Advances in the axenic isolation methods of Blastocystis sp. and their applications.

Blastocystis sp. is a prevalent protistan parasite found globally in the gastrointestinal tract of humans and various animals. This review aims to elucidate the advancements in research on axenic isolation techniques for Blastocystis sp. and their diverse applications. Axenic isolation, involving the culture and isolation of Blastocystis sp. free from any other organisms, necessitates the application of specific media and a series of axenic treatment methods. These methods encompass antibiotic treatment, monoclonal culture, differential centrifugation, density gradient separation, micromanipulation and the combined use of culture media. Critical factors influencing axenic isolation effectiveness include medium composition, culture temperature, medium characteristics, antibiotic type and dosage and the subtype (ST) of Blastocystis sp. Applications of axenic isolation encompass exploring pathogenicity, karyotype and ST analysis, immunoassay, characterization of surface chemical structure and lipid composition and understanding drug treatment effects. This review serves as a valuable reference for clinicians and scientists in selecting appropriate axenic isolation methods.

Molecular prevalence and subtypes distribution of Blastocystis sp. among outpatients and inpatients in north and south areas of Henan Province, China.

Blastocystis sp. is one of the most common intestinal parasites in humans and many animals. To further understand the infection of Blastocystis hominis (B. hominis) and the distribution of its genotype in some areas of Henan Province, China, 793 stool samples from outpatients and inpatients in Xinxiang City and Xinyang City, Henan Province were collected from April 2020 to July 2022. The samples were detected by polymerase chain reaction and analyzed by univariate analysis and logistic regression analysis. The results showed that the infection rates of B. hominis in Xinxiang and Xinyang were 10.97% (51/465) and 10.98% (36/328), respectively. Although there were no significant differences in B. hominis infection between gender, age, residence, and disease background, the incidence of hematochezia significantly differed from the incidence of abdominal pain, diarrhea, and constipation among participants (χ2  = 15.795, p = 0.002). A total of 87 positive samples were sequenced and compared with Basic Local Alignment Search Tool, and five subtypes (ST1, ST3, ST4, ST6, and ST7) were identified, of which ST3 was the dominant subtype (63.22%, 55/87), followed by ST7 (17.24%, 15/87) and ST1 (16.09%, 14/87). This is the first study that analyzed the prevalence and subtype distribution of B. hominis in southern and northern Henan Province, thus providing new insights into the epidemiology of B. hominis.

Construction a novel detection method for Trichomonas vaginalis based on recombinant enzyme polymerase amplification targeting the Actin gene.

Trichomoniasis is a common and curable sexually transmitted disease worldwide. The rapid, convenient, and accurate diagnosis of trichomoniasis is an important link in the prevention and treatment of the disease. The current detection methods of Trichomonas vaginalis are mainly wet mount microscopy, culture, nested PCR, and loop-mediated isothermal amplification. However, these detection methods have some shortcomings. In this study, a recombinant enzyme polymerase amplification (RPA) assay had been conducted to detect T. vaginalis. The target gene and the corresponding primers were screened, and the reaction system and conditions were optimized in the assay of RPA. The sensitivity and specificity of this detection method were analyzed. The detection efficiency of wet mount microscopy, culture, nested PCR, and RPA was compared by testing 53 clinical samples from vaginal secretions. By screening, the actin gene of T. vaginalis could be used as a target gene for RPA detection of T. vaginalis, and the optimum reaction condition to amplify the actin gene by RPA was at 39°C for 30 min. The detection limit of T. vaginalis DNA using RPA was 1 pg, corresponding to a sensitivity of approximately five trophozoites. The RPA assay demonstrated high specificity for T. vaginalis, and there was no cross-reactivity with Giardia lamblia, Escherichia coli, Lactobacillus, Toxoplasma gondii, Staphylococcus aureus, and Candida albicans. Of the 53 clinical samples, the positive rates of T. vaginalis detected by wet mount microscopy, culture, nested PCR and RPA were 50.9 4% (27/53), 71.7% (38/53), 71.7% (38/53), and 69.81% (37/53), respectively. Compared with culture which was used as the gold standard for diagnosing trichomoniasis, testing clinical samples by wet mount microscopy showed 71.05% sensitivity, 100% specificity, and moderate diagnostic agreement with the culture (K = 0.581, Z = 4.661, p < 0.001). The nested PCR showed 100% sensitivity, 100% specificity, and excellent diagnostic agreement (K = 1, Z = 7.28, p < 0.001), while RPA displayed 97.37% sensitivity, 100% specificity, and excellent diagnostic agreement (K = 0.954, Z = 6.956, p < 0.001). At the present study, rapid amplification of actin gene by RPA could be used as a tool for detection of T. vaginalis. The detection method of RPA was more sensitive than wet mount microscopy and displayed excellent specificity. Moreover, RPA amplification of actin gene did not require a PCR instrument and the amplification time was shorter than that of ordinary PCR. Therefore, the RPA assay was proposed in this study as a point-of-care examination and a diagnostic method of T. vaginalis infection, which exhibited the potential value in the treatment and prevention of trichomoniasis.

