Azadirachtin Induces Fat Body Apoptosis by Suppressing Caspase-8 in the Fall Armyworm, Spodoptera frugiperda.

Azadirachtin is a widely used botanical pesticide for agricultural pest control worldwide. However, the molecular mechanisms of azadirachtin in insects are not fully understood. In this study, histological analysis and RNA sequencing were conducted to investigate the impact of azadirachtin on the larval development of Spodoptera frugiperda. Under azadirachtin exposure, the development was completely inhibited, and the major internal tissues, fat body, and midgut were strongly damaged under histological analysis. Differential gene expression analysis demonstrated that nutrient absorption and detoxification metabolism-related genes are differentially expressed. Interestingly, the expression of the apoptosis-related gene, caspase-8, was significantly inhibited under exposure to azadirachtin. In addition, after knocking down the expression of the caspase-8 gene, the fat body displayed a similar apoptotic phenotype as azadirachtin treatment; the distribution of chromatin and lipid droplets was uneven in the fat body cells. Thus, the results in this study demonstrated that exposure to azadirachtin rapidly activates apoptosis, resulting in innate tissue disruption, ultimately arresting larval development in S. frugiperda.

LINC00525 enhances ZNF460-regulated CD24 expression through the sponge miR-125a-5p to promote malignant progression of breast cancer.

INTRODUCTION: CD24 is a highly glycosylated glycosylphosphatidylinositol anchored membrane protein that plays an important role in tumor progression. The aim of this study was to investigate the effect of abnormal expression of CD24 on the proliferation, migration and invasion of breast cancer (BC) cells, and the molecular mechanism of regulating CD24 expression in breast cancer. METHODOLOGY: The bioinformatics method was used to predict the expression level of CD24 in BC and its relationship with the occurrence and development of BC. IHC, RT-qPCR and WB were used to detect the expression of CD24 in BC tissues and cells. The proliferation of CD24 was evaluated by CCK-8 and colony formation assay, and the migration and invasion of CD24 were evaluated by wound healing and transwell. In addition, the effect of CD24 on the malignancy of BC in vivo was further evaluated by subcutaneous tumorigenesis assay. Molecular mechanisms were measured by luciferase reporter assays, biotin-labeled miRNA pull-down assay, RIP, and western blotting. RESULTS: The results show that CD24 is highly expressed in breast cancer tissues and cell lines, and knockdown of CD24 in vivo and in vitro can inhibit the proliferation, migration and invasion of BC cells. Mechanistically, the transcription factor ZNF460 promotes its expression by binding to the CD24 promoter, and the expression of ZNF460 is regulated by miR-125a-5p, which inhibits its expression by targeting the 3'UTR of ZNF460. In addition, LINC00525 acts as a ceRNA sponge to adsorb miR-125a-5p and regulate its expression. CONCLUSIONS: Overexpression of CD24 is involved in the development and poor prognosis of BC, which can be used as a potential target for the treatment of BC and provide a theoretical basis for the treatment of BC.

Comparing the effects of different acupoint-stimulating therapies in mitigating post-stroke spasticity and motor dysfunction in older stroke survivors: A network meta-analysis of randomized trials.

Acupoint-stimulating therapies have often been used to manage stroke-related spasticity and motor dysfunction. However, the effects of different acupoint-stimulating therapies in older stroke survivors have been unclear. This systematic review and network meta-analysis compared the effects of different acupoint-stimulating therapies in managing spasticity and motor dysfunction in older stroke survivors. The study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We searched 7 databases for studies published up to July 2023. Inclusion criteria were: (1) older adults with strokes; (2) treatments were acupoint-stimulating therapies; (3) a control group did not receive acupoint-stimulating therapy, or the study compared different acupoint-stimulating therapies; and (4) outcomes included spasticity and motor function. Methodological quality was assessed with Risk-of-bias tool for randomized trials version 2, while R and Metainsight were used to conduct the network meta-analysis. We analyzed 27 studies and the results showed that non-invasive electroacupuncture and warm acupuncture were more effective in reducing spasticity than conventional acupuncture (standardized mean difference and 95 % confidence intervals = 1.35/1.19 [0.57; 2.13/0.54; 1.83]) and invasive electroacupuncture (standardized mean difference and 95 % confidence intervals = 0.96/0.80 [0.12; 1.80/0.08; 1.51]). Conventional acupuncture and invasive electroacupuncture were effective in improving motor function (standardized mean difference and 95 % confidence intervals = 0.99/1.41 [0.42; 1.56/0.54; 2.28]). However, there was significant inconsistency for the effects of invasive electroacupuncture between studies. Our findings suggest that for older stroke survivors with spasticity, non-invasive electroacupuncture and warm acupuncture are appropriate, whereas conventional acupuncture is more appropriate for patients aiming for motor recovery. SYSTEMATIC REVIEW REGISTRATION: This study was registered in the PROSPERO database (CRD42023442202).

