A low testosterone level impairs erectile function by increasing endocan expression in rat penile corpus cavernosum.

BACKGROUND: The mechanism by which a state of low testosterone leads to erectile dysfunction (ED) has not been determined. Endocan is a novel marker of endothelial function. However, whether endocan is involved in the regulation of erectile function under low testosterone levels remains unclear. AIM: In this study we sought to determine whether a low-testosterone state inhibits erectile function by regulating endocan expression in the endothelial cells of the rat penile corpus cavernosum. METHODS: Thirty-six male Sprague-Dawley rats aged 8 weeks were randomly assigned to 6 groups (n = 6 per group) as follows: (1) control, (2) castration, (3) castration + testosterone treatment (treated with 3 mg/kg testosterone propionate per 2 days), (4) control + transfection (4 weeks after castration, injected with lentiviral vector (1 × 108 transduction units/mL, 10 µL), (5) castration + transfection, or (6) castration + empty transfection. One week after the injection, we measured the maximal intracavernous pressure/mean arterial pressure (ICPmax/MAP), serum testosterone and nitric oxide (NO) levels, and the expression of endocan, phospho-endothelial NO synthase (p-eNOS), eNOS, phospho-protein kinase B (p-AKT), and AKT in the rat penile corpus cavernosum. OUTCOMES: Under a low-androgen state, the expression of endocan in the rat penile corpus cavernosum was significantly increased, which inhibited the AKT/eNOS/NO signaling pathway and resulted in ED. RESULTS: In the castration group, the expression of endocan in the rat penile corpus cavernosum was significantly higher than that in the control group (P < .05). Additionally, the levels of p-AKT/AKT, p-eNOS/eNOS, and NO in the rat penile corpus cavernosum and ICPmax/MAP were significantly lower in the castration group than in the control group (P < .05). In the castration + transfection group compared with the castration group there was a significant decrease in the expression of endocan (P < .05) and an increase in the ratios of p-AKT/AKT, p-eNOS/eNOS, and ICPmax/MAP (P < .05) in the rat penile corpus cavernosum. CLINICAL IMPLICATIONS: Downregulating the expression of endocan in the penile corpus cavernosum may be a feasible approach for treating ED caused by hypoandrogenism. STRENGTHS AND LIMITATIONS: The results of this study indicte that endocan may affect NO levels and erectile function through multiple signaling pathways, but further experiments are needed to clarify the relationship between endocan and androgens. CONCLUSION: A low-testosterone state inhibits the AKT/eNOS/NO signaling pathway by increasing the expression of endocan in the rat penile corpus cavernosum and impairing erectile function in rats. Decreasing the expression of endocan in the penile corpus cavernosum can improve erectile function in rats with low testosterone levels.

Molecular characterization of Cryptosporidium spp. in Bactrian camels (Camelus bactrianus) from Yili Kazak Autonomous Prefecture of Xinjiang, China.

Introduction: Cryptosporidium spp. is a significant zoonotic parasite. The prevalence and infection characteristics of Cryptosporidium spp. in Bactrian camels in Yili Kazak Autonomous Prefecture have yet to be fully understood. Thus, the molecular epidemiology of cryptosporidiosis in camels was investigated in this region. Methods: A total of 1,455 fecal samples were collected from 6 counties in three regions (Altay, Tacheng, and Yili) in Yili Prefecture. Nested PCR targeting the small subunit ribosomal RNA (ssu rRNA) gene was used to identify the species or genotypes of Cryptosporidium infection in camels. For C. parvum positive samples, the subtypes were identified using the 60-kDa glycoprotein (gp60) gene. Results and discussion: The overall infection rate was 8.7% (126/1,455), ranging from 5.6% to 11.7% in different regions, and 4.2% to 15.8% in different counties. A significant difference was observed amongst the counties (p < 0.001). Three species were detected, namely C. andersoni (65.1%, 82/126), C. parvum (34.1%, 43/126), and C. occultus (0.8%, 1/126). Three C. parvum subtypes, If-like-A15G2 (n = 29), IIdA15G1 (n = 4), and IIdA19G1(n = 1) were detected, with If-like-A15G2 being the most prevalent subtype. Camels aged 3-12 months exhibited the highest infection rate (11.4%, 44/387), with no significant difference among age groups (p > 0.05). C. parvum was predominant in camels under 3 months, while C. andersoni prevailed in camels over 3 months. There was an extremely significant difference observed among seasons (p < 0.001), summer had the highest infection rates (16.9%, 61/360). This study collected nearly 1,500 samples and, for the first time, investigated Cryptosporidium spp. infection in camels based on different age groups and seasons. All three Cryptosporidiumspecies identified were zoonotic, posing a potential threat to human health and requiring close attention.

