Cultivation, cryopreservation, and transcriptomic studies of host-adapted Cryptosporidium parvum and Cryptosporidium hominis using enteroids.

Cryptosporidium hominis and Cryptosporidium parvum are major causes of severe diarrhea. Comparative studies of them are hampered by the lack of effective cultivation and cryopreservation methods, especially for C. hominis. Here, we describe adapted murine enteroids for the cultivation and complete development of host-adapted C. parvum and C. hominis subtypes, producing oocysts infectious to mice. Using the system, we developed a cryopreservation method for Cryptosporidium isolates. In comparative RNA-seq analyses of C. hominis cultures, the enteroid system generated significantly more host and pathogen responses than the conventional HCT-8 cell system. In particular, the infection was shown to upregulate PI3K-Akt, Ras, TNF, NF-κB, IL-17, MAPK, and innate immunity signaling pathways and downregulate host cell metabolism, and had significantly higher expression of parasite genes involved in oocyst formation. Therefore, the enteroid system provides a valuable tool for comparative studies of the biology of divergent Cryptosporidium species and isolates.

Investigating the effects of Laggera pterodonta on H3N2-Induced inflammatory and immune responses through network pharmacology, molecular docking, and experimental validation in a mice model.

For centuries, Laggera pterodonta (LP), a Chinese herbal medicine, has been widely employed for treating respiratory infectious diseases; however, the mechanism underlying LP's effectiveness against the influenza A/Aichi/2/1968 virus (H3N2) remains elusive. This study aims to shed light on the mechanism by which LP combats influenza in H3N2-infected mice. First, we conducted quasi-targeted metabolomics analysis using liquid chromatography-mass spectrometry to identify LP components. Subsequently, network pharmacology, molecular docking, and simulation were conducted to screen candidate targets associated with AKT and NF-κB. In addition, we conducted a series of experiments including qPCR, hematoxylin-eosin staining, flow cytometry, immunohistochemistry, and enzyme-linked immunosorbent assay to provide evidence that LP treatment in H3N2-infected mice can reduce pro-inflammatory cytokine levels (TNF-α, IL-6, IL-1ß, and MCP-1) while increasing T cells (CD3+, CD4+, and CD8+) and syndecan-1 and secretory IgA expression. This, in turn, aids in the prevention of excessive inflammation and the fortification of immunity, both of which are compromised by H3N2. Finally, we utilized a Western blot assay to confirm that LP indeed inhibits the AKT/NF-κB signaling cascade. Thus, the efficacy of LP serves as a cornerstone in establishing a theoretical foundation for influenza treatment.

Constitutive upregulation of transcription factors underlies permissive bradyzoite differentiation in a natural isolate of Toxoplasma gondii.

Toxoplasma gondii bradyzoites play a critical role in pathology due to their long-term persistence in intermediate hosts and their potential to reactivate, resulting in severe diseases in immunocompromised individuals. Currently there is no effective treatment for eliminating bradyzoites. Hence, better in vitro models of T. gondii cyst development would facilitate identification of therapeutic targets for bradyzoites. Herein we characterized a natural isolate of T. gondii, called Tg68, which showed slower in vitro replication of tachyzoites, and permissive bradyzoite development under stress conditions in vitro. Transcriptional analysis revealed constitutive expression in Tg68 tachyzoites of the key regulators of bradyzoite development including BFD1, BFD2, and several AP2 factors. Consistent with this finding, Tg68 tachyzoites expressed high levels of bradyzoite-specific genes including BAG1, ENO1, and LDH2. Moreover, after stress induced differentiation, Tg68 bradyzoites exhibited gene expression profiles of mature bradyzoites, even at early time points. These data suggest that Tg68 tachyzoites exist in a pre-bradyzoite stage primed to readily develop into mature bradyzoites under stress conditions in vitro. Tg68 presents a novel model for differentiation in vitro that will serve as a useful tool for investigation of bradyzoite biology and development of therapeutics.

Revolutionizing breast cancer treatment: Harnessing the related mechanisms and drugs for regulated cell death (Review).

