twenty-one kinds of proteins were downregulated, and five kinds were upregulated, among which eight proteins in protein-protein interactions (PPI) network had direct interaction. These proteins are mainly involved in the MAPK signaling pathway, pantothenic acid and coenzyme A (CoA) biosynthesis, PPAR signaling pathway, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. The top four DEPs in PPI (APOC3, APOC4, SAA4, PHLD) and one important protein (VNN1) by literature review were further verified by ELISA and Western blot. The expression levels of APOC3, VNN1, and SAA4 were significantly lower than those of healthy controls by ELISA. VNN1 was significantly lower than those of healthy controls by Western blot. VNN1 is closely related to dairy cow subclinical VE deficiency and can be a potential biomarker. It lays a foundation for further research on the lack of pathological mechanism and antioxidative stress of VE.

Antibiofilm Efficacy of Luteolin Against Single and Dual Species of Candida albicans and Enterococcus faecalis.

Candida albicans and Enterococcus faecalis biofilm-associated infections have been a huge challenge to the medical community. However, the efficacy of natural products against mixed biofilms of C. albicans and E. faecalis still remains largely unexploited. The aim of this study was to evaluate the efficacy of luteolin against planktonic cell growth, adhesion, and biofilm formation of C. albicans and E. faecalis in single and mixed cultures in vitro. The results showed that the minimum inhibitory concentrations of luteolin against planktonic cells of C. albicans, E. faecalis, and mixed cultures were 32 and 64 µg ml-1, respectively. The results displayed that a remarkable variation in biofilm biomass, viability, structure, and composition of single and dual-species biofilms formed by mono- and dual-species biofilms of C. albicans and E. faecalis in the presence of luteolin was confirmed by mainly crystal violet staining assay (CVSA), optical microscope, field emission scanning electron microscope (FESEM), and confocal laser scanning microscope (CLSM). The tolerance of luteolin-treated single- and dual-species biofilms to antibiotics was found to obviously decrease, and the loss of biofilm matrix components (mainly polysaccharides and proteins) was revealed by CLSM. Moreover, luteolin was effective at inactivating biofilm cells, as well as destructing preformed biofilm structures by single and dual species by CVSA, FESEM, and CLSM. Collectively, these data indicate the potential of luteolin as a promising antibiofilm agent for the therapeutic management of biofilm-related infections induced by single and dual species of C. albicans and E. faecalis.

CRISPR-Cas12a combined with reverse transcription recombinase polymerase amplification for sensitive and specific detection of human norovirus genotype GII.4.

Human norovirus (NOV) is a common and serious virus that accounts for sporadic cases and outbreaks of gastroenteritis. This study aimed to develop rapid, reliable and portable detection systems by coupling reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a (RT-RPA-Cas12a) for NOV genotype GII.4. Here, three primers for RNA-dependent RNA polymerase gene of NOV were designed and screened. Then, RT-RPA products were detected using CRISPR-Cas12a system by combing with fluorescence or lateral flow (LF). RT-RPA-Cas12a-based fluorescence or LF assay can be completed within 40 min, with the detection limit of up to 9.65 × 102copies/mL and no cross-reactivity with metapneumovirus, bocavirus, seoul virus, and respiratory syncytial virus. Furthermore, the detection coincidence rates of RT-RPA-Cas12a-based fluorescence and LF with qRT-PCR were 98.3%. Therefore, the present study suggests that both RT-RPA-Cas12a-based fluorescence and LF are promising sensitive, specific and alternative method for diagnosis of NOV genotype GII.4 without ancillary equipment.