Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii.

Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within  30-40 min, comprising a 25-30 min RPA reaction and a 5-10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33%. In conclusion, a novel, rapid, sensitive, and cost-effective RPA-Cas12a-based detection method was developed and demonstrates significant potential for on-site detection of P. jirovecii in resource-limited settings.

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 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.

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.

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.

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.

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.

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.

Antibacterial and anti-biofilm activities of paeonol against Klebsiella pneumoniae and Enterobacter cloacae.

Paeonol, the active ingredient of Paeonia lactiflora root bark, is widely used in traditional Chinese medicine. Few studies have reported the antibacterial activity of paeonol against bacterial pathogens. In this study, the antibacterial and anti-biofilm performance of paeonol against Klebsiella pneumoniae and Enterobacter cloacae was investigated as well as its mechanisms of action. Paeonol effectively inhibited the growth of K. pneumoniae and E. cloacae with a minimum inhibitory concentration of 64 µg ml-1 and it was shown to disrupt the integrity of bacterial cell membranes, and alter cell morphology. Moreover, paeonol exhibited a potent inhibitory effect against adhesion and biofilm formation by K. pneumoniae and E. cloacae. In particular, paeonol efficiently compromised cells within biofilms, and dispersed mature biofilms. Therefore, the present study suggests that paeonol is a promising alternative antibacterial and anti-biofilm agent for combating infections caused by planktonic and biofilm cells of K. pneumoniae and E. cloacae.

In vitro anti-biofilm efficacy of sanguinarine against carbapenem-resistant Serratia marcescens.

Sanguinarine, a plant-derived benzophenanthridine alkaloid, was studied in terms of its anti-biofilm effects against carbapenem-resistant Serratia marcescens (CRSM). Minimum inhibitory concentrations (MICs) and cell membrane integrity were measured to investigate the antimicrobial mechanism of sanguinarine. Additionally, the extent of biofilm formation by CRSM exposed to sanguinarine was measured by crystal violet staining and visualized via field emission scanning electron microscopy and confocal laser scanning microscopy. Sanguinarine displayed moderate activity against CRSM, with a MIC90 of 32 µg ml-1. Moreover, cell membrane integrity was severely disrupted by sanguinarine at 64 µg ml-1, and biofilm formation was sharply inhibited at 32 µg ml-1. The minimum biofilm eradication concentration was 512 µg ml-1 against mature CRSM biofilms. The overall results suggest that sanguinarine is a potential anti-biofilm agent that can be explored to treat CRSM infections.

Equivalent effect of extracellular proteins and polysaccharides on biofilm formation by clinical isolates of Staphylococcus lugdunensis.

Biofilm formation by Staphylococcus lugdunensis involves formation of an extracellular matrix; however, the identity of the constituents responsible for the structure of biofilms fabricated by different clinical strains is largely unclear. Here, biofilms produced by 24 clinical isolates of S. lugdunensis were characterized. The optimal medium for S. lugdunensis was selected, and the biofilm-forming capacity was assessed. Extracelullar polymeric substances (EPS) contributing to biofilm robustness were determined by evaluating the susceptibility of biofilms to EPS-degrading agents using field emission scanning electron microscopy and confocal laser scanning microscopy. Biofilm formation by the clinical isolates of S. lugdunensis was augmented by glucose supplementation. Further, extracellular DNA (eDNA), proteins, and polysaccharides were present in the 24 clinical isolates. Proteins and polysaccharides were the most common components within the S. lugdunensis biofilms, whereas the eDNA content was marginal in biofilm formation. Therefore, proteins and polysaccharides within biofilms may be used as the primary targets for developing eradication strategies to prevent S. lugdunensis biofilm formation.

Antimicrobial activity of eugenol against carbapenem-resistant Klebsiella pneumoniae and its effect on biofilms.