Breast cancer arises from the malignant transformation of mammary epithelial cells under the influence of various carcinogenic factors, leading to a gradual increase in its prevalence. This disease has become the leading cause of mortality among female malignancies, posing a significant threat to the health of women. The timely identification of breast cancer remains challenging, often resulting in diagnosis at the advanced stages of the disease. Conventional therapeutic approaches, such as surgical excision, chemotherapy and radiotherapy, exhibit limited efficacy in controlling the progression and metastasis of the disease. Regulated cell death (RCD), a process essential for physiological tissue cell renewal, occurs within the body independently of external influences. In the context of cancer, research on RCD primarily focuses on cuproptosis, ferroptosis and pyroptosis. Mounting evidence suggests a marked association between these specific forms of RCD, and the onset and progression of breast cancer. For example, a cuproptosis vector can effectively bind copper ions to induce cuproptosis in breast cancer cells, thereby hindering their proliferation. Additionally, the expression of ferroptosis­related genes can enhance the sensitivity of breast cancer cells to chemotherapy. Likewise, pyroptosis­related proteins not only participate in pyroptosis, but also regulate the tumor microenvironment, ultimately leading to the death of breast cancer cells. The present review discusses the unique regulatory mechanisms of cuproptosis, ferroptosis and pyroptosis in breast cancer, and the mechanisms through which they are affected by conventional cancer drugs. Furthermore, it provides a comprehensive overview of the significance of these forms of RCD in modulating the efficacy of chemotherapy and highlights their shared characteristics. This knowledge may provide novel avenues for both clinical interventions and fundamental research in the context of breast cancer.

Mendelian segregation and high recombination rates facilitate genetic analyses in Cryptosporidium parvum.

Very little is known about the process of meiosis in the apicomplexan parasite Cryptosporidium despite the essentiality of sex in its life cycle. Most cell lines only support asexual growth of Cryptosporidium parvum (C. parvum), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis in C. parvum, we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures or Ifngr1-/- mice with mCherry and GFP parasites produced "yellow" oocysts generated by cross-fertilization. Outcrossed oocysts from the F1 generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally (~25%) in the population, indicating that C. parvum chromosomes exhibit a Mendelian inheritance pattern. Unexpectedly, the most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses in Ifngr1-/- mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed in Cryptosporidium spp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors.

Sequence introgression from exogenous lineages underlies genomic and biological differences among Cryptosporidium parvum IOWA lines.

The IOWA strain of Cryptosporidium parvum is widely used in studies of the biology and detection of the waterborne pathogens Cryptosporidium spp. While several lines of the strain have been sequenced, IOWA-II, the only reference of the original subtype (IIaA15G2R1), exhibits significant assembly errors. Here we generated a fully assembled genome of IOWA-CDC of this subtype using PacBio and Illumina technologies. In comparative analyses of seven IOWA lines maintained in different laboratories (including two sequenced in this study) and 56 field isolates, IOWA lines (IIaA17G2R1) with less virulence had mixed genomes closely related to IOWA-CDC but with multiple sequence introgressions from IOWA-II and unknown lineages. In addition, the IOWA-IIaA17G2R1 lines showed unique nucleotide substitutions and loss of a gene associated with host infectivity, which were not observed in other isolates analyzed. These genomic differences among IOWA lines could be the genetic determinants of phenotypic traits in C. parvum. These data provide a new reference for comparative genomic analyses of Cryptosporidium spp. and rich targets for the development of advanced source tracking tools.

Bergapten exerts a chondroprotective effect in temporomandibular joint osteoarthritis by combining intestinal flora alteration and reactive oxygen species reduction.

Bergapten, a furanocoumarin naturally occurring in bergamot essential oil, has been demonstrated to have the potential to alleviate osteoarthritis-related symptoms via its anti-inflammatory activity. Although its systemic bioavailability is limited, its precise mechanisms of action and effects on temporomandibular joint osteoarthritis (TMJOA) and its relationship with the intestinal flora remain unclear. Here, we explored the anti-TMJOA effect of BGT combined with the interleukin-1ß-induced inflammatory response of chondrocytes in a monosodium iodoacetate (MIA)-induced TMJOA rat model. It was confirmed that BGT effectively reduced proinflammatory mediators and increased type II collagen, bone volume, and trabecular number of condyles in TMJOA rats. Importantly, the oral administration of BGT altered the intestinal flora of rats by increasing the relative abundances of nine prebiotic species and decreasing the relative abundance of one potential species. In addition, BGT considerably reduced reactive oxygen species (ROS) levels by suppressing glutathione, oxidized glutathione, and superoxide dismutase in the serum and malondialdehyde in urine. These results suggest that BGT exerts a chondroprotective effect, most likely by improving the intestinal flora and reducing ROS production associated with TMJOA in rats. This finding indicates a novel beneficial effect of BGT on the prevention and treatment of TMJOA.