Development and application of recombinase polymerase amplification assay for rapid detection of Blastocystis sp.

Blastocystis sp. is a common parasite in the intestinal tract of humans and animals. The clinical diagnosis of Blastocystis sp. mainly depends on the microscopic observation of parasite, which can lead to false-negative results. An accurate and convenient diagnostic approach for Blastocystis sp. infection is crucial for effectively preventing and controlling blastocystosis. Herein, we developed a recombinase polymerase amplification (RPA) method for detecting Blastocystis sp. The results showed that the DNA amplification by RPA established in this study could be performed within 5 min at 37°C, with maximum band intensity observed at 30 min. The minimum detection limit of RPA was 100 fg µL−1, consistent with conventional polymerase chain reaction (cPCR). Furthermore, the RPA method exhibited no cross-reactivity with 7 other non-target pathogens in the intestinal tract. Next, the newly established RPA method was used to analyse 40 fecal samples collected clinically, and the detection results were consistent with cPCR. These results corroborate that the newly developed RPA method has good sensitivity and specificity and offers the advantage of short detection times, which can be harnessed for differential diagnosis and rapid detection of Blastocystis sp.

Association between the infections of Trichomonas vaginalis and uterine cervical human papillomavirus: a meta-analysis.

Trichomonas vaginalis (TV) may have an impact on other reproductive tract infections. Studies on the connection between the infection of TV and human papillomavirus (HPV) have been inconsistent. We performed a systematic review of the relevant articles through keywords that satisfy the criteria and filtered the articles according to the inclusion and exclusion criteria. A total of 16 eligible studies were screened for the meta-analysis, involving a total of 150,605 women. RevMan 5.4 software was used for meta-analysis of the selected literatures. The results showed that the papers included in this study had good homogeneity and no significant publication bias was found in the current analysis. The pooled estimates using a fixed-effects model showed that TV was more prevalent in HPV-infected women than in non-infected women [odds ratio (OR): 1.51, 95% confidence interval (CI): 1.29-1.75]; In turn, HPV was more widespread in TV-infected women than in uninfected women (OR: 3.62, 95% CI: 2.71-4.85). Moreover, the interaction between TV and HPV infection was insensitive to the deletion of some studies and correlation coefficients, consequently, the results were robust and reliable. These results suggested that TV is positively associated with HPV infection, and HPV is also a risk factor for TV infection.

Prevalence and molecular subtyping of Blastocystis in domestic pigeons in Henan Province, Central China.

Blastocystis is an anaerobic intestinal protozoan parasite found in humans and many kinds of animals that mainly causes diarrhea, abdominal pain, and other clinical symptoms. At present, research on the prevalence and subtype diversity of Blastocystis in domestic pigeons is very limited. The purpose of this study was to detect the infection rate and gene subtype distribution of Blastocystis in domestic pigeons in Henan Province, Central China, to provide a foundation for preventing and controlling Blastocystis in domestic pigeons. Fecal DNA was extracted from 504 fresh fecal samples of pigeons collected from four areas in Henan Province, Central China. All DNA samples were investigated by polymerase chain reaction, and positive samples were sequenced to analyze the gene subtypes based on small ribosomal subunit (SSU rRNA) gene. The overall infection rate of Blastocystis in pigeons in Henan Province was 7.7% (39/504). Four subtypes (STs) of Blastocystis were identified including ST1 (2/39, 5.1%), ST3 (16/39, 41%), ST4 (1/39, 2.6%), and ST7 (20/39, 51.3%), all of which belonged to zoonotic subtypes, and ST7 was the dominant gene subtype. The results show that Blastocystis infection is common in domestic pigeons in Henan Province, Central China, and the pathogens were zoonotic subtypes. Particular attention should be given to reducing the risk of transmission of Blastocystis from domestic pigeons to humans.