Microbiota Profiles of Hen Eggs from the Different Seasons and Different Sectors of Shanghai, China.

Hen eggs are one of the most popular foods worldwide, and their safety is critical. Employing 16S rRNA full-length sequencing is an effective way to identify microorganisms on or in eggs. Here, hen eggs collected from poultry farms over four seasons, as well as from markets in Shanghai, were analyzed with third-generation sequencing. Firmicutes (44.46%) and Proteobacteria (35.78%) were the two dominant phyla, and Staphylococcus, Acinetobacter, Aerococcus, Psychrobacter, and Lactobacillus were the dominant genera. The dominant genera on the eggshell surfaces from the farms varied with the seasons, and the highest contamination of Staphylococcus (32.93%) was seen in the eggs collected during the summer. For the market samples, Pseudomonas was the most abundant in content, with Staphylococcus being the most-often genera found on the eggshell surfaces. Moreover, several potential pathogenic bacteria including Riemerella anatipestifer (species), Klebsiella (genus), and Escherichia/shigella (genus) were detected in the samples. The results revealed the impacts of weather on the microbiota deposited on an eggshell's surface, as well as the impacts due to the differences between the contents and the surface. The results can help disinfect eggs and guide antibiotic selection.

EgSeverin and Eg14-3-3zeta from Echinococcus granulosus are potential antigens for serological diagnosis of echinococcosis in dogs and sheep.

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the metacestode larva of Echinococcus granulosus. In this study, two-dimensional gel electrophoresis (2-DE) coupled with immunoblot analysis revealed that E. granulosus severin and 14-3-3zeta proteins (named EgSeverin and Eg14-3-3zeta, respectively) might be two potential biomarkers for serological diagnosis of echinococcosis. The recombinant EgSeverin (rEgSeverin, 45 kDa) and Eg14-3-3zeta (rEg14-3-3zeta, 35 kDa) were administered subcutaneously to BALB/c mice to obtain polyclonal antibodies for immunofluorescence analyses (IFAs). And IFAs showed that both proteins were located on the surface of protoscoleces (PSCs). Western blotting showed that both proteins could react with sera from E. granulosus-infected sheep, dog, and mice. Indirect ELISAs (rEgSeverin- and rEg14-3-3zeta-iELISA) were developed, respectively, with sensitivities and specificities ranging from 83.33% to 100% and a coefficient of variation (CV %) of less than 10%. The rEgSeverin-iELISA showed cross-reaction with both E. granulosus and E. multilocularis, while the rEg14-3-3zeta-iELISA showed no cross-reaction with other sera except for the E. granulosus-infected ones. The field sheep sera from Xinjiang and Qinghai were analyzed using rEgSeverin-iELISA, rEg14-3-3zeta-iELISA, and a commercial kit respectively, and no significant differences were found among the three methods (p > 0.05). However, the CE positive rates in sheep sera from Qinghai were significantly higher than those from Xinjiang (p < 0.01). Overall, the results suggest that EgSeverin and Eg14-3-3zeta could be promising diagnostic antigens for E. granulosus infection.

Transcriptome analysis of a newly established mouse model of Toxoplasma gondii pneumonia.