Influence of SPIO labelling on the function of BMSCs in chemokine receptors expression and chemotaxis.

Bone marrow-derived mesenchymal stem cells (BMSCs) are increasingly being used in bone marrow transplantation (BMT) to enable homing of the allogeneic hematopoietic stem cells and suppress acute graft versus host disease (aGVHD). The aim of this study was to optimize the labelling of BMSCs with superparamagnetic iron oxide particles (SPIOs), and evaluate the impact of the SPIOs on the biological characteristics, gene expression profile and chemotaxis function of the BMSCs. The viability and proliferation rates of the SPIO-labeled BMSCs were analyzed by trypan blue staining and CCK-8 assay respectively, and the chemotaxis function was evaluated by the transwell assay. The expression levels of chemokine receptors were measured by RT-PCR and flow cytometry. The SPIOs had no effect on the viability of the BMSCs regardless of the labelling concentration and culture duration. The labelling rate of the cells was higher when cultured for 48 h with the SPIOs. Furthermore, cells labeled with 25 µg/ml SPIOs for 48 h had the highest proliferation rates, along with increased expression of chemokine receptor genes and proteins. However, there was no significant difference between the chemotaxis function of the labeled and unlabeled BMSCs. To summarize, labelling BMSCs with 25 µg/ml SPIOs for 48h did not affect their biological characteristics and chemotaxis function, which can be of significance for in vivo applications.

Catalytic activity of H2O2 by goethite and lepidocrocite: Insight from 5-bromosalicylic acid removal mechanism and density functional theory calculation (ID:CHEM114760).

We have compared the elimination of 5-bromosalicylic acid (BSA) in the systems of goethite (α-FeOOH)/H2O2 and lepidocrocite (γ-FeOOH)/H2O2. The results demonstrated that BSA (10 mg L-1) could be successfully adsorbed on α- and γ-FeOOH (0.5 g L-1) and then effectively degraded after the addition of H2O2 (14.7 mM). BSA adsorption on both α- and γ-FeOOH followed pseudo-second order adsorption kinetic models, with γ-FeOOH having greater adsorption ability than α-FeOOH. In the α-FeOOH/H2O2 system, BSA degradation was well fitted with the pseudo-second order kinetics, whereas the oxidation in γ-FeOOH/H2O2 system had a two-stage pseudo-first order kinetics. Electron paramagnetic resonance (EPR) results for these two systems revealed the presence of •OH and •OOH, and further tests with radical captures demonstrated their dominance in degrading BSA. Based on the electronic structure analysis, electrons were more easily transferred from the H2O2 molecule to the Fe atoms of α-FeOOH, explaining the density functional theory (DFT) calculation results, which showed that α-FeOOH performed better in catalyzing the decomposition of H2O2. However, the free radicals are more likely to desorb from γ-FeOOH, which made the γ-FeOOH/H2O2 system more efficient in degrading BSA.

NLRP3 inflammasome activation in response to metals.

Implant surgery is followed by a series of inflammatory reactions that directly affect its postoperative results. The inflammasome plays a vital role in the inflammatory response by inducing pyroptosis and producing interleukin-1ß, which plays a critical role in inflammation and tissue damage. Therefore, it is essential to study the activation of the inflammasome in the bone healing process after implant surgery. As metals are the primary implant materials, metal-induced local inflammatory reactions have received significant attention, and there has been more and more research on the activation of the NLRP3 (NOD-like receptor protein-3) inflammasome caused by these metals. In this review, we consolidate the basic knowledge on the NLRP3 inflammasome structures, the present knowledge on the mechanisms of NLRP3 inflammasome activation, and the studies of metal-induced NLRP3 inflammasome activation.

Multiple introductions and recombination events underlie the emergence of a hyper-transmissible Cryptosporidium hominis subtype in the USA.