A preliminary study found that eugenol expressed an antibacterial activity against Klebsiella pneumoniae. However, the mechanism of action of eugenol against K. pneumoniae still remains unexplored. The aim of this study was to gain further insight into the antibacterial effect of eugenol against carbapenem-resistant Klebsiella pneumoniae (CRKP) and possible mode of action. Here, minimum inhibitory concentration (MIC) of eugenol against CRKP strains was determined using the agar dilution method. Moreover, variations in intracellular ATP concentration, intracellular pH (pHin), membrane potential and membrane integrity were measured to evaluate the effect of eugenol on cell membrane. Besides, changes in cell structure and biofilm formation of CRKP as well as biofilm-associated cell damage were determined using field emission scanning electron microscope (FESEM), transmission electron microscope (TEM) and confocal laser scanning microscopy (CLSM). Finally, gene expression of biofilm-related biosynthesis was investigated. The results showed that MICs of eugenol against four tested CRKP were 0.2 mg/mL. Eugenol damaged the cell membrane of CRKP, as evidenced by decreased intracellular ATP concentration, reduced pHin and cell membrane hyperpolarization, coupled with enhanced membrane permeability. Furthermore, eugenol compromised cell structure and induced loss of intracellular components of CRKP. Additionally, eugenol inhibited biofilm formation and inactivated biofilm CRKP cells. Finally, eugenol presented strong inhibitory effects on biofilm formation and biofilm-associated gene expression, and inactivated CRKP cells growing in biofilms. These findings suggest that eugenol exhibits antimicrobial effect against CRKP strains and could be potentially used to control CRKP-related infections.

Antimicrobial mechanism of luteolin against Staphylococcus aureus and Listeria monocytogenes and its antibiofilm properties.

Luteolin (LUT) is a naturally occurring compound found in a various of plants. Few recent studies have reported LUT antimicrobial activities against bacterial pathogens, however, the fundamental LUT mediated antimicrobial mechanism has never been elucidated. This study aimed to investigate the antimicrobial activities of LUT and its mode of action against Staphylococcus aureus and Listeria monocytogenes, either as planktonic cells or as biofilms. Here, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of LUT against S. aureus and L. monocytogenes were determined using the broth microdilution method, and the antimicrobial mode of LUT was elucidated by evaluating the variations in both cell membrane integrity and cell morphology. Moreover, the biofilm inhibition was measured by crystal violet staining assay, while its qualitative imaging was achieved by confocal laser scanning microscope and field emission scanning electron microscope. MIC and MBC values of LUT against S. aureus were 16-32 and 32-64 µg/mL, and 32-64 and 64-128 µg/mL for L. monocytogenes. LUT destroyed the cell membrane integrity, as evidenced by a significant increase in the number of non-viable cells, and well-defined variations in cell morphology. Moreover, LUT presented robust inhibitory effects on the biofilm formation, enhanced antibiotics diffusion within biofilms and killed efficiently mono- and dual-species biofilm cells. Overall, LUT demonstrates potent antimicrobial properties on planktonic and biofilm cells, and the biofilm formation, and thus has the potential use as a natural food preservative in foods.

Antimicrobial and Antibiofilm Activities of Citral Against Carbapenem-Resistant Enterobacter cloacae.

Citral that is produced in the essential oils of plants is an isomeric mixture of geranial and neral. Few recent studies exhibited robust antibacterial activity of citral against several pathogens. However, little is reported about effects of citral on carbapenem-resistant Enterobacter cloacae (CREC). The purpose of this study was to assess antibacterial and antibiofilm activities of citral against CREC. Here, minimum inhibitory concentrations (MICs) of citral against CREC were determined by the agar dilution method. Antibacterial mode of citral against CREC was elucidated by evaluating changes in intracellular adenosine triphosphate (ATP) concentration, intracellular pH (pHin), membrane potential, membrane integrity, and cell morphology. In addition, CREC cell damage within biofilms was examined by confocal laser scanning microscopy (CLSM). The results showed that the MIC of citral against CREC was 1000 µg/mL. Citral inhibited CREC growth and destroyed its cell membrane integrity, as measured by the decrease of intracellular ATP, pH, and membrane potential as well as distinctive deformation in cellular morphology. CLSM images demonstrated that citral could inactivate CREC cells within biofilms. These results revealed that citral exhibits potent antibacterial and antibiofilm activity against CREC, and thus has potential to be used as natural food preservatives to control CREC-associated infection spread.

