Rapid Detection of blaKPC in Carbapenem-Resistant Enterobacterales Based on CRISPR/Cas13a.

Klebsiella pneumoniae carbapenemase (KPC) is a crucial enzyme that causes carbapenem resistance in Enterobacterales, and infections by these "superbugs" are extremely challenging to treat. Therefore, there is a pressing need for a rapid and accurate KPC detection test to control the prevalence of carbapenem-resistant Enterobacterales (CREs). In this study, we established a novel method for detection of blaKPC, the gene responsible for encoding KPC, based on a recombinase polymerase amplification (RPA) and a CRISPR/Cas13a reaction coupled to fluorophore activation (termed RPA-Cas13a assay). We carefully selected a pair of optimal amplification primers for blaKPC and achieved a lower limit of detection of approximately 2.5 copies/µL by repeatedly amplifying a recombinant plasmid containing blaKPC. The RPA-Cas13a assay demonstrated a sensitivity of 96.5% and specificity of 100% when tested on 57 blaKPC-positive CRE strains, which were confirmed by DNA sequencing. Moreover, in 311 sputum samples, the theoretical antibiotic resistance characteristics of blaKPC-positive strains obtained by the RPA-Cas13a assay were highly consistent with the results of antibiotic susceptibility test (Kappa = 0.978 > 0.81, P < 0.01). In conclusion, the RPA-Cas13a system is a simple and one-hour efficient technology for the detection of a potentially fatal antibiotic resistance gene.

PHF20L1 mediates PAX2 expression to promote angiogenesis and liver metastasis in colorectal cancer through regulating HIC1.

Colorectal cancer (CRC) is a common cancer with poor prognosis. The research was designed to explore the role of PHF20L1 in angiogenesis and liver metastasis in CRC and discuss its molecular mechanism. Expression levels of PHF20L1, HIC1 and PAX2 in CRC tissues collected from CRC patients were detected using qRT-PCR, WB and immunohistochemical staining. CRC cells were transfected with PHF20L1, HIC1 and PAX2 overexpression or knockdown vectors and the proliferation, apoptosis, EMT and angiogenesis of the cells were determined. WB was utilized to assess protein levels of PHF20L1, HIC1, PAX2 and angiogenesis factor (ANGPT2, FGF1, PDGFA and VEGFA). The role of PHF20L1 regulating tumor formation and liver metastasis in vivo was detected as well. PHF20L1 was observed to express at a high level of CRC tissues. PHF20L1 promoted CRC cell growth, EMT and angiogenesis, and inhibited cell apoptosis. Knockdown of PHF20L1 had opposite effects on CRC cells. PHF20L1 negatively regulated HIC1 expression to promote PAX2 expression, thus promoting CRC cell progression. The in vivo results showed that PHF20L1 contributed to tumor formation and liver metastasis. PHF20L1 increases PAX2 expression to promote angiogenesis in CRC by inhibiting HIC1, therefore facilitating CRC cell EMT and liver metastasis. Our finding may provide a novel insight for CRC pathogenesis.

Directed evolution and secretory expression of a pyrethroid-hydrolyzing esterase with enhanced catalytic activity and thermostability.

BACKGROUND: Pyrethroids are potentially harmful to human health and ecosystems. It is necessary to develop some efficient strategies to degrade pyrethroid residues. Biodegradation is generally considered as a safe, efficient, and inexpensive way to eliminate environmental contaminants. To date, although several pyrethroid-hydrolyzing esterases have been cloned, there has been no report about a pyrethroid hydrolase with high hydrolytic activity, good stability, and high productivity, indispensable enzymatic properties in practical biodegradation. Almost all pyrethroid hydrolases are intracellular enzymes, which require complex extraction protocols and present issues in terms of easy inactivation and low production. RESULTS: In this study, random mutagenesis was performed on one pyrethroid-hydrolyzing esterase, Sys410, to enhance its activity and thermostability. Two beneficial mutations, A171V and D256N, were obtained by random mutagenesis and gave rise to the mutant M2. The mutant displayed ~1.5-fold improvement in the kcat/Km value and 2.46-fold higher catalytic activity. The optimal temperature was 10 °C higher than that of the wild-type enzyme (55 °C). The half-life at 40-65 °C was 3.3-310 times longer. It was surprising that M2 has a half-life of 12 h at 70 °C while Sys410 was completely inactivated at 70 °C. In addition, the desired gene was extracellularly expressed in a Pichia pastoris host system. The soluble expression level reached up to 689.7 mg/L. Remarkably, the enzyme could efficiently degrade various pyrethroids at moderate temperature for 15 min, exceeding a hydrolysis rate of 98%, which is the highest value ever reported. CONCLUSIONS: This is the first report about random mutagenesis and secretory expression of pyrethroid-hydrolyzing esterase with high-level productivity and purity in P. pastoris. Broad substrate specificity, enhanced activity and thermostability make M2 an ideal candidate for the biodegradation of pyrethroid residues.