The parasite Cryptosporidium hominis is a leading cause of the diarrheal disease cryptosporidiosis, whose incidence in the United States has increased since 2005. Here, we show that the newly emerged and hyper-transmissible subtype IfA12G1R5 is now dominant in the United States. In a comparative analysis of 127 newly sequenced and 95 published C. hominis genomes, IfA12G1R5 isolates from the United States place into three of the 14 clusters (Pop6, Pop13, and Pop14), indicating that this subtype has multiple ancestral origins. Pop6 (IfA12G1R5a) has an East Africa origin and has recombined with autochthonous subtypes after its arrival. Pop13 (IfA12G1R5b) is imported from Europe, where it has recombined with the prevalent local subtype, whereas Pop14 (IfA12G1R5c) is a progeny of secondary recombination between Pop6 and Pop13. Selective sweeps in invasion-associated genes have accompanied the emergence of the dominant Pop14. These observations offer insights into the emergence and evolution of hyper-transmissible pathogens.

Development of a Serum-Free Culture Method for Endothelial Cells of the Stria Vascularis and Their Pro-Inflammatory Secretome Changes Induced by Oxidative Stress.

OBJECTIVES: Reactive oxygen species in the stria vascularis (SV) of the cochlea may be involved in the pathogenesis of sensorineural hearing loss. However, the effects of oxidative stress on SV endothelial cells (SV-ECs) remain largely unknown, and no feasible in vitro cell culture model exists for the functional study of SV-ECs. METHODS: We isolated primary SV-ECs from the SV of neonatal mice. The apoptosis-reducing effects of fibronectin in SV-ECs cultured with serum-free medium were determined using ß-galactosidase staining and flow cytometry. SV-ECs incubated in serum-free medium were treated with various H2O2 concentrations to evaluate the effects of H2O2 on their viability. The secretome of SV-ECs treated with or without H2O2 (100 µM or 500 µM) was analyzed using high-resolution mass spectrometry. The function of the SV-EC secretome was evaluated by a macrophage assay. RESULTS: We successfully isolated and characterized the SV-ECs. Treatment with H2O2 at concentrations up to 500 µM for 2 hours and further incubation with serum-free medium in plates precoated with fibronectin showed no significant effect on apoptosis. Compared to the control SV-ECs, the amount of differential proteins in the secretome of SV-ECs stimulated with 500 µM H2O2 was much higher than in those treated with 100 µM H2O2. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses suggested that the proteins differentially expressed in SV-ECs treated with 500 µM H2O2 were involved in the regulation of multiple signaling pathways and cellular processes. The secretome of H2O2-stimulated SV-ECs exhibited significant pro-inflammatory effects on macrophages. CONCLUSION: We successfully established an in vitro serum-free culture method, identified the differential proteins released by oxidative stress-induced ECs and their functions, and revealed the pro-inflammatory effects of the secretome of H2O2-stimulated SV-ECs. Therefore, SV-ECs might elicit immunoregulatory effects on bystander cells in the microenvironment of oxidative stress-induced cochlea, especially cochlear macrophages.

Identification of nine mutant genes and establishment of three prediction models of organ tropism metastases of non-small cell lung cancer.

BACKGROUND: Most Non-small cell lung cancer (NSCLC) patients tend to have metastases at the initial diagnosis. However, limited knowledge has been established regarding which factors, are associated with its metastases. This study aims to identify more biomarkers associated with its organ tropism metastasis and to establish models for prediction of its metastatic organs. METHODS: We performed targeted next-generation sequencing (NGS) to detect genes related to lung cancer in 272 patients with primary advanced NSCLC from Northeast China. We adopted Fisher test, multivariate logistic regression analysis to identify metastasis-related gene mutations and to establish prediction models. RESULTS: Mutations of EGFR (p = 0.0003, OR = 2.554) (especially EGFR L858R [p = 0.02, OR = 2.009]), ATM (p = 0.008, OR = 11.032), and JAK2 (p = 0.009, OR = Inf) were positively and of TP53 exon4mut (p = 0.001, OR = 0.173) was negatively correlated with lung metastasis, and those of CSF1R (p = 0.01, OR = Inf), KIT (p = 0.03, OR = 4.746), MYC (p = 0.05, OR = 7.938), and ERBB2 (p = 0.02, OR = 2.666) were positively correlated with pleural dissemination; those of TP53 (p = 0.01, OR = 0.417) was negatively, while of SMAD4 (p = 0.03, OR = 4.957) was positively correlated with brain metastasis of NSCLC. Additionally, smoking history (p = 0.004, OR = 0.004) was negatively correlated with pleural dissemination of NSCLC. Furthermore, models for prediction of lung metastasis (AUC = 0.706), pleural dissemination (AUC = 0.651), and brane metastasis (AUC = 0.629) were established. CONCLUSION: Taken together, this study revealed nine mutant genes and smoking history associated with organ tropism metastases of NSCLC and provided three models for the prediction of metastatic organs. This study enables us to predict the organs to which non-small cell lung cancer metastasizes before it does develop.

