Meta-analysis identifying gut microbial biomarkers of Qinghai-Tibet Plateau populations and the functionality of microbiota-derived butyrate in high-altitude adaptation.

The extreme environmental conditions of a plateau seriously threaten human health. The relationship between gut microbiota and human health at high altitudes has been extensively investigated. However, no universal gut microbiota biomarkers have been identified in the plateau population, limiting research into gut microbiota and high-altitude adaptation. 668 16s rRNA samples were analyzed using meta-analysis to reduce batch effects and uncover microbiota biomarkers in the plateau population. Furthermore, the robustness of these biomarkers was validated. Mendelian randomization (MR) results indicated that Tibetan gut microbiota may mediate a reduced erythropoietic response. Functional analysis and qPCR revealed that butyrate may be a functional metabolite in high-altitude adaptation. A high-altitude rat model showed that butyrate reduced intestinal damage caused by high altitudes. According to cell experiments, butyrate may downregulate hypoxia-inducible factor-1α (HIF-1α) expression and blunt cellular responses to hypoxic stress. Our research found universally applicable biomarkers and investigated their potential roles in promoting human health at high altitudes.

Results of a Pilot External Quality Assessment Scheme for Genetic Testing of Newborns with Spinal Muscular Atrophy.

BACKGROUND: This study aims to evaluate the ability of laboratories to perform spinal muscular atrophy (SMA) genetic testing in newborns based on dried blood spot (DBS) samples, and to provide reference data and advance preparation for establishing the pilot external quality assessment (EQA) scheme for SMA genetic testing of newborns in China. METHODS: The pilot EQA scheme contents and evaluation principles of this project were designed by National Center for Clinical Laboratories (NCCL), National Health Commission. Two surveys were carried out in 2022, and 5 batches of blood spots were submitted to the participating laboratory each time. All participating laboratories conducted testing upon receiving samples, and test results were submitted to NCCL within the specified date. RESULTS: The return rates were 75.0% (21/28) and 95.2% (20/21) in the first and second surveys, respectively. The total return rate of the two examinations was 83.7% (41/49). Nineteen laboratories (19/21, 90.5%) had a full score passing on the first survey, while in the second survey twenty laboratories (20/20, 100%) scored full. CONCLUSIONS: This pilot EQA survey provides a preliminary understanding of the capability of SMA genetic testing for newborns across laboratories in China. A few laboratories had technical or operational problems in testing. It is, therefore, of importance to strengthen laboratory management and to improve testing capacity for the establishment of a national EQA scheme for newborn SMA genetic testing.

Tricholoma matsutake polysaccharides suppress excessive melanogenesis via JNK-mediated pathway: Investigation in 8- methoxypsoralen induced B16-F10 melanoma cells and clinical study.

Skin hyperpigmentation is a worldwide condition associated with augmented melanogenesis. However, conventional therapies often entail various adverse effects. Here, we explore the safety range and depigmentary effects of polysaccharides extract of Tricholoma matsutake (PETM) in an in vitro model and further evaluated its efficacy at the clinical level. An induced-melanogenesis model was established by treating B16-F10 melanoma cells with 8-methoxypsoralen (8-MOP). Effects of PETM on cell viability and melanin content were examined and compared to a commonly used depigmentary agent, α-arbutin. Expressions of key melanogenic factors and upstream signaling pathway were analysed by quantitative PCR and western blot. Moreover, a placebo-controlled clinical study involving Chinese females with skin hyperpigmentation was conducted to measure the efficacy of PETM on improving facial pigmented spots, melanin index, and individual typology angle (ITA°). Results demonstrated that PETM (up to 0.5 mg/mL) had little effect on the viability and motility of B16-F10 cells. Notably, it significantly suppressed the melanin content and expressions of key melanogenic factors induced by 8-MOP in B16-F10 melanoma cells. Western blotting results revealed that PETM inhibited melanogenesis by inactivating c-Jun N-terminal kinase (JNK), and this inhibitory role could be rescued by JNK agonist treatment. Clinical findings showed that PETM treatment resulted in a significant reduction of facial hyperpigmented spot, decreased melanin index, and improved ITA° value compared to the placebo-control group. In conclusion, these in vitro and clinical evidence demonstrated the safety and depigmentary efficacy of PETM, a novel polysaccharide agent. The distinct mechanism of action of PETM on melanogenic signaling pathway positions it as a promising agent for developing alternative therapies.