Infectious factors in myocarditis: a comprehensive review of common and rare pathogens.

BACKGROUND: Myocarditis is a significant health threat today, with infectious agents being the most common cause. Accurate diagnosis of the etiology of infectious myocarditis is crucial for effective treatment. MAIN BODY: Infectious myocarditis can be caused by viruses, prokaryotes, parasites, and fungi. Viral infections are typically the primary cause. However, some rare opportunistic pathogens can also damage heart muscle cells in patients with immunodeficiencies, neoplasms and those who have undergone heart surgery. CONCLUSIONS: This article reviews research on common and rare pathogens of infectious myocarditis, emphasizing the complexity of its etiology, with the aim of helping clinicians make an accurate diagnosis of infectious myocarditis.

Misdiagnosis and delayed diagnosis of atypical autoimmune pancreatitis: Experience from clinical cases.

Autoimmune pancreatitis (AIP) is a fibro-inflammatory disease characterized by inflammation and fibrosis of the pancreas. It is a systemic disease that can affect multiple organs, including the bile ducts, kidneys, lungs, and other organs. However, due to its complex presentation, AIP is often challenging to diagnose, and misdiagnosis with pancreatic tumors can occur. In our study, we reviewed three cases of atypical AIP where patients had normal serum IgG4 levels, leading to initial misdiagnosis with pancreatic tumors. Delayed diagnosis resulted in irreversible pathologies such as retroperitoneal fibrosis. All three patients had bile duct involvement, and imaging findings were similar to those of tumors, further complicating the diagnosis. The correct diagnosis was confirmed only after diagnostic therapy. Our study aims to raise awareness of atypical AIP and improve diagnostic efficiency by analyzing the clinical characteristics of these patients.

The E3 ubiquitin ligase SCF (FBXW10)-mediated LATS2 degradation regulates angiogenesis and liver metastasis in colorectal cancer.

F-box and WD repeat domain containing 10 (FBXW10) is a member of the FBXW subgroup that contains the WD40 domain. FBXW10 has been rarely reported in colorectal cancer (CRC) and its mechanism is unclear. To investigate the role of FBXW10 in CRC, we conducted in vitro and in vivo experiments. Through the database and our clinical samples, we found that FBXW10 expression was up-regulated in CRC, and it was positively correlated with CD31 expression. CRC patients with high FBXW10 expression levels had a poor prognosis. Overexpression of FBXW10 up-regulated cell proliferation, migration and vascular formation, while knockdown of FBXW10 had the opposite effects. Studies on the mechanism of FBXW10 in CRC showed that FBXW10 could ubiquitinate large tumor suppressor kinase 2 (LATS2) and promote its degradation with the Fbox region of FBXW10 played an essential role in this process. In vivo studies demonstrated that knockout of FBXW10 inhibited tumor proliferation and reduced liver metastasis. In conclusion, our study proved that FBXW10 was significantly overexpressed in CRC and was involved in the pathogenesis of CRC by affecting angiogenesis and liver metastasis. Mechanistically, FBXW10 degraded LATS2 through ubiquitination. Therefore, FBXW10-LATS2 can be used as a therapeutic target for CRC in subsequent studies.

Aii810, a Novel Cold-Adapted N-Acylhomoserine Lactonase Discovered in a Metagenome, Can Strongly Attenuate Pseudomonas aeruginosa Virulence Factors and Biofilm Formation.

The pathogen Pseudomonas aeruginosa uses quorum sensing (QS) to control virulence and biofilm formation. Enzymatic disruption of quorum sensing is a promising anti-infection therapeutic strategy that does not rely on antibiotics. Here, a novel gene (aii810) encoding an N-acylhomoserine lactonase was isolated from the Mao-tofu metagenome for the first time. Aii810 encoded a protein of 269 amino acids and was expressed in Escherichia coli BL21 (DE3) in soluble form. It showed the highest activity at 20°C, and it maintained 76.5% of activity at 0°C and more than 50% activity at 0-40°C. The optimal pH was 8.0. It was stable in both neutral and slightly alkaline conditions and at temperatures below 40°C. The enzyme hydrolyzed several ρ-nitrophenyl esters, but its best substrate was ρ-nitrophenyl acetate. Its kcat and Km values were 347.7 S-1 and 205.1 µM, respectively. It efficiently degraded N-butyryl-L-homoserine lactone and N-(3-oxododecanoyl)-L-homoserine lactone, exceeding hydrolysis rates of 72.3 and 100%, respectively. Moreover, Aii810 strongly attenuated P. aeruginosa virulence and biofilm formation. This enzyme with high anti-QS activity was the most cold-adapted N-acylhomoserine lactonase reported, which makes it an attractive enzyme for use as a therapeutic agent against P. aeruginosa infection.