Novel strategy to quantify the viability of oocysts of Cryptosporidium parvum and C. hominis, a risk factor of the waterborne protozoan pathogens of public health concern.

While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.

CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm.

Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.

Unlocking the mystery of the feeder organelle and versatile energy metabolism in Cryptosporidium parvum.

Xu and colleagues recently revealed the critical role of Cryptosporidium's feeder organelle in nutrient uptake, showcasing the parasite's ability to harness glucose and glucose-6-phosphate from host cells. This illuminates the sophisticated energy metabolism and survival strategies of the parasite, highlighting potential therapeutic targets.

Requirement of microtubules for secretion of a micronemal protein CpTSP4 in the invasive stage of the apicomplexan Cryptosporidium parvum.

The zoonotic Cryptosporidium parvum is a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised or weakened individuals. Like other apicomplexans, it possesses several specialized secretory organelles, including micronemes, rhoptry, and dense granules. However, the understanding of cryptosporidial micronemal composition and secretory pathway remains limited. Here, we report a new micronemal protein in C. parvum, namely, thrombospondin (TSP)-repeat domain-containing protein-4 (CpTSP4), providing insights into these ambiguities. Immunostaining and enzyme-linked assays show that CpTSP4 is prestored in the micronemes of unexcysted sporozoites but secreted during sporozoite excystation, gliding, and invasion. In excysted sporozoites, CpTSP4 is also distributed on the two central microtubules unique to Cryptosporidium. The secretion and microtubular distribution could be completely blocked by the selective kinesin-5 inhibitors SB-743921 and SB-715992, resulting in the accumulation of CpTSP4 in micronemes. These support the kinesin-dependent microtubular trafficking of CpTSP4 for secretion. We also localize γ-tubulin, consistent with kinesin-dependent anterograde trafficking. Additionally, recombinant CpTSP4 displays nanomolar binding affinity to the host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and validated biochemically for its contribution to the adhesive property of CpTSP4 by peptide competition assays and site-directed mutagenesis. These findings shed light on the mechanisms of intracellular trafficking and secretion of a cryptosporidial micronemal protein and the interaction of a TSP-family protein with host cells.IMPORTANCECryptosporidium parvum is a globally distributed apicomplexan parasite infecting humans and/or animals. Like other apicomplexans, it possesses specialized secretory organelles in the zoites, in which micronemes discharge molecules to facilitate the movement and invasion of zoites. Although past and recent studies have identified several proteins in cryptosporidial micronemes, our understanding of the composition, secretory pathways, and domain-ligand interactions of micronemal proteins remains limited. This study identifies a new micronemal protein, namely, CpTSP4, that is discharged during excystation, gliding, and invasion of C. parvum sporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the two Cryptosporidium-unique microtubes that could be blocked by kinesin-5/Eg5 inhibitors. Additionally, a novel heparin-binding motif is identified and biochemically validated, which contributes to the nanomolar binding affinity of CpTSP4 to host cells. These findings indicate that kinesin-dependent microtubular trafficking is critical to CpTSP4 secretion, and heparin/heparan sulfate is one of the ligands for this micronemal protein.

Dynamic biomechanical changes of clear aligners during extraction space closure: Finite element analysis.

INTRODUCTION: Clear aligners (CAs) have recently become popular and widely used orthodontic appliances. Research on CA biomechanics has become a focal point in orthodontics to improve the efficiency of CA treatment and address challenging issues, such as extraction. The biomechanical characteristics of CAs in space closure have been reported. However, previous studies have mainly focused on static biomechanical analysis that cannot demonstrate the dynamic biomechanical changes in CAs during space-closing. Given that these biomechanical changes can be significant and have considerable clinical value, this study aimed to investigate these characteristics. METHODS: Sequential extraction space-closing models were derived from included patient data and refined using modeling and CA design software. A finite element analysis was performed to obtain biomechanical raw data. This study introduced a dual coordinate system and space geometry analysis to demonstrate the biomechanical properties accurately. RESULTS: As space closure progressed, the instantaneous tooth displacements increased, indicating an enhanced space closure force because of the increased strain in the CA extraction area. Meanwhile, the central axis of rotation of the anterior teeth continuously moved toward the labial-apical direction, showing a gradually enhanced vertical and torque control effect. CONCLUSIONS: During space closure, CAs undergo specific biomechanical changes, including increased contraction and control forces on both sides of the gap. These biomechanical effects are beneficial to alleviate the roller coaster effect gradually. Meanwhile, more reasonable staging design strategies can be proposed on the basis of this biomechanical mechanism.