Splicing factor SRSF1 is essential for homing of precursor spermatogonial stem cells in mice.

Spermatogonial stem cells (SSCs) are essential for continuous spermatogenesis and male fertility. The underlying mechanisms of alternative splicing (AS) in mouse SSCs are still largely unclear. We demonstrated that SRSF1 is essential for gene expression and splicing in mouse SSCs. Crosslinking immunoprecipitation and sequencing data revealed that spermatogonia-related genes (e.g. Plzf, Id4, Setdb1, Stra8, Tial1/Tiar, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes. Specific deletion of Srsf1 in mouse germ cells impairs homing of precursor SSCs leading to male infertility. Whole-mount staining data showed the absence of germ cells in the testes of adult conditional knockout (cKO) mice, which indicates Sertoli cell-only syndrome in cKO mice. The expression of spermatogonia-related genes (e.g. Gfra1, Pou5f1, Plzf, Dnd1, Stra8, and Taf4b) was significantly reduced in the testes of cKO mice. Moreover, multiomics analysis suggests that SRSF1 may affect survival of spermatogonia by directly binding and regulating Tial1/Tiar expression through AS. In addition, immunoprecipitation mass spectrometry and co-immunoprecipitation data showed that SRSF1 interacts with RNA splicing-related proteins (e.g. SART1, RBM15, and SRSF10). Collectively, our data reveal the critical role of SRSF1 in spermatogonia survival, which may provide a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying homing of precursor SSCs.

TMEM225 Is Essential for Sperm Maturation and Male Fertility by Modifying Protein Distribution of Sperm in Mice.

Nonobstructive azoospermia is the leading cause of male infertility. Abnormal levels of transmembrane protein 225 (TMEM225), a testis-specific protein, have been found in patients with nonobstructive azoospermia, suggesting that TMEM225 plays an essential role in male fertility. Here, we generated a Tmem225 KO mouse model to explore the function and mechanism of TMEM225 in male reproduction. Male Tmem225 KO mice were infertile. Surprisingly, Tmem225 deletion did not affect spermatogenesis, but TMEM225-null sperm exhibited abnormalities during epididymal maturation, resulting in reduced sperm motility and an abnormal hairpin-loop configuration. Furthermore, proteomics analyses of cauda sperm revealed that signaling pathways related to mitochondrial function, the glycolytic pathway, and sperm flagellar morphology were abnormal in Tmem225 KO sperm, and spermatozoa lacking TMEM225 exhibited high reactive oxygen species levels, reduced motility, and flagellar folding, leading to typical asthenospermia. These findings suggest that testicular TMEM225 may control the sperm maturation process by regulating the expression of proteins related to mitochondrial function, glycolysis, and sperm flagellar morphology in epididymal spermatozoa.

BCAS2 regulates oocyte meiotic prophase I by participating in mRNA alternative splicing.

Oocyte meiotic prophase I (MI) is an important event in female reproduction. Breast cancer amplified sequence 2 (BCAS2) is a component of the spliceosome. Previous reports have shown that BCAS2 is critical in male germ cell meiosis, oocyte development, and early embryo genome integrity. However, the role of BCAS2 in oocyte meiosis has not been reported. We used Stra8-GFPCre mice to knock out Bcas2 in oocytes during the pachytene phase. The results of fertility tests showed that Bcas2 conditional knockout (cKO) in oocytes results in infertility in female mice. Morphological analysis showed that the number of primordial follicles in the ovaries of 2-month-old (M) mice was significantly reduced and that follicle development was blocked. Further analysis showed that the number of primordial follicles decreased and that follicle development was slowed in 7-day postpartum (dpp) ovaries. Moreover, primordial follicles undergo apoptosis, and DNA damage cannot be repaired in primary follicle oocytes. Meiosis was abnormal; some oocytes could not reach the diplotene stage, and more oocytes could not develop to the dictyotene stage. Alternative splicing (AS) analysis revealed abnormal AS of deleted in azoospermia like (Dazl) and diaphanous related formin 2 (Diaph2) oogenesis-related genes in cKO mouse ovaries, and the process of AS was involved by CDC5L and PRP19.