LncRNA LINC00680 promotes lung adenocarcinoma growth via binding to GATA6 and canceling GATA6-mediated suppression of SOX12 expression.

Lung adenocarcinoma (LUAD) is a major subtype of non-small-cell lung cancers (NSCLC). LINC00680 has been characterized as a novel oncogenic lncRNA in LUAD, but its regulatory mechanisms remain largely unclear. This study aimed to explore the subcellular localization of LINC00680 in LUAD and its regulation on the transcriptional process. LUAD cell lines (A549, H1650, and H1299) were used for in vitro and in vivo studies. Results showed LINC00680 depletion resulted in G0/G1 phase arrest of LUAD cells and reduced CDK4 and cyclin D1 expression in H1650 and H1299 cells. LINC00680 overexpression promoted A549 cell proliferation and increased CDK4 and cyclin D1 expression. RNA-fluorescence in situ hybridization (FISH) assay showed that LINC00680 has both cytoplasmic and nuclear distribution in LUAD cells. RNA pulldown and western blotting assays confirmed a physical interaction between LINC00680 and GATA6. In LUAD cells, GATA6 overexpression only slightly suppressed SOX12 transcription. ChIP-qPCR and dual-luciferase assay showed that GATA6 only weakly bound to the SOX12 promoter and decreased its activity. However, when LINC00680 was depleted, these transcriptional suppressive effects were significantly enhanced. These findings suggested that LINC00680 forms a complex with GATA6 and weakens its transcriptional suppressive effect on SOX12 expression. In the nude mice model, LINC00680 overexpression partly abrogated the growth-suppressive effects of GATA6 on A549 derived tumors. In summary, this study revealed a novel LINC00680-GATA6-SOX12 axis in promoting LUAD cell cycle progression and proliferation. Future studies should be conducted for a better understanding of the complex networking of LINC00680 in LUAD.

Characteristics of Biotin lipoyl attachment and 2-oxoacid dehydrogenase acyltransferase of the parasitic nematode Haemonchus contortus and its modulatory functions on goat PBMCs in vitro.

Biotin lipoyl attachment and 2-oxoacid dehydrogenase acyltransferase (BLAODA), as an essential excretion of Haemonchus contortus (HcESPs), was identified to have antigenic functions. T helper-9 (Th9) cells secrete interleukin (IL)-9, a signature cytokine associated with tumour immunology, allergy and autoimmunity. Nonetheless, the understanding of modulatory functions of BLAODA on Th9 and other immune cells is limited. In this study, the BLAODA gene was cloned, and the recombinant (r) protein of BLAODA (rHcBLAODA) was expressed and immunoblotting was performed. The results revealed that HcBLAODA gene was successfully cloned and rHcBLAODA protein was expressed. The localization of rHcBLAODA was confirmed on the surface of gut sections from adult H. contortus. The rHcBLAODA protein capability to react precisely with anti-H. contortus antibodies were confirmed by immunoblotting and immunofluorescence assay (IFA). Further functional analysis showed that interaction of rHcBLAODA with host cells significantly enhanced Th9 cells generation, IL-9 expression, nitric oxide production and cell apoptosis while suppressing the cells proliferation and cells migration depending on the concentration. Overall, these findings suggest that rHcBLAODA protein could modulate the host immune response by inducing Th9 cells to secrete IL-9 cytokine in vitro.

Protection studies of an excretory-secretory protein HcABHD against Haemonchus contortus infection.