High infectivity and unique genomic sequence characteristics of Cryptosporidium parvum in China.

Zoonotic Cryptosporidium parvum infections are mainly caused by IIa and IId subtypes. As most biological characterizations have been performed on IIa subtypes, the biological and genetic characteristics of IId subtypes in China are not clear. We evaluated the infection and genetic characteristics of IId isolates in interferon-γ-knockout mice using qPCR to quantify oocyst shedding, histological examination to monitor pathological changes and comparative genomic analyses to identify infectivity and virulence-associated differences. Compared with the reference IIa isolate, mice infected with the IId isolates had significantly higher and longer oocyst shedding and lower body weight gain. In addition, the four IId isolates examined differed significantly in infectivity (as indicated by the median infective dose), oocyst shedding duration, and pathogenicity. Comparative genomic analysis indicated that the IId isolates had three more subtelomeric genes than the reference IIa isolate and 5385-5548 nucleotide substitutions, with the hypervariable genes mostly in two blocks on chromosome 1. In contrast, the four IId isolates differed from each other by 77-1,452 nucleotides, with virulence-associated sequence differences mainly in nine genes within a 28-kb block on chromosome 6. These data indicate the newly emerged C. parvum IId subtypes in China have high animal infectivity and unique genomic characteristics.

Biodegradability and Cytocompatibility of 3D-Printed Mg-Ti Interpenetrating Phase Composites.

Orthopedic hybrid implants combining both titanium (Ti) and magnesium (Mg) have gained wide attraction nowadays. However, it still remains a huge challenge in the fabrication of Mg-Ti composites because of the different temperatures of Ti melting point and pure Mg volatilization point. In this study, we successfully fabricated a new Mg-Ti composite with bi-continuous interpenetrating phase architecture by infiltrating Mg melt into Ti scaffolds, which were prepared by 3D printing and subsequent acid treatment. We attempted to understand the 7-day degradation process of the Mg-Ti composite and examine the different Mg2+ concentration composite impacts on the MC3T3-E1 cells, including toxicity, morphology, apoptosis, and osteogenic activity. CCK-8 results indicated cytotoxicity and absence of the Mg-Ti composite during 7-day degradation. Moreover, the composite significantly improved the morphology, reduced the apoptosis rate, and enhanced the osteogenic activity of MC3T3-E1 cells. The favorable impacts might be attributed to the appropriate Mg2+ concentration of the extracts. The results on varying Mg2+ concentration tests indicated that Mg2+ showed no cell adverse effect under 10-mM concentration. The 8-mM group exhibited the best cell morphology, minimum apoptosis rate, and maximum osteogenic activity. This work may open a new perspective on the development and biomedical applications for Mg-Ti composites.

Development and validation of the Chinese version of the perceived partner responsiveness scale (C-PPRS).

BACKGROUND: Perceived partner responsiveness (PPR) refers to the belief that the relational partner knows and is sensitive and supportive. Instead of translating the English version of the Perceived Partner Responsiveness Scale (PPRS) into Chinese, this study aimed to construct and analyze the psychometric properties of the Chinese version of the Perceived Partner Responsiveness Scale (C-PPRS). On the one hand, some words in the original scale are inappropriate for the Chinese due to cultural differences. On the other hand, we intended the scale to apply just to persons in romantic relationships, not to friends or roommates. METHOD: We conducted two studies. In the first study, 441 participants who completed the C-PPRS were randomly divided into two samples for exploratory factor analysis and confirmatory factor analysis. Concurrent validity was assessed in a group of 224 participants who completed the C-PPRS and the Quality of Relationship Index in the second study. RESULTS: The results indicated that the four-factor model (understanding, intimacy, acceptance, and trust) was a feasible representation of the C-PPRS factor structure (χ2/df = 2.27, CFI = 0.94, TLI = 0.93, RMSEA = 0.08, SRMR = 0.05) and had robust internal consistency reliability (alpha = 0.90) and concurrent validity (moderately correlated with the Quality of Relationship Index, r = 0.66, p < 0.001). CONCLUSION: PPR is a concept to understand the psychological manifestations of a person who believes that his or her partner is concerned with core characteristics of the self. The C-PPRS has good psychometric characteristics to evaluate such manifestations and can be applied to future intimacy research.

