AMB Express最新文献

The development of rapid, sensitive, and user-friendly diagnostic methods for Chlamydia psittaci (C. psittaci) is critical for early diagnosis and effective control of infections. Current techniques, such as qPCR and next-generation sequencing, face limitations in accessibility and practicality for point-of-care testing (POCT). In this study, we present the WPTTS platform (one-step recombinase polymerase amplification (RPA)-CRISPR/Cas12b assay using weak protospacer adjacent motif (PAM) recognition with two-temperature shifting), which leverages the temperature-dependent PAM stringency of Alicyclobacillus acidophilus Cas12b (AapCas12b) to overcome the challenges of one-step CRISPR detection. By employing weak PAM sequences and a two-temperature protocol (37 °C for amplification, 60 °C for detection), WPTTS minimizes interference between RPA amplification and CRISPR cleavage, achieving a sensitivity of 10² copies/reaction-equivalent to two-step methods-while maintaining operational simplicity. The platform also demonstrates enhanced specificity, enabling single-base resolution at critical target positions. Clinical validation with 40 samples confirmed its superior performance, with 95% positive agreement and 100% negative agreement compared to qPCR. This study highlights the potential of WPTTS as a robust, rapid, and highly sensitive tool for POCT applications in C. psittaci detection and broader infectious disease diagnostics.

Chinese hamster ovary (CHO) cells are the predominant host for recombinant protein production. While transient gene expression (TGE) systems provide a faster, more cost-effective alternative to stable gene expression (SGE), their application is often hampered by low protein yields. Here, we optimized TGE systems based on high-density suspension transfection using 40 kDa polyethylenimine (PEI) by engineering vector regulatory elements (Kozak, polyA, introns, leader) and optimizing transfection conditions. Vector optimization led to a substantial enhancement in recombinant protein, with EGFP and SEAP levels increased by 5.33-fold and 4.13-fold, respectively (p < 0.05). By applying the optimized vector in combination with identified optimal conditions, a DNA concentration of 4 µg/mL, a DNA: PEI mass ratio of 1:5, and a repeated transfection strategy, we achieved a transfection efficiency of 76.34% while maintaining cell viability above 85%. Moreover, the combined optimization of vectors and transfection conditions robustly enhance the production of therapeutically relevant proteins, increased the expression of recombinant type III human collagen and human serum albumin by 4.86-fold and 5.50-fold, respectively (p < 0.05). In summary, high-density suspension transfection markedly boosts transient recombinant protein expression in CHO cells by improving transfection efficiency, expanding the population of recombinant cells, and maintaining high cell viability. This approach presents a promising and effective strategy for high yield transient transgene expression.

Bone disorders are marked by leg deformities, and disruption in the gut microbiota have been associated with degradation of bone structure, which increases the susceptibility of fractures. Perennial ryegrass has antimicrobial, antioxidant and anti-inflammatory properties to improve bone health by regulating the gut microbiota. In this study, we investigated the anti-inflammatory potential of perennial ryegrass to modulate the inflammatory bone loss in meat geese through regulating gut microbiota. Perennial ryegrass (GD) significantly reduced (p < 0.05) the inflammatory response (IL-1β and TNF-α) by improving the gut barrier function. Perennial ryegrass (GD) improved tibia bone mineral density (BMD), ash% and tibia bone mineral contents (p < 0.05). Results of gut microbiota and micro-computed tomography analysis showed that the beneficial effect of GD on bone mass might be associated with higher relative abundance of short-chain fatty acids (SCFAs) producing gut microbiota in the cecum by improving microbial diversity as compared to commercial feeding (CD). Regarding bone turnover, GD increased bone formation markers such alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), osteocalcin (OCN), mothers against decapentaplegic homolog (SMAD) expression by reducing the nuclear factor-kappa B (NF-κB) expression and bone resorption markers i.e. nuclear factor of activated T-cells, cytoplasmic 1 (NFATC1) and tumor necrosis factor receptor associated factor 6 (TRAF-6). Perennial ryegrass exhibited an increase in antioxidant potential by reducing the malondialdehyde (MDA) and reactive oxygen species (ROS) production leading to increased catalase (CAT) and glutathione per oxidase (GSH-PX) levels (P < 0.05). Western blot and Immunofluorescence analysis of tibia showed reduced expression of NF-κB in GD group. These results indicate the importance of perennial ryegrass in alleviating bone loss through gut microbiota via NF-κB signaling mechanism. These findings emphasize the significance of the gut-bone axis and offer new insights into the role of perennial ryegrass in promoting bone health.