Unlike the successful immunization of native H. contortus antigens that contributed to the realization of the first commercial vaccine Barbervax, not many studies revealed the encouraging protective efficacies of recombinant H. contortus antigens in laboratory trials or under field conditions. In our preliminary study, H. contortus α/ß-hydrolase domain protein (HcABHD) was demonstrated to be an immunomodulatory excretory-secretory (ES) protein that interacts with goat T cells. We herein evaluated the protective capacities of two HcABHD preparations, recombinant HcABHD (rHcABHD) antigen and anti-rHcABHD IgG, against H. contortus challenge via active and passive immunization trials, respectively. Parasitological parameter, antibody responses, hematological pathology and cytokine profiling in unchallenged and challenged goats were monitored and determined throughout both trials. Subcutaneous administration of rHcABHD with Freund adjuvants elicited protective immune responses in challenged goats, diminishing cumulative fecal egg counts (FEC) and total worm burden by 54.0% and 74.2%, respectively, whereas passive immunization with anti-rHcABHD IgG conferred substantial protection to challenged goats by generating a 51.5% reduction of cumulative FEC and a 73.8% reduction of total worm burden. Additionally, comparable changes of mucosal IgA levels, circulating IgG levels, hemoglobin levels, and serum interleukin (IL)-4 and IL-17A levels were observed in rHcABHD protein/anti-rHcABHD IgG immunized goats in both trials. Taken together, the recombinant version of HcABHD might have further application under field conditions in protecting goats against H. contortus infection, and the integrated immunological pipeline of ES antigen identification, screening and characterization may provide new clues for further development of recombinant subunit vaccines to control H. contortus.

The excretory-secretory antigen HcADRM1 to generate protective immunity against Haemonchus contortus.

The prevention, treatment and control of Haemonchus contortus have been increasingly problematic due to its widespread occurrence and anthelmintic resistance. There are very few descriptions of recombinant antigens being protective for H. contortus, despite the success of various native antigen preparations, including Barbervax. We recently identified an H. contortus excretory­secretory antigen, H. contortus adhesion-regulating molecule 1 (HcADRM1), that served as an immunomodulator to impair host T-cell functions. Given the prophylactic potential of HcADRM1 protein as a vaccine candidate, we hereby assessed the efficacies of HcADRM1 preparations against H. contortus infection. Parasitological and immunological parameters were evaluated throughout all time points of the trials, including fecal egg counts (FEC), abomasal worm burdens, complete blood counts, cytokine production profiles and antibody responses. Active vaccination with recombinant HcADRM1 (rHcADRM1) protein induced protective immunity in inoculated goats, resulting in reductions of 48.9 and 58.6% in cumulative FEC and worm burdens. Simultaneously, passive administration of anti-HcADRM1 antibodies generated encouraging levels of protection with 46.7 and 56.2% reductions in cumulative FEC and worm burdens in challenged goats. In addition, HcADRM1 preparations-immunized goats showed significant differences in mucosal and serum antigen-specific immunoglobulin G (IgG) levels, total mucosal IgA levels, haemoglobin values and circulating interferon-γ, interleukin (IL)-4 and IL-17A production compared to control goats in both trials. The preliminary data of these laboratory trials validated the immunoprophylactic effects of rHcADRM1 protein. It can be pursued as a potential vaccine antigen to develop an effective recombinant subunit vaccine against H. contortus under field conditions.

Proteomic analysis revealed T cell hyporesponsiveness induced by Haemonchus contortus excretory and secretory proteins.

Haemonchus contortus has evolved highly integrated and sophisticated mechanisms to promote coexistence with hosts. The excretory-secretory (ES) products generated by this parasite contribute to the regulation of the host immune response to facilitate immune evasion and induce chronicity, but the proteins responsible for this process and the exact cellular mechanisms have yet to be defined. In this study, we identified 114 H. contortus ES proteins (HcESPs) interacting with host T cells and 15 T cell binding receptors via co-immunoprecipitation and shotgun liquid chromatography-tandem mass spectrometry analysis. Based on bioinformatics analysis, we demonstrated that HcESPs could inhibit T cell viability, induce cell apoptosis, suppress T cell proliferation and cause cell cycle arrest. Furthermore, the stimulation of HcESPs exerted critical control effects on T cell cytokine production profiles, predominantly promoting the secretion of interleukin (IL)-10, IL-17A and transforming growth factor-ß1 and inhibiting IL-2, IL-4 and interferon-γ production. Collectively, these findings may provide insights into the interaction between ES proteins and key host effector cells, enhancing our understanding of the molecular mechanism underlying parasite immune evasion and providing new clues for novel vaccine development.