BACKGROUND: Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii. Toxoplasma gondii infection of the lungs can lead to severe pneumonia. However, few studies have reported Toxoplasma pneumonia. Most reports were clinical cases due to the lack of a good disease model. Therefore, the molecular mechanisms, development, and pathological damage of Toxoplasma pneumonia remain unclear. METHODS: A mouse model of Toxoplasma pneumonia was established by nasal infection with T. gondii. The model was evaluated using survival statistics, lung morphological observation, and lung pathology examination by hematoxylin and eosin (H&E) and Evans blue staining at 5 days post-infection (dpi). Total RNA was extracted from the lung tissues of C57BL/6 mice infected with T. gondii RH and TGME49 strains at 5 dpi. Total RNA was subjected to transcriptome analysis by RNA sequencing (RNA-seq) followed by quantitative real-time polymerase chain reaction (qRT-PCR) validation. Transcript enrichment analysis was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to assess the biological relevance of differentially expressed transcripts (DETs). RESULTS: C57BL/6 mice infected with T. gondii via nasal delivery exhibited weight loss, ruffled fur, and respiratory crackles at 5 dpi. The clinical manifestations and lethality of RH strains were more evident than those of TGME49. H&E staining of lung tissue sections from mice infected with T. gondii at 5 dpi showed severe lymphocytic infiltration, pulmonary edema, and typical symptoms of pneumonia. We identified 3167 DETs and 1880 DETs in mice infected with the T. gondii RH and TGME49 strains, respectively, compared with the phosphate-buffered saline (PBS) control group at 5 dpi. GO and KEGG enrichment analyses of DETs showed that they were associated with the immune system and microbial infections. The innate immune, inflammatory signaling, cytokine-mediated signaling, and chemokine signaling pathways displayed high gene enrichment. CONCLUSION: In this study, we developed a new mouse model for Toxoplasma pneumonia. Transcriptome analysis helped to better understand the molecular mechanisms of the disease. These results provided DETs during acute T. gondii lung infection, which expanded our knowledge of host immune defenses and the pathogenesis of Toxoplasma pneumonia.

Temporal metabolic profiling of erythrocytes in mice infected with Babesia microti.

BACKGROUND: Babesiosis is an emerging zoonosis worldwide that is caused by tick-borne apicomplexans, Babesia spp., which threatens the health of domesticated and wild mammals and even humans. Although it has done serious harm to animal husbandry and public health, the study of Babesia is still progressing slowly. Until now, no effective anti-Babesia vaccines have been available, and administration of combined drugs tends to produce side effects. Therefore, non-targeted metabolomics was employed in the present study to examine the temporal dynamic changes in the metabolic profile of the infected erythrocytes. The goal was to obtain new insight into pathogenesis of Babesia and to explore vaccine candidates or novel drug targets. METHODS: C57BL/6 mice were infected with B. microti and erythrocytes at different time points (0, 3, 6 , 9, 12, and 22-days post-infection) were subjected to parasitemia surveillance and then metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analyses were performed to clearly separate and identify dysregulated metabolites in Babesia-infected mice. The analyses included principal components analysis (PCA) and orthogonal partial least squares-discrimination analysis (OPLS-DA). The time-series trends of the impacted molecules were analyzed using the R package Mfuzz and the fuzzy clustering principle. The temporal profiling of amino acids, lipids, and nucleotides in blood cells infected with B. microti were also investigated. RESULTS: B. microti infection resulted in a fast increase of parasitemia and serious alteration of the mouse metabolites. Through LC-MS metabolomics analysis, 10,289 substance peaks were detected and annotated to 3,705 components during the analysis period. There were 1,166 dysregulated metabolites, which were classified into 8 clusters according to the temporal trends. Consistent with the trend of parasitemia, the numbers of differential metabolites reached a peak of 525 at 6-days post-infection (dpi). Moreover, the central carbon metabolism in cancer demonstrated the most serious change during the infection process except for that observed at 6 dpi. Sabotage occurred in components involved in the TCA cycle, amino acids, lipids, and nucleotide metabolism. CONCLUSION: Our findings revealed a great alteration in the metabolites of Babesia-infected mice and shed new light on the pathogenesis of B. microti at the metabolic level. The results might lead to novel information about the mechanisms of pathopoiesis, babesisosis, and anti-parasite drug/vaccine development in the future.

Corrigendum: Wildlife is a potential source of human infections of Enterocytozoon bieneusi and Giardia duodenalis in southeastern China.

[This corrects the article DOI: 10.3389/fmicb.2021.692837.].

Rapid and visual detection of Staphylococcus aureus in milk using a recombinase polymerase amplification-lateral flow assay combined with immunomagnetic separation.