SRSF1 is essential for primary follicle development by regulating granulosa cell survival via mRNA alternative splicing.

Granulosa cell abnormalities are characteristics of premature ovarian insufficiency (POI). Abnormal expression of serine/arginine-rich splicing factor 1 (SRSF1) can cause various diseases, but the role of SRSF1 in mouse granulosa cells remains largely unclear. In this study, we found that SRSF1 was expressed in the nuclei of both mouse oocytes and granulosa cells. The specific knockout of Srsf1 in granulosa cells led to follicular development inhibition, decreased granulosa cell proliferation, and increased apoptosis. Gene Ontology (GO) analysis of RNA-seq results revealed abnormal expression of genes involved in DNA repair, cell killing and other signalling pathways. Alternative splicing (AS) analysis showed that SRSF1 affected DNA damage in granulosa cells by regulating genes related to DNA repair. In summary, SRSF1 in granulosa cells controls follicular development by regulating AS of genes associated with DNA repair, thereby affecting female reproduction.

BCAS2 Participates in Insulin Synthesis and Secretion via mRNA Alternative Splicing in Mice.

Insulin secreted by pancreatic ß cells is essential for maintaining blood glucose levels. Diabetes is caused primarily by a loss of ß cells or impairment of ß-cell function. A previous whole-transcriptome analysis of islets from a type 2 diabetes group and a control group showed that a splicing disorder occurred in approximately 25% of splicing events. Breast carcinoma amplified sequence 2 (BCAS2) is a spliceosome component whose function in islet ß cells is unclear. Here, we report that knockdown of Bcas2 decreased glucose- and KCl-stimulated insulin secretion in the NIT-1 cell line. Pancreas weight, glucose tolerance, and insulin sensitivity were measured in normal chow-fed Bcas2 f/f-ßKO mice, and ß-cell mass and islet size were analyzed by immunohistochemistry. Glucose intolerance developed in Bcas2 f/f-ßKO mice, but there were no significant differences in pancreas weight, insulin sensitivity, ß-cell mass, or islet size. Furthermore, observation of glucose-stimulated insulin secretion and insulin secretion granules in normal chow-fed mice revealed that the insulin level in serum and the number of insulin secretion granules were decreased in Bcas2 f/f-ßKO mice. These differences were related to abnormal splicing of Syt7 and Tcf7l2 pre-mRNA. Taken together, these results demonstrate that BCAS2 is involved in alternative splicing during insulin synthesis and secretion.

BCAS2 regulates granulosa cell survival by participating in mRNA alternative splicing.

BACKGROUND: Granulosa cell proliferation and differentiation are essential for follicle development. Breast cancer amplified sequence 2 (BCAS2) is necessary for spermatogenesis, oocyte development, and maintaining the genome integrity of early embryos in mice. However, the function of BCAS2 in granulosa cells is still unknown. RESULTS: We show that conditional disruption of Bcas2 in granulosa cells caused follicle development failure; the ratio of the positive cells of the cell proliferation markers PCNA and Ki67 were unchanged in granulosa cells. Specific deletion of Bcas2 caused a decrease in the BrdU-positive cell ratio, cell cycle arrest, DNA damage, and an increase in apoptosis in granulosa cells, and RPA1 was abnormally stained in granulosa cells. RNA-seq results revealed that knockout of Bcas2 results in unusual expression of cellular senescence genes. BCAS2 participated in the PRP19 complex to mediate alternative splicing (AS) of E2f3 and Flt3l mRNA to inhibit the cell cycle. Knockout of Bcas2 resulted in a significant decrease in the ratio of BrdU-positive cells in the human granulosa-like tumour (KGN) cell line. CONCLUSIONS: Our results suggest that BCAS2 may influence the proliferation and survival of granulosa cells through regulating pre-mRNA splicing of E2f3 and Flt3l by forming the splicing complex with CDC5L and PRP19.

SRSF2 in Sertoli cells is essential for testicular development and spermatogenesis in mice.