Y75B8A.8 (HC8) protein of Haemonchus contortus: A functional inhibitor of host IL-2.

Interleukin 2 (IL-2) is an important immune regulatory factor in the immune response of the host. However, little is known about the inhibitor of host IL-2 in Haemonchus contortus infection. In this study, we found that globin domain-containing protein (HCGB) and Protein Y75B8A.8 (HC8) from H contortus excretory and secretory products are two binding proteins of IL-2 in goats. The yeast two-hybrid screening further validated the positive interactions of IL-2 with HCGB and HC8. Meanwhile, we found that HC8 had inhibitory effects on IL-2-induced peripheral blood mononuclear cell (PBMC) proliferation, while HCGB did not. Furthermore, transcriptional analysis revealed that HC8 could block the IL-2-activated signalling pathway. Our results showed that HC8 was a functional inhibitor of goat IL-2.

HcTTR: a novel antagonist against goat interleukin 4 derived from the excretory and secretory products of Haemonchus contortus.

Haemonchus contortus (H. contortus) has evolved sophisticated evasion mechanisms to ensure their survival, including generating excretion and secretion products (ESPs) to regulate the secretion of host cytokines. Interleukin 4 (IL4) is a classic T-helper cell type 2 (Th2)-type cytokine that plays an irreplaceable role against nematode infection. In this study, three proteins, glutathione S-transferase domain containing protein (HcGST), transthyretin domain containing protein (HcTTR) and calponin actin-binding domain containing protein (HcCab), were identified to bind to goat IL4 by co-immunoprecipitation (Co-IP) assays and yeast two-hybrid screening. Additionally, cell proliferation analysis showed that HcTTR blocked the IL4-induced proliferation of peripheral blood mononuclear cells in goats, while HcGST and HcCab did not. In addition, HcTTR could also downregulate the transcription of candidate genes in the IL4-induced JAK/STAT pathway. These results indicated that HcTTR is a novel antagonist against goat IL4 from HcESPs, and this information could improve our understanding of the relationship between host cytokines and parasite infections.

Self-Assembly of TiO2 Nanofiber-Based Microcapsules by Spontaneously Evolved Multiple Emulsions.

We demonstrate hierarchical nest/crust-like colloidosomes composed of interlocked titanium dioxide (TiO2) nanofibers using spontaneously evolved n-butanol/water/ n-butanol (B/W/B) emulsions. We find two mechanisms to produce colloidosomes from B/W/B droplets due to their mutual solubility and dewetting discrepancy. Porous TiO2 colloidal capsules with loosely intertwined nanofibers were obtained after the dewetting of nanofiber-coated B/W/B droplets, while crustlike TiO2 colloidosomes with a thin shell and large hollow interior are developed from amphiphilic polymer-stabilized B/W/B droplets. We further investigate the effect of experimental parameters, including the initial droplet size, the nanofiber concentration, and the water/butanol ratios in butanol phases, on the droplet-to-colloidosome evolution and resultant morphology of colloidosomes. Our simple and versatile approach for fabricating TiO2 colloidosomes can be extended to a range of irregular colloidal particles, and the products have great potential to act as host systems in electrochemical catalysis, photothermal therapy, or filtration materials.

Molecular cloning and characterization of a novel carotenoid cleavage dioxygenase 1 from Lycium chinense.

Carotenoids are key precursor for aroma compounds in plants. Although the fruit of Lycium chinense contains numerous carotenoids, the formation mechanism of aroma compounds in L. chinense is still poorly understood. In this study, a new carotenoid cleavage dioxygenase (termed LmCCD1) was identified from the leaves of L. chinense. Expression analysis by semiquantitative PCR reveals that LmCCD1 gene is expressed in different tissues of L. chinense, and dominant expression of LmCCD1 gene was found in leaves, flowers, and ripe fruits. In addition, the expression level of LmCCD1 in fruits is in accordance with the content of ß-ionone. Finally, recombinantly expressed LmCCD1 can cleave ß-carotene and lycopene to produce ß-ionone and pseudoionone in in vitro assays. These results indicate that LmCCD1 a novel carotenoids cleavage dioxygenase gene that may regulate the metabolic pathways responsible for aroma metabolite production (such as ß-ionone and pseudoionone) in L. chinense has been identified.