AIMS: The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA-LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk. METHODS AND RESULTS: Under optimum conditions, the average capture efficiency values for S. aureus strains (104 colony-forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7 mg immunomagnetic beads. The RPA-LF assay, which comprised DNA amplification via RPA at 39°C for 10 min and visualization of the amplicons through LF strips for 5 min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA-LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36 CFU of S. aureus), which was about 16.7-fold more sensitive than that of the conventional polymerase chain reaction method. When RPA-LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40 CFU per reaction. CONCLUSIONS: The newly developed IMS-RPA-LF method enabled detection of S. aureus at levels as low as 40 CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed IMS-lateral flow RPA-LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS-RPA-LF assay will have important implications for the rapid detection of S. aureus in contaminated food.

Biological and transcriptional studies reveal VmeL is involved in motility, biofilm formation and virulence in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a marine pathogen thought to be the leading cause of seafood-borne gastroenteritis globally, urgently requiring efficient management methods. V. parahaemolyticus encodes 12 resistance/nodulation/division (RND) efflux systems. However, research on these systems is still in its infancy. In this study, we discovered that the inactivation of VmeL, a membrane fusion protein within the RND efflux systems, led to reduction of the ability of biofilm formation. Further results displayed that the decreased capacity of Congo red binding and the colony of ΔvmeL is more translucent compared with wild type strains, suggested reduced biofilm formation due to decreased production of biofilm exopolysaccharide upon vmeL deletion. In addition, the deletion of vmeL abolished surface swarming and swimming motility of V. parahaemolyticus. Additionally, deletion of vmeL weakened the cytotoxicity of V. parahaemolyticus towards HeLa cells, and impaired its virulence in a murine intraperitoneal infection assay. Finally, through RNA-sequencing, we ascertained that there were 716 upregulated genes and 247 downregulated genes in ΔvmeL strain. KEGG enrichment analysis revealed that quorum sensing, bacterial secretion systems, ATP-binding cassette transporters, and various amino acid metabolism pathways were altered due to the inactivation of vmeL. qRT-PCR further confirmed that genes accountable to the type III secretion system (T3SS1) and lateral flagella were negatively affected by vmeL deletion. Taken together, our results suggest that VmeL plays an important role in pathogenicity, making it a good target for managing infection with V. parahaemolyticus.

Corrigendum: Anti-Tumoral Effect and Action Mechanism of Exosomes Derived From Toxoplasma gondii-Infected Dendritic Cells in Mice Colorectal Cancer.

[This corrects the article DOI: 10.3389/fonc.2022.870528.].

Fibrillarin RNA methylase is an interacting protein of Cryptosporidium parvum calmodulin-like protein (CpCML).

Cryptosporidium parvum is an obligate protozoan parasite invading epithelial cells of small intestine of human and animals, and causing diarrheal disease. In apicomplexan parasites, calcium signaling can regulate many essential biological processes such as invasion and migration. As the main intracellular receptor for calcium ions, calmodulins control the activities of hundreds of enzymes and proteins. Calmodulin-like protein (CML) is an important member of the calmodulin family and may play a key role in C. parvum, however, the actual situation is still not clear. The present study aimed to identify the parasite interaction partner proteins of C. parvum calmodulin-like protein (CpCML). By constructing the cpcml bait plasmid, 5 potential CpCML - interacting proteins in C. parvum oocyst were screened by yeast-two-hybrid system (Y2H). Bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) were performed as subsequent validations. Fibrillarin RNA methylase (FBL) was identified via this screening method as CpCML interacting protein in C. parvum. The identification of this interaction made it possible to get a further understanding of the function of CpCML and its contribution to the pathogenicity of C. parvum.

Hypothetical bromodomain-containing protein 5 is required for the growth of Toxoplasma gondii.

Bromodomain (BRD) is a highly conserved structural module domain, found in various proteins, including chromatin-related proteins, nucleus acetyltransferases, and transcription-associated proteins. Toxoplasma gondii, a zoonotic protozoan, encodes at least 12 predicted BRD-containing proteins (BDPs). Here, we investigated the subcellular location and regulatory role of a hypothetical protein BDP that we named TgBDP5. The BRD of TgBDP5 did not contain the conserved Asn and Tyr residues required for acetyl-lysine recognition. TgBDP5 localized in the nucleus of the parasite and remained unchanged during parasite replication. Conditional ablation of TgBDP5 through an auxin-inducible degron-based knockdown strategy caused a growth defect in parasite replication. Depletion of TgBDP5 led to changes in the expression level of 179 genes, suggesting it as an important target for drugs acting against T. gondii.