Sertoli cells are essential for testis development and normal spermatogenesis by providing support and nutrients. Pre-messenger RNA (pre-mRNA) processing is the basic mechanism required for gene expression, and members of the serine/arginine-rich protein (SR) family are key components of the machines that perform these basic processing events. Serine/arginine-rich splicing factor 2 (SRSF2) is an important member of the SR family; however, the physiological functions of SRSF2 in Sertoli cells are still unclear. Here, we found that SRSF2 was localized in the nuclei of Sertoli and germ cells in male mice at all stages by breeding Amh-Cre mice obtained with Srsf2-specific knockout in Sertoli cells to define the function of SRSF2 in Sertoli cells. The experimental results showed that specific deletion of SRSF2 impaired fetal Sertoli cell proliferation and induced abnormal apoptosis and severe DNA damage in seminiferous tubules, resulting in severe testicular dysplasia, seminiferous tubule atrophy, and almost no normal seminiferous tubules at postnatal day 14. Eventually, these changes resulted in failure to produce normal sperm and infertility. Further RNA-seq results showed that many key genes related to proliferation and apoptosis were downregulated; Racgap1 mRNA undergoes exon skipping. Thus, SRSF2-dependent Sertoli cells are essential for testicular development and male reproduction.

SRSF1 regulates primordial follicle formation and number determination during meiotic prophase I.

BACKGROUND: Ovarian folliculogenesis is a tightly regulated process leading to the formation of functional oocytes and involving successive quality control mechanisms that monitor chromosomal DNA integrity and meiotic recombination. A number of factors and mechanisms have been suggested to be involved in folliculogenesis and associated with premature ovarian insufficiency, including abnormal alternative splicing (AS) of pre-mRNAs. Serine/arginine-rich splicing factor 1 (SRSF1; previously SF2/ASF) is a pivotal posttranscriptional regulator of gene expression in various biological processes. However, the physiological roles and mechanism of SRSF1 action in mouse early-stage oocytes remain elusive. Here, we show that SRSF1 is essential for primordial follicle formation and number determination during meiotic prophase I. RESULTS: The conditional knockout (cKO) of Srsf1 in mouse oocytes impairs primordial follicle formation and leads to primary ovarian insufficiency (POI). Oocyte-specific genes that regulate primordial follicle formation (e.g., Lhx8, Nobox, Sohlh1, Sohlh2, Figla, Kit, Jag1, and Rac1) are suppressed in newborn Stra8-GFPCre Srsf1Fl/Fl mouse ovaries. However, meiotic defects are the leading cause of abnormal primordial follicle formation. Immunofluorescence analyses suggest that failed synapsis and an inability to undergo recombination result in fewer homologous DNA crossovers (COs) in the Srsf1 cKO mouse ovaries. Moreover, SRSF1 directly binds and regulates the expression of the POI-related genes Six6os1 and Msh5 via AS to implement the meiotic prophase I program. CONCLUSIONS: Altogether, our data reveal the critical role of an SRSF1-mediated posttranscriptional regulatory mechanism in the mouse oocyte meiotic prophase I program, providing a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying primordial follicle formation.

Casein Kinase 1α Regulates Testosterone Synthesis and Testis Development in Adult Mice.

Casein kinase 1α (CK1α) is a main component of the Wnt/ß-catenin signaling pathway, which participates in multiple biological processes. Our recent study demonstrated that CK1α is expressed in both germ cells and somatic cells of mouse testes and regulates spermatogenesis. However, little information is known about the role of CK1α in regulating the development of somatic cells in mouse testes. Our results demonstrated that conditional disruption of CK1α in murine Leydig cells sharply decreased testosterone levels; markedly affected testis development, sperm motility, and sperm morphology; and caused subfertility. The germ cell population was partially decreased in CK1α conditional knockout (cKO) mice, while the proliferation of Leydig cells and Sertoli cells was not affected. Furthermore, in vitro results verified that luteinizing hormone upregulates CK1α through the luteinizing hormone/protein kinase/Epidermal Growth Factor Receptor/extracellular regulated protein kinases/2 signaling pathway and that CK1α interacts with and phosphorylates EGFR, which subsequently activates the phosphorylation of ERK1/2, thereby promoting testosterone synthesis. In addition, high-dose testosterone propionate partially rescued the phenotype observed in cKO mice. This study provides new insights into the role of CK1α in steroidogenesis and male reproduction.

RAF1 mediates the FSH signaling pathway as a downstream molecule to stimulate estradiol synthesis and secretion in mouse ovarian granulosa cells.