Heteroplasmic mitochondrial genomes of a Raillietina tapeworm in wild Pangolin.

BACKGROUND: Raillietina species belong to the family Davaineidae, which parasitizes in a wide variety of mammals and birds, causing stunted growth, lethargy, emaciation, and digestive tract obstruction. However, only a limited number of Raillietina species have been identified in wild animals. METHODS: We analyzed and annotated the complete mitochondrial (mt) genome of a worm from the intestine of a wild pangolin using Illumina sequencing of whole genomic DNA. RESULTS: These findings showed the presence of two mtDNA sequences in Raillietina sp., designated as mt1 and mt2, with the lengths of 14,331 bp and 14,341 bp, respectively. Both the mts genomes of Raillietina sp. comprised 36 genes, containing 12 protein-coding genes (PCGs), 2 ribosomal RNAs, and 22 transfer RNAs. Gene arrangements of both mt genomes of Raillietina sp. were similar to those of most flatworms, except for taeniids, which shift positions between tRNAL1 and tRNAS2 genes. Twenty of 22 tRNA secondary structures of Raillietina sp. had a typical cloverleaf structure similar to Raillietina tetragona. Sequence differences between the mt1 and mt2 genomes were 4.4%, and this difference arises from the mtDNA heteroplasmic mutations. Moreover, heteroplasmic mtDNA mutations were detected in PCGs, tRNAs, rRNAs, NCRs, and intergenes, but the highest proportion of heteroplasmy of 79.0% was detected in PCGs, indicating the occurrence of mtDNA heteroplasmy in Raillietina sp. To our knowledge, this is the first report of mtDNA heteroplasmy in tapeworm parasites. Phylogenetic analyses of 18S rRNA, ITS2, and 12 PCG sequences demonstrated that the worm was clustered with other Raillietina species in the Davaneidae family. CONCLUSIONS: We found a novel Raillietina species in wild pangolin with the existence of mitochondrial DNA heteroplasmy. Thus, these findings provide insights into the heterogeneity of the mt genome in parasitic cestodes, and mt genome data contributes to the understanding of pangolin-parasitic cestodes in terms of their molecular biology, epidemiology, diagnosis, and taxonomy.

Effect of Electroacupuncture at Zusanli (ST36) on Intestinal Microbiota in Rats With Chronic Atrophic Gastritis.

Background: Electroacupuncture is a common treatment for chronic atrophic gastritis (CAG) in China. We aimed to determine the effects of electroacupuncture at zusanli (ST36) on intestinal microbiota in CAG rats. Methods: In total, 42 SD rats were randomly divided into normal (NC, 10 rats) and model (MG, 32 rats) groups. Rats in the MG group were established as CAG disease models. After that, the rats in the MG group were randomly divided into CAG (10 rats), electroacupuncture (EA, 10 rats), and Vitacoenzyme (Vit, 10 rats) groups. Rats in the NC and CAG groups were subjected to a 30-min/d confinement for 4 weeks. Rats in the EA group were given electroacupuncture at zusanli for 30 min/d for 4 weeks. Rats in the Vit group were given Vitacoenzyme solution 10 ml/(kg d) for 4 weeks. Histopathological changes in the gastric mucosa were observed with hematoxylin and eosin staining, and the gene expression level of p53, Bcl-2, and c-myc was determined using the qPCR method. The 16S rDNA sequencing technique was used to determine structural changes and relative abundance expression of intestinal flora. Results: Compared with the NC group, gastric mucosal pathology in the CAG group revealed significant inflammatory infiltration, and the gastric mucosal lesions in the electroacupuncture group were improved remarkably; the expression of p53 and c-myc genes in the CAG group increased (p < 0.05), while the expression of Bcl-2 genes decreased (p < 0.05) in the EA group, that of p53 and c-myc genes decreased (p < 0.05), and that of Bcl-2 genes increased (p < 0.05). The abundance of bacteria such as Lactobacillus, Desulfobacterota, and Bacteroides pectinophilus group in the CAG group increased (p < 0.05), while that of bacteria such as Gastranaerophilales, Romboutsia, and Blautia decreased (p < 0.05). The relative abundance of Desulfobacterota and Helicobacter in the EA group decreased (p < 0.05), while that of probiotic bacteria such as Oscillospirales, Romboutsia, and Christensenellaceae increased (p < 0.05). Conclusion: Electroacupuncture at zusanli can promote the repair of pathological damage to the gastric mucosa in rats with CAG, and the mechanism might relate to the reduction in the relative abundance of harmful bacteria, increase in the relative abundance of intestinal probiotics, and regulation of the intestinal microbiota.