Necrophagous flies play a critical role in decomposition and serve as bioindicators of environmental health and pollution. Malaysia's tropical rainforest ecosystems may host many necrophagous fly species, including unique blowflies and their associated bacteria. However, in many forest reserve areas, the diversity of these flies remains poorly studied. This study examines the diversity of necrophagous flies and their associated surface bacteria community, with a particular focus on Phumosia promittens (Walker in J Proc Linn Soc Lond Zool 4:90-96, 1859) in the Bangi Forest Reserve, Universiti Kebangsaan Malaysia. Sampling was conducted across three plots using baited traps, and collected flies were morphologically identified and processed under sterile conditions. Surface bacteria from P. promittens were isolated and analysed through metagenomic analysis targeting the 16S rRNA (V3-V4) amplicon sequencing gene to characterise their microbial communities comprehensively. Among 2,528 individuals collected, Chrysomya megacephala (Fabricius) was the most dominant species overall, while P. promittens was the most abundant among native forest species, suggesting their ecological adaptability and potential as a bioindicator of healthy rainforest. Shannon-Wiener and Simpson's diversity of flies in the study location were 0.67 ± 0.11 and 0.29 ± 0.06, respectively. Meanwhile, the Shannon-Wiener and Simpson's diversity of bacteria from P. promittens were 5.64 ± 0.70 and 0.96 ± 0.02, respectively. Bacterial microbiome analysis revealed the presence of core genera, including Wohlfahrtiimonas, Dysgonomonas, Vagococcus, and Ignatzschineria, which are implicated in both ecological symbiosis and public health concerns. These bacteria may contribute to nutrient cycling, such as heavy metals and antibiotics. Notably, several of these genera are emerging zoonotic pathogens with antimicrobial resistance, highlighting the dual role of necrophagous flies as ecosystem contributors and disease vectors. The findings underscore the importance of monitoring native fly species and their microbiota to assess the integrity of forest ecosystems and potential public health risks.

In 2004, 59 cases of food poisoning linked to the consumption of the mushroom Pleurocybella porrigens were reported in Japan, resulting in 17 fatalities due to acute encephalopathy. In 2023, we found three components of this mushroom-a lectin (PPL), pleurocybelline (PC), and pleurocybellaziridine (PA)-which are collectively implicated in its toxicity in mice. Although we reported the genomic data for P. porrigens in 2013, detailed annotation was not provided at that time. In addition, the biosynthetic pathway of PA, the compound responsible for acute encephalopathy, has not previously been reported in any Basidiomycetes species, including P. porrigens. In this study, we obtained the genome sequence of P. porrigens using the PacBio Revio System. De novo assembly with hifiasm resulted in a higher N50 value compared to previous assemblies, and gene annotation was conducted through BLAST search, GO analysis, KEGG pathway analysis, and carbohydrate-active enzyme identification. In addition, differential gene expression analysis revealed a significant up-regulation of 1427 genes in the fruiting bodies and 1436 genes in the mycelia. Although the gene encoding TqaL, a PA biosynthetic enzyme reported in the genus Penicillium, was not found in P. porrigens, a detailed examination of the BLASTx search identified genes with Fe/αKG oxygenase activity similar to TqaL. Three-dimensional structural modeling using AlphaFold3 revealed that the active site cavities of TqaL and the identified protein were in close proximity. Furthermore, the validity of the differential gene expression analysis between the fruiting bodies and the mycelia was confirmed by quantitative reverse-transcription PCR.