Leptospirosis is a re-emerging tropical infectious disease caused by pathogenic Leptospira spp. The different host innate immune responses are partially related to the different severities of leptospirosis. In this study, we employed transcriptomics and cytokine arrays to comparatively calculate the responses of murine peritoneal macrophages (MPMs) and human peripheral blood monocytes (HBMs) to leptospiral infection. We uncovered a series of different expression profiles of these two immune cells. The percentages of regulated genes in several biological processes of MPMs, such as antigen processing and presentation, membrane potential regulation, and the innate immune response, etc., were much greater than those of HBMs (>2-fold). In MPMs and HBMs, the caspase-8 and Fas-associated protein with death domain (FADD)-like apoptosis regulator genes were significantly up-regulated, which supported previous results that the caspase-8 and caspase-3 pathways play an important role in macrophage apoptosis during leptospiral infection. In addition, the key component of the complement pathway, C3, was only up-regulated in MPMs. Furthermore, several cytokines, e.g. interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-alpha), were differentially expressed at both mRNA and protein levels in MPMs and HBMs. Some of the differential expressions were proved to be pathogenic Leptospira-specific regulations at mRNA level or protein level. Though it is still unclear why some animals are resistant and others are susceptible to leptospiral infection, this comparative study based on transcriptomics and cytokine arrays partially uncovered the differences of murine resistance and human susceptibility to leptospirosis. Taken together, these findings will facilitate further molecular studies on the innate immune response to leptospiral infection. Although pathogenic Leptospira is not an obligate intracellular pathogen, recent studies have shown that phagocytosis and innate immunity play important roles in leptospirosis. The Leptospira-macrophage interaction is a common model used to elucidate the initial response in leptospiral infection. Our previous research has shown that there is little difference in the transcriptomics of pathogenic Leptospira infecting murine or human macrophage cell lines. Contrarily, in this study, we observed significant differences of murine and human primary macrophages infected by L. interrogans as shown in several processes, such as antigen processing and presentation, Toll-like receptor signaling pathway and innate immune response, complement and coagulation cascades, expression of major cytokines and chemokines, etc. These results suggested that different immune responses explain the major disparities in the murine and human Leptospira-macrophage infection models. This study added to the former leptospiral transcriptomics research on the Leptospira-macrophage interaction model and laid a foundation for further investigation in the pathogenesis of leptospirosis.

Background: Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo on its own. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages. Methods: The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and PCR, Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-sequencing and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2^--3D7 parasites. Results: Pfhrp2^- transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512 and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, respectively, with 373, 520, 1499, 353, 1253 and 742 of these DEGs upregulated and 591, 741, 1639, 711, 1259 and 1036 of them downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups at six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding delta-aminolevulinic acid synthetase and ferrochelatase, both related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and those encoding haem oxygenase, stromal-processing peptidase and porphobilinogen deaminase were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value = 1). Conclusion: Our data revealed changes in the transcriptome expression profile of the Pfhrp2^--3D7 parasite during the intraerythrocytic stages. The findings provide insight at the gene transcript level that will facilitate further research on and development of anti-malaria drugs. Graphical

Botulinum neurotoxins (BoNTs), produced by the anaerobic spore-forming bacterium Clostridium botulinum, are among the most potent known biological toxins. BoNTs cause lethal botulism via contaminated food, wound infections, or infant intestinal colonization, posing significant threats to public health. Although the mouse bioassay is still being considered as the gold standard for detecting BoNTs, its drawbacks, including the lengthy experimental duration, high costs, and ethical issues, highlight the urgent need to develop alternative methods to fulfill the detection requirements. In recent years, frequent botulism poisoning incidents haves put forward higher requirements for detection technology. On-site detection is expected to be rapid and immediate, while laboratory detection requires high sensitivity and serotype discrimination capabilities. This review comprehensively introduces current detection approaches, including mouse bioassay, cell-based assays, immunological methods, endopeptidase–mass spectrometry, biosensors, chromatography, and mass spectrometry techniques. Notably, cell-based assays have been used for the potency testing of commercialized botulinum toxin type A and are considered the most promising alternative to the mouse bioassay. Biosensors based on nanomaterials demonstrate advantages in real-time detection due to their rapid response and portability, while endopeptidase–mass spectrometry achieves high sensitivity and effective serotype identification by specifically recognizing toxin-cleaved substrates. Future works shall aim to completely replace MBA, developing a detection system suitable for multiple scenarios such as clinical diagnosis, food safety monitoring, and environmental monitoring. The detection methods should also have matrix compatibility and serotype discrimination capabilities.

(1) Background: At present, the only potency assay approved in China for the in-country testing of botulinum toxin type A for injection products is the mouse bioassay (MBA). The Chinese market for neurotoxin products is rapidly expanding, but MBAs are subject to high variability due to individual variations in mice, as well as variations in injection sites, in addition to the limited number of batches tested for one MBA. Compared with the mLD₅₀ method, the cell-based potency assay (CBPA) developed for the potency testing of onabotulinumtoxinA (BOTOX) by AbbVie not only does not use any experimental animals but also allows for significant time and cost savings. Due to the significant benefits conferred by the replacement of the mLD₅₀ assay with CBPA in China, the CBPA method has been transferred, validated, and cross-validated to demonstrate the equivalence of the two potency methods. (2) Methods: The differentiated SiMa cells were treated with both BOTOX samples and the reference standard, and the cleaved SNAP25₁₉₇ in the cell lysates was quantified using Chemi-ECL ELISA. A 4-PL model was used for the data fit and sample relative potency calculation. The method accuracy, linearity, repeatability, and intermediate precision were determined within the range of 50% to 200% of the labeled claim. A statistical equivalence of the two potency methods (CBPA and mLD₅₀) was initially demonstrated by comparing the AbbVie CBPA data with NIFDC mLD₅₀ data on a total of 167 commercial BOTOX lots (85 50U lots and 82 100U lots). In addition, six lots of onabotulinumtoxinA (three 50U and three 100U) were re-tested as cross-validation by these two methods for equivalence. (3) Results: The overall assay’s accuracy and intermediate precision were determined as 104% and 9.2%, and the slope, R-square, and Y-intercept for linearity were determined as 1.071, 0.998, and 0.036, respectively. The repeatability was determined as 6.9%. The range with the acceptable criteria of accuracy, linearity, and precision was demonstrated as 50% to 200% of the labeled claim. The 95% equivalence statistic test using margins [80%, 125%] indicates that CBPA and mLD₅₀ methods are equivalent for both BOTOX strengths (i.e., 50U and 100U). The relative potency data from cross-validation were within the range of ≥80% to ≤120%. (4) Conclusions: The CBPA meets all acceptance criteria and is equivalent to mLD₅₀. The replacement of mLD₅₀ with CBPA is well justified in terms of ensuring safety and efficacy, as well as for animal benefits.