Therapeutic Effect of CD4+CD25+ Regulatory T Cells Amplified In Vitro on Experimental Autoimmune Neuritis in Rats.

BACKGROUND/AIMS: This study aimed to explore whether the adoptive transfusion of autologous CD4+CD25+ regulatory T cells (CD4+CD25+ Tregs) has a therapeutic effect on Experimental autoimmune neuritis (EAN) model rats, and it provides new experimental and theoretical bases for the immunotherapy of Guillain-Barre syndrome (GBS). METHODS: CD4+CD25+ Tregs were sorted from the spleens of rats using immunomagnetic bead separation techniques combined with flow cytometry. Their in vitro inhibitory function was determined using a lymphocyte proliferation inhibition test, and their purity was confirmed by flow cytometry. Cells were stimulated using CD3/CD28 monoclonal antibodies and were cultured in culture medium containing interleukin 2 (IL-2), transforming growth factor-ß (TGF-ß) and rapamycin. After 15 days of amplification, CD4+CD25+ Tregs were collected and transfused into EAN model rats. Changes in the pathology and electron microscopical morphology of rat sciatic nerves in the normal group, untreated group, low-dose group (2 × 107) and high-dose group (4 × 107) were observed, and the expression of CD4+CD25+FOXP3 in peripheral blood in the four groups of rats was detected by flow cytometry. RESULTS: Compared with rats in the untreated group, rats in the treatment groups had significantly reduced infiltration of inflammatory cells in the sciatic nerve, as well as myelin and axonal damage. Additionally, the CD4+CD25+ Tregs levels in peripheral blood were significantly higher than those in the untreated group (P< 0. 05). Moreover, the therapeutic effect became more significant with an increase in the dose of adoptive transfusion. CONCLUSION: Adoptive transfusion of CD4+CD25+ Tregs into EAN model rats has significant therapeutic effects.

Role of the inositol pyrophosphate multikinase Kcs1 in Cryptococcus inositol metabolism.

Cryptococcus neoformans is the most common cause of deadly fungal meningitis. This fungus has a complex inositol acquisition and utilization system, and our previous studies have shown the importance of inositol utilization in cryptococcal development and virulence. However, how inositol utilization is regulated in this fungus remains unknown. In this study, we found that inositol, irrespective of the presence of glucose in the media, represses the expression of C. neoformans genes involved in inositol pyrophosphate biosynthesis, including the gene encoding inositol hexakisphosphate kinase Kcs1. Kcs1 was recently reported to regulate inositol metabolism in Saccharomyces cerevisiae and to impact virulence in C. neoformans. To examine the potential role of Kcs1 in inositol regulation in C. neoformans, we generated the kcs1Δ mutant and compared its phenotype with the wild type strain. We found that Kcs1 negatively regulates inositol uptake and catabolism in C. neoformans, but, in contrast to Kcs1 function in S. cerevisiae, does not appear to regulate inositol biosynthesis. Together, these results show that Kcs1 functions to fine-tune inositol acquisition to maintain inositol homeostasis in C. neoformans.

Heightened stress response and cognitive impairment after repeated neonatal sevoflurane exposures might be linked to excessive GABAAR-mediated depolarization.