Background: The v-raf-leukemia viral oncogene 1 (RAF1) plays an essential physiological role in reproduction and development through the mediation of steroid hormone synthesis. Follicle-stimulating hormone (FSH) signaling pathway was not involved in the majority of RAF1 studies, whether RAF1 takes part in the signaling events of gonadotropic hormones such as FSH in ovarian tissue is unknown. Methods: The process is blocked by treating granulosa cells (GCs) with the RAF1 inhibitor, RAF709. Inhibition of RAF1 activity by RAF709 decreased extracellular regulated protein kinases (ERK) phosphorylation and suppressed the expression of the cytochrome P450 subfamily 19 member 1 (CYP19A1), which is a major rate-limiting enzyme that participates in the last step of E2 biosynthesis. Results: We found that RAF1, acting as a downstream molecule, mediates FSH signalling to stimulate estradiol (E2) synthesis and secretion in mouse ovarian GCs. Gene expression of RAF1 was induced by FSH and the secretion of E2 increased into the bloodstream of mice and into the supernatant of primary GCs. Our in vitro and in vivo studies clearly illustrate RAF1 plays an important medium adjusting role in the FSH signaling pathway, and RAF1 acting as a downstream molecule to trigger ERK phosphorylation to stimulate GC E2 synthesis and secretion. Conclusions: RAF1 plays a pivotal mediating role in the FSH signaling pathway by inducing the phosphorylation of ERK and promoting E2 synthesis.

Key Factors in the Application of Sharestart to Enhance the Learning Attitude of Students.

Under the background of globalization and the popularity of distance learning ande-learning channels provided on the Internet, teaching methods that encourage the self-directed learning of students are becoming popular. There is an increasing number of domestic teachers joining in the practice for change. The various teaching methods that make the students acquire critical thinking skills can be summarized as learning by doing, critical thinking learning, multiple assessments, team discussion teaching, and cooperative learning. With the teachers of the universities in Shanghai as the questionnaire analysis objects, a total of 360 copies of questionnaires were distributed, and 256 valid copies were retrieved, with the retrieval rate of 71%. The research results are summarized as follows. (1) The "mental adaptation and engagement of students" is the most emphasized dimension, followed by the "professional development of teachers," "administration and parent support," and "material and teaching strategy." (2) The top five emphasized indicators, among 14, are the ordered cultivation of self-study and thinking habits, the development of the professional community for the collaborative lesson study of teachers, the support and cooperation of the president and the administration, adoption of heterogeneous grouping, and co-learning, discussion and cooperative learning. According to the results, it is expected to propose more definite practice directions for teachers intending to attempt such a teaching method, as well as provide some specific suggestions for the first movers of Sharestart.

Cyclin-dependent kinase inhibitor 1B acts as a novel molecule to mediate testosterone synthesis and secretion in mouse Leydig cells by luteinizing hormone (LH) signaling pathway.

Cyclin-dependent kinase inhibitor 1B (Cdkn1b, p27) plays important regulatory roles in many cellular processes. p27 is highly expressed in the mouse testis, but its roles and underlying mechanisms for testosterone synthesis and secretion remain not well understood. In the current study, we found that p27 located in Leydig cells and Sertoli cells of adult mouse testis. To explore the function of p27 in Leydig cells, p27 inhibitor and activator were injected into the adult mice, primary Leydig cells and TM3 cells. Our in vivo and in vitro results showed that change in the expression of p27 significantly alters the testosterone in both globe serum and culture medium. Meanwhile, the steroidogenesis-related gene expression was significantly regulated too. Moreover, our in vitro study showed that luteinizing hormone (LH) significantly increased p27 mRNA levels. Furthermore, our results proved that altering the mRNA expression of p27 leads to the synchronized changes of Lhcgr, Star, Cyp11a1, Hsd3b6, Cyp11a1, and Hsd17b3. Alterations of p27 also result in synchronously changes of RAF1 and ERK1/2 phosphorylation. These findings indicate that p27 plays vital roles in LH-induced testosterone production, providing a novel mechanism that p27 acts as an upstream molecule to elevate ERK1/2 phosphorylation to promote the expression of StAR and other cholesterol-metabolizing enzymes.

Screening of different sample types associated with sheep and cattle for the presence of nematophagous fungi in China.