Disordered Gut Microbiota in Colorectal Tumor-Bearing Mice Altered Serum Metabolome Related to Fufangchangtai.

Purpose: This study aimed to investigate the relationship between gut microbiota (GM) and serum metabolism using antineoplastic Fufangchangtai (FFCT) as the model prescription in the treatment of colorectal cancer (CRC). Methods: Tumor-bearing mice and normal mice were administered different doses of FFCT. The tumor volume of tumor-bearing mice was observed. The levels of CD4+ and CD8+ T cells in the blood, spleen, and tumor of mice were determined using a flow cytometer. The bacterial microbiota in stool samples from mice and the serum metabolomics of FFCT-treated mice and fecal microbiota transplantation mice were detected using 16s RNA sequencing and liquid chromatography-mass spectrometry (LC/MS), respectively. Results: The tumor volume of mice showed no significant decrease after FFCT intervention. The levels of CD4+ and CD8+T lymphocytes showed a significant increase under the intervention of FFCT. GM of colorectal tumor-bearing mice and healthy mice were determined, and the diversity and abundance of Firmicutes, Deferribacteres, Bacteroidetes, and Proteobacteria were significantly different between the two groups. Furthermore, we found that the levels of matrine, isogingerenone B, and armillaripin were significantly decreased in tumor-bearing mice after FFCT intervention, indicating that the tumor-induced dysbiosis of gut bacteria may affect the absorption and metabolism of FFCT. Under the intervention of FFCT, serum metabolism of mice transplanted with feces from CRC patients showed less metabolites related to FFCT than that from healthy people, indicating that GM could be a single factor affecting the metabolism of FFCT. Furthermore, we found that different doses of FFCT-treated mice had higher abundance of Roseburia, Turicibacter, and Flexispira than that in the non-intervention control group. Firmicutes and Bacteroidetes in FFCT-treated groups showed a similar trend compared to the healthy group, indicating that FFCT might correct the intestinal microenvironment by modulating gut microbiota in colorectal tumor-bearing mice. Conclusion: The dysbiosis of GM in tumor-bearing mice reduced the serum metabolites related to FFCT, and FFCT could correct the disordered GM of colorectal tumor-bearing mice to exert efficacy.

Identification of novel biofilm genes in avian pathogenic Escherichia coli by Tn5 transposon mutant library.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen for new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR. The reported genes include 3 flagella genes (fliS, fliD, and fliR), 4 curli fimbriae genes (csgD, csgA, csgF, and csgG) and 3 type 1 fimbriae genes (fimA, fimD, and fimC). The novel genes include 3 coenzyme genes (gltA, bglX, and mltF) and 4 putative protein genes (yehE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted from APEC strain APEC81. The results showed that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P < 0.05). The result of rdar (red, dry and rough) colony morphology showed that curli fimbriae genes (csgD, csgA, csgF, and csgG) and other functional genes (fimC, glxK, yehE, 07045, and 11255) affected the colony morphology. In particular, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as the type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, and the fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence, the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P < 0.05). This study identified 5 novel genes (gltA, bglX, mltF, yehE, and 07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit further study of the mechanism of biofilm regulation in APEC.

Isolation and identification of sporozoite membrane protein of Cryptosporidium parvum and evaluation of calmodulin-like protein immune protection.

Until now, no completely effective parasite-specific drugs or vaccines have been approved for the treatment of cryptosporidiosis. Through the separation and identification of the sporozoite membrane protein of Cryptosporidium parvum (C. parvum), 20 related proteins were obtained. Among them, a calmodulin-like protein (CML) has a similar functional domain-exchange factor hand (EF-hand) motif as calmodulin proteins (CaMs), so it may play a similarly important role in the invasion process. A 663 bp full gene encoding the C. parvum calmodulin-like protein (CpCML) was inserted in pET28a vector and expressed in Escherichia coli. An immunofluorescence assay showed that CpCML was mainly located on the surface of the sporozoites. Three-week-old female BALB/c mice were used for modelling the immunoreactions and immunoprotection of recombinant CpCML (rCpCML) against artificial Cryptosporidium tyzzeri infections. The results indicated a significantly increased in anti-CpCML antibody response, which was induced by the immunized recombinant protein. Compared to rP23 (recombinant P23), GST6P-1 (expressed by pGEX-6P-1 transfected E. coli), GST4T-1 (expressed by pGEX-4T-1 transfected E. coli), glutathione (GSH), adjuvant and blank control groups, rCpCML-immunized mice produced specific spleen cell proliferation in addition to different production levels of IL-2, IFN-γ, TNF-α, IL-4 and IL-5. Additionally, immunization with rCpCML led to 34.08% reduction of oocyst shedding in C. tyzzeri infected mice faeces which was similar to rP23. These results suggest that CpCML may be developed as a potential vaccine candidate antigen against cryptosporidiosis.