OBJECTIVE: Children with repeated exposures to anesthesia at an early age are at an increased risk of cognitive impairment. Data in the literature link increased developmental depolarizing γ-aminobutyric acid (GABA) type A receptor (GABAAR) at younger age to neurodevelopmental disorders. Here we investigated the involvement of GABAergic signaling during development in mediating the adverse effects of repeated sevoflurane exposures. METHODS: Sprague-Dawley male rats received repeated exposures to 3 % sevoflurane for 2 h daily for 3 consecutive days on postnatal days (P) 4, 5, and 6; maternally separated and unseparated rats served as controls. A subgroup of rats received three injections of the Na(+)-K(+)-2Cl(-) cotransporter inhibitor, bumetanide (1.82 mg/kg, intraperitoneally) 15 min prior to initiation of each sevoflurane exposure. RESULTS: The results showed that repeated neonatal sevoflurane exposures contribute to learning and memory impairment in the Morris water maze (MWM) at P60. The corticosterone level was significantly increased immediately after repeated neonatal sevoflurane exposures. Repeated neonatal sevoflurane exposures heightened the secretion of corticosterone in response to stress in P7 and P60 rats. Pretreatment of male rats prior to each sevoflurane exposure with bumetanide attenuated the corticosterone level immediately after repeated neonatal sevoflurane exposures, normalized endocrine response to stress at P7 and P60, and attenuated the sevoflurane-induced learning and memory impairment in the MWM. CONCLUSION: These data suggested that the heightened stress response and cognitive impairment after repeated neonatal sevoflurane exposures might be linked to excessive GABAAR-mediated depolarization.

Imbalance in circulating T lymphocyte subsets contributes to Hu antibody-associated paraneoplastic neurological syndromes.

Paraneoplastic neurological syndromes (PNS) are associated with small cell lung cancer (SCLC) and Hu antibodies, which are considered to have an immune-mediated etiology. As a pathogenic role for Hu antibodies (Hu-Ab) in PNS could not be demonstrated, the cellular immune response against the Hu proteins has been further investigated. To delve deeper into the hypothesized cell-mediated immune pathogenesis of these syndromes, imbalances within circulating T lymphocyte subsets were investigated to determine their significance in Hu antibody-associated PNS. The circulating T lymphocyte subsets were analyzed in untreated patients with SCLC, PNS and Hu-Ab (n=10), SCLC without PNS (n=10) and healthy controls (n=12) using flow cytometry. Patients with PNS and SCLC, had a variety of changes within their circulating T lymphocyte subsets, which included; lymphopenia of the CD3(+)and CD4(+) T cells, increased proportions of total activated T cells and activated CD4(+) T cells, and reduced numbers of CD4(+) and CD25(+) regulatory T cells (Treg). These results suggest that the excessive activation of T cells and dysfunction of Treg contribute to Hu antibody-associated PNS.

Establishment of a Nucleic Acid Detection Method for Norovirus GII.2 Genotype Based on RT-RPA and CRISPR/Cas12a-LFS.

To establish a rapid detection method for norovirus GII.2 genotype, this study employed reverse transcription recombinase polymerase amplification (RT-RPA) combined with CRISPR/Cas12a and lateral flow strip (RT-RPA-Cas12a-LFS). Here, the genome of norovirus GII.2 genotype was compared to identify highly conserved sequences, facilitating the design of RT-RPA primers and crRNA specific to the conserved regions of norovirus GII.2. Subsequently, the reaction parameters of RT-RPA were optimized and evaluated using agar-gel electrophoresis and LFS. The results indicate that the conserved sequences of norovirus GII.2 were successfully amplified through RT-RPA at 37°C for 25 minutes. Additionally, CRISPR/Cas12a-mediated cleavage detection was achieved through LFS at 37°C within 10 minutes using the amplification products as templates. Including the isothermal amplification reaction time, the total time is 35 minutes. The established RT-RPA-Cas12a-LFS method demonstrated specific detection of norovirus GII.2, yielding negative results for other viral genomes, and exhibited an excellent detection limit of 10 copies/µl. The RT-RPA-Cas12a-LFS method was further compared with qRT-PCR by analyzing 60 food-contaminated samples. The positive conformity rate was 100%, the negative conformity rate was 95.45%, and the overall conformity rate reached 98.33%. This detection method for norovirus GII.2 genotype is cost-effective, highly sensitive, specific, and easy to operate, offering a promising technical solution for field-based detection of the norovirus GII.2 genotype.