Targeting RNA N6-methyladenosine modification-- a novel therapeutic target for HER2- positive gastric cancer.

Gastric cancer is one of the most common cancers and is considered the 5th most frequent occurring cancer worldwide. It has gained great attention from the clinicians and researchers because of high mortality rate. It is generally treated with chemotherapy, radiotherapy, and surgery. Recently, additional treatment options including immunotherapy and targeted therapy and immunotherapy have been developed. However, poor prognosis, limited survival rate of patients, and drug resistance to treatment remain critical problems. To improve treatment options or to overcome the bottleneck of treatment, identification of diagnostic and prognostic markers, determining the most effective therapeutic options, and uncovering the molecular regulations associated with treatment strategies are required. In this regard n6-methyladenosine (m6A) regulation is considered important. This reversible modification plays a crucial role in progression, development and treatment of HER2-positive gastric cancer. Here, we discuss the role of m6A modification in HER2-positive gastric cancer progression through collecting related studies at present. We further discuss the association of m6A modification with therapeutic efficacy in HER2-positive gastric cancer and list some examples. We conclude that modification of m6A can be a new strategy for improving the prognosis and survival rate of HER2-positive gastric cancer patients.

Identification and characterization of the receptors of a microneme adhesive repeat domain of Eimeria maxima microneme protein 3 in chicken intestine epithelial cells.

Eimeria maxima microneme protein 3 (EmMIC3) is pivotal in the initial recognition and attachment of E. maxima sporozoites to host cells. EmMIC3 comprises 5 tandem Type I microneme adhesive repeat (MAR) domains, among which MAR2 of EmMIC3 (EmMAR2) has been identified as the primary determinant of EmMIC3-mediated tissue tropism. Nonetheless, the mechanisms through which EmMAR2 guides the parasite to its invasion site through interactions with host receptors remained largely uncharted. In this study, we employed yeast two-hybrid (YTH) screening assays and shotgun LC-MS/MS analysis to identify EmMAR2 receptors in chicken intestine epithelial cells. ATPase H+ transporting V1 subunit G1 (ATP6V1G1), receptor accessory protein 5 (REEP5), transmembrane p24 trafficking protein (TMED2), and delta 4-desaturase sphingolipid 1 (DEGS1) were characterized as the 4 receptors of EmMAR2 by both assays. By blocking the interaction of EmMAR2 with each receptor using specific antibodies, we observed varying levels of inhibition on the invasion of E. maxima sporozoites, and the combined usage of all 4 antibodies resulted in the most pronounced inhibitory effect. Additionally, the spatio-temporal expression profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 were assessed. The tissue-specific expression patterns of EmMAR2 receptors throughout E. maxima infection suggested that ATP6V1G1 and DEGS1 might play a role in early-stage invasion, whereas TMED2 could be involved in middle and late-stage invasion and REEP5 and DEGS1 may participate primarily in late-stage invasion. Consequently, E. maxima may employ a multitude of ligand-receptor interactions to drive invasion during different stages of infection. This study marks the first report of EmMAR2 receptors at the interface between E. maxima and the host, providing insights into the invasion mechanisms of E. maxima and the pathogenesis of coccidiosis.