A total of 1502 samples, including feces of sheep (793) and cattle (348), pasture soil (118), dung compost (147) and barn soil (96), were examined between October 2012 and August 2014 to discover potential strains of nematophagous fungi for the biological control of livestock-parasitic nematodes. These samples were collected from 87 sites located in 48 counties of 20 provinces (autonomous regions/municipalities) of China. Fungi were identified down to a species level. Four hundred and seventy-seven isolates, which were distributed in 8 genera and 28 taxa, were identified as nematophagous fungi. Nematode-trapping fungi included 17 species and one unidentified species of Arthrobotrys, two of Dactylella, Drechslerella dactyloides, and Duddingtonia flagrans. Five identified species and two unidentified species of endoparasitic fungi were isolated. The predominant species from all regions were Arthrobotrys oligospora, followed by Arthrobotrys musiformis, Arthrobotrys (Monacrosporium) thaumasiun, and Arthrobotrys (Monacrosporium) microscaphoides. Species with adhesive networks were the most frequently isolated. Among the endoparasitic fungi, Podocrella harposporifera (Harposporium anguillulae) was the most common species, followed by Harposporium lilliputanum and Harposporium arcuatum. Based on Shannon diversity index, the diversity levels of nematophagous fungi were relatively higher in samples associated with cattle, barn soil, and subtropical monsoon climate zone. Three species isolated from this study, namely, Duddingtonia flagrans, Arthrobotrys salina (Monacrosporium salinum), and Arthrobotrys oligospora var. sarmatica, are newly recorded in China, and 20 species (including one unidentified species) are newly recorded in sheep and cattle barn soils worldwide.

Isolation and Characterization of China Isolates of Duddingtonia flagrans, a Candidate of the Nematophagous Fungi for Biocontrol of Animal Parasitic Nematodes.

The nematophagous fungus Duddingtonia flagrans has been investigated as a biological agent for the control of gastrointestinal nematodes infecting domestic animals in other countries. However, D. flagrans has not been detected in China. In this study 1,135 samples were examined from 2012 to 2014; 4 D. flagrans isolates (SDH 035, SDH 091, SFH 089, SFG 170) were obtained from the feces of domestic animals and dung compost. The 4 isolates were then characterized morphologically. The SDH 035 strain was characterized by sequencing the ITS1-5.8S rDNA-ITS2 region. A BLAST search showed that the SDH 035 strain (GenBank KP257593) was 100% identical to Arthrobotrys flagrans (AF106520) and was identified as D. flagrans. The morphological plasticity of the isolated strain and the interaction of this strain with the nematode targets were observed by subjecting the infected trichostrongylide L3 to scanning electron microscopy. At 6 and 8 hr after trichostrongylide L(3) was added, hyphal ramifications were observed and L(3) were captured, respectively. Scanning electron micrographs were obtained at 0, 6, 12, 18, 24, 30, 36, 42, and 48 hr, where 0 is the time when trichostrongylide L(3) were first captured by the fungus. The details of the capture process by the fungus are also described. Chlamydospores were observed in the body of L(3) in the late stage of digestion. A sticky substance and bacteria could be observed in contact areas between predation structures and nematode cuticle.

Isolation, identification, and characterization of the nematophagous fungus Monacrosporium salinum from China.

Nematophagous fungi are considered to have the best potential as biological agents for the control of gastrointestinal nematodes in domestic animals. However, relatively few studies have been conducted with the genus Monacrosporium, especially with strains native to China. In the present study, we isolated and identified nematophagous fungi from fresh sheep feces. A pure fungal strain was molecularly characterized, and its nematophagous activity was evaluated. The morphological plasticity of the isolated strain, as well as its interaction with the nematode targets, was observed by scanning electron microscopy of the infected Trichostrongylus colubriformis L3 and the free-living nematode Caenorhabditis elegans. Three isolated fungal strains from the 30 fresh fecal samples of sheep from Inner Mongolia, China exhibited predatory activity; however, only a single strain was successfully purified (SF 0459). The SF 0459 strain was characterized by morphological analysis of its conidia and sequencing of its ITS1-5.8S rDNA-ITS2 region. This strain was identified to be Monacrosporium salinum (GenBank ID: KP036623). Nematophagous fungus helper bacteria were found at the interaction points between fungi and nematodes. The percentage of live T. colubriformis L3 was reduced by 83.79-88.69% based on the in vitro assay.