Age and episode-associated occurrence of Cryptosporidium species and subtypes in a birth-cohort of dairy calves.

The role of species-specific immunity in infection patterns of Cryptosporidium spp. in humans and farm animals is not well understood. In the present study, the dynamics of Cryptosporidium infections in a natural cryptosporidiosis model was examined using genotyping, subtyping and whole genome sequencing tools. In a cross-sectional survey of Cryptosporidium spp. in 934 dairy cattle on one farm, marked age-associated differences in the distribution of Cryptosporidium species and C. bovis subtypes were observed. In a closely followed longitudinal birth cohort study of 81 calves over a 9-month period, shedding of C. parvum oocysts by the IIdA19G1 subtype started at 4 days, peaked at 2 weeks and ended mostly by 4 weeks. In contrast, the shedding of C. bovis oocysts started at 2 weeks, peaked initially at 6 weeks and had a second wave during 15th to 23rd weeks. For C. ryanae, calves had mostly only one episode of infection by one subtype, with accumulative infection increasing much slower than C. parvum and C. bovis. Overall, the accumulative infection rates and mean duration of oocyst shedding for calves in the cohort were 97.4% (76/78) and 2.3 weeks, 100.0% (80/80) and 3.9 weeks, and 78.7% (63/80) and 3.2 weeks for C. parvum, C. bovis and C. ryanae, respectively. The oocyst shedding intensity was much lower in C. bovis and C. ryanae infections compared with C. parvum infection, and in the second episode of C. bovis infection compared with the first episode. The two episodes of C. bovis infections were caused by different genome types that differed mostly in nine genes. Cryptosporidium parvum infection was associated with the occurrence of watery diarrhoea. Data from the natural history study of cryptosporidiosis indicate that despite the existence of acquired immunity against homologous pathogens, neonatal animals experience waves of Cryptosporidium infections by different species and genome types.

Molecular detection of a novel Ancylostoma sp. by whole mtDNA sequence from pangolin Manis javanica.

BACKGROUND: Ancylostoma species are hematophagous parasites that cause chronic hemorrhage in various animals and humans. Pangolins, also known as scaly anteaters, are mammals that live in soil environments where they are readily exposed to soil-borne parasitic nematodes. However, only a limited number of helminth species have been identified in this animal host so far. METHODS: Ancylostoma sp. was isolated from a wild pangolin, and the complete mitochondrial (mt) genome of Ancylostoma sp. was obtained by Illumina sequencing of total genomic DNA. RESULTS: The circular complete mt genome that was assembled had a total length of 13,757 bp and comprised 12 protein-coding genes (PCGs), 22 transfer ribosomal RNAs, two ribosomal RNAs (rRNAs), two non-coding regions and one AT-rich region, but lacked the gene coding for ATPase subunit 8 (atp8). The overall AT content of the mt genome of Ancylostoma sp. was 76%, which is similar to that of other nematodes. The PCGs used two start codons (ATT and TTG) and three stop codons (TAA, TAG, and T). The nucleotide identity of the 12 PCGs ranged from 83.1% to 89.7% and had the highest sequence identity with Ancylostoma caninum among species in the Ancylostomatidae family. Also, the pangolin-derived Ancylostoma sp. lacked repeat sequences in the non-coding regions and in the unique sequence of the short non-coding regions, which differentiated it from other Ancylostoma species. In addition, phylogenetic analyses of 18S rRNA and mtDNA sequences revealed that the Ancylostoma sp. was positioned in a separate branch in the subfamily Ancylostomatinae along with other Ancylostoma species. CONCLUSIONS: The Ancylostoma sp. isolated from a pangolin in this study was identified as a possible new Ancylostoma species. The identification of this Ancylostoma sp. from pangolin enriches our knowledge of the species in the Ancylostomatidae family and provides information that will lead to a better understanding of the taxonomy, diagnostics, and biology of hookworms.