A novel detection method based on MIRA-CRISPR/Cas13a-LFD targeting the repeated DNA sequence of Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis is a protozoan parasite, widely recognized as the most prevalent non-viral sexually transmitted infection (STI) globally. This infection is linked to various complications, including pelvic inflammatory disease, adverse pregnancy outcomes, and an increased risk of acquiring HIV. Current molecular detection methods for T. vaginalis are often costly and technically challenging. METHODS: We developed a novel detection method for T. vaginalis using a multi-enzyme isothermal rapid amplification-clustered regularly interspaced short palindromic repeats (MIRA-CRISPR)/Cas13a-lateral flow device (LFD). This assay targets the repeated DNA sequence (GenBank: L23861.1) of T. vaginalis and is performed at a constant temperature of 37 °C for approximately 1 hour. RESULTS: The detection limit of genomic DNA (gDNA) using our protocol was 1 × 10-4 ng/µl. Specificity was confirmed by the absence of cross-reaction with gDNA from various other microorganisms such as Staphylococcus aureus, Lactobacillus taiwanensis, Escherichia coli, Monilia albicans, Giardia lamblia, or Toxoplasma gondii. Among 30 clinical samples tested, the positive rates of T. vaginalis detection were 33.33% (10/30) by wet mount microscopy, 40% (12/30) by nested polymerase chain reaction (PCR), 40% (12/30) by MIRA-CRISPR/Cas13a-LFD, and 40% (12/30) by the culture method. Compared with the culture method, the gold standard for diagnosing trichomoniasis, wet mount microscopy showed a sensitivity of 83.3% and moderate diagnostic agreement (kappa value = 0.87). Both nested PCR and MIRA-CRISPR/Cas13a-LFD exhibited 100% sensitivity and excellent diagnostic agreement (kappa value = 1). CONCLUSIONS: The MIRA-CRISPR/Cas13a-LFD method is a convenient, rapid, stable, and accurate diagnostic tool for detecting T. vaginalis. This method has the potential to enhance the diagnosis and management of vaginitis, offering a significant improvement over existing diagnostic techniques.

Evaluation of Protective Efficacy of Recombinant Toxoplasma gondii DDX39 Protein Vaccine against Acute and Chronic T. gondii Infection in Mice.

Toxoplasma gondii, a pervasive parasite responsible for toxoplasmosis, poses significant health risks to humans and animals. In this study, we investigated the immunogenicity and protective efficacy of the recombinant T. gondii DDX39 protein formulated with ISA201 adjuvant (rTgDDX39) as a candidate vaccine against toxoplasmosis. The full-length of TgDDX39 gene was successfully amplified, cloned into the pET-30a vector, and expressed in BL21 (DE3) competent cells, which was purified and identified as a 57.1 kDa protein via sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis confirmed that rTgDDX39 was specifically recognized by serum from T. gondii-infected mice. Furthermore, immunization of rats with rTgDDX39 generated antiserum that could specifically recognize the native TgDDX39 protein in T. gondii tachyzoite lysates. Immunofluorescence assay revealed that TgDDX39 was primarily located in the nucleus and perinuclear region of tachyzoites. Our vaccination strategy significantly increased T cell proliferation, with CD4+ T cells rising by 21.9% and CD8+ T cells by 57.8% by the sixth week compared to the adjuvant control group. Additionally, high titers of anti-rTgDDX39 IgG antibodies were detected in vaccinated mice, with a notable induction of IgG1 and IgG2a isotypes, and IgG1/IgG2a > 1 suggests a Th2-biased immune response. Furthermore, in vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize this parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a potential candidate vaccine against toxoplasmosis. The protective efficacy of the vaccine was evaluated in mice challenged with acute (RH) and chronic (PRU) strains of T. gondii, showing a survival time extended to 17 days in the acute model, compared to 13.5 and 14 days in the control groups, and a significant 34% reduction in cyst burden in the chronic model. Additionally, the survival rate in the PRU-infected mice increased from 15-20% in the control groups to 45% in the vaccinated group. In vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize the parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a candidate vaccine against toxoplasmosis.

Autophagy induced by the withdrawal of mitogens promotes neurite extension in rat neural stem cells.

Neural stem cells (NSCs) are usually affected by a number of biological functions in neural traumatic and degenerative diseases. Autophagy may be involved in these diseases. However, whether autophagy could affect NSCs is largely unknown. Therefore, we aimed to investigate intracellular microstructures, proliferation, axon extension, and Beclin-1 expression of rat NSCs by basic culture medium and conditioned medium without epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Transmission electron microscopy showed the formation of autophagic vacuoles in conditioned medium. Compared to the control group with normal medium, the number of secondary neurosphere was significantly reduced whereas the expression of Beclin-1 was enhanced. The majority of NSCs were nestin-positive when EGF/bFGF was withdrawn for 3 days and showed enhanced neurite extension, which was suppressed by autophagy antagonist 3-methyladenine. Our findings revealed that a short-term paucity of mitogens in microenvironments could induce autophagy of NSCs, which facilitated NSCs' axonal growth.