Anti-Tumoral Effect and Action Mechanism of Exosomes Derived From Toxoplasma gondii-Infected Dendritic Cells in Mice Colorectal Cancer.

Toxoplasma gondii is an obligate intracellular protozoan with anti-tumor activity against a variety of cancers. However, the therapeutic effect of T. gondii on colorectal cancer is unclear, and using direct Toxoplasma infection in immunotherapy involves safety concerns. This study investigated the anti-tumoral effect and mechanism of exosomes derived from dendritic cells (DCs) infected with T. gondii (Me49-DC-Exo). We used differential ultracentrifugation to isolate exosomes from uninfected DCs (DC-Exo) and T. gondii Me49-infected DCs (Me49-DC-Exo). The isolated exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Me49-DC-Exo significantly inhibited the tumor growth and reduced the proportion of M2 macrophages in the blood of tumor-bearing mice. In vitro, Me49-DC-Exo suppressed macrophage (RAW264.7) polarization to M2 phenotype. miRNA sequencing revealed that multiple miRNAs in Me49-DC-Exo were differentially expressed compared with DC-Exo, among which miR-182-5p, miR-155-5p, miR-125b-2-3p, and miR-155-3p were up-regulated, while miR-9-5p was significantly down-regulated. Transfecting mimics or inhibitors of these differential miRNAs into RAW264.7 cells showed that miR-155-5p promoted M1 macrophage polarization while inhibiting M2 macrophage polarization. Bioinformatics prediction and dual-luciferase reporter assay confirmed the suppressor of cytokine signaling 1 (SOCS1) as a direct target of miR-155-5p. Silencing SOCS1 gene expression in RAW264.7 cells increased CD86 + CD206 - M1 macrophage proportion, and inducible nitric oxide synthase and tumor necrosis factor-α mRNA levels. However, arginase-1 and transglutaminase 2 expression levels decreased. These results suggest that the exosomes inhibit macrophage polarization to M2 phenotype and regulate SOCS1 expression by delivering functional miR-155-5p. These findings provide new ideas for colorectal cancer immunotherapy.

Exosomes Derived From Dendritic Cells Infected With Toxoplasma gondii Show Antitumoral Activity in a Mouse Model of Colorectal Cancer.

Pathogen-based cancer therapies have been widely studied. Parasites, such as Toxoplasma gondii have elicited great interest in cancer therapy. Considering safety in clinical applications, we tried to develop an exosome-based immunomodulator instead of a live parasite for tumor treatment. The exosomes, called DC-Me49-exo were isolated from culture supernatants of dendritic cells (DCs) infected with the Me49 strain of T. gondii and identified. We assessed the antitumoral effect of these exosomes in a mouse model of colorectal cancer (CRC). Results showed that the tumor growth was significantly inhibited after treatment with DC-Me49-exo. Proportion of polymorphonuclear granulocytic bone marrow-derived suppressor cells (G-MDSCs, CD11b+Ly6G+) and monocytic myeloid-derived suppressor cells (M-MDSCs, CD11b+Ly6C+) were decreased in the DC-Me49-exo group compared with the control groups in vitro and in vivo. The proportion of DCs (CD45+CD11c+) increased significantly in the DC-Me49-exo group. Levels of interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly decreased after treatment with DC-Me49-exo. Furthermore, we found that DC-Me49-exo regulated the lever of MDSC mainly by inhibiting the signal transducer and activator of transcription (STAT3) signaling pathway. These results indicated that exosomes derived from DCs infected with T. gondii could be used as part of a novel cancer therapeutic strategy by reducing the proportion of MDSCs.

The GntR-like transcriptional regulator HutC involved in motility, biofilm-forming ability, and virulence in Vibrio parahaemolyticus.

As a halophilic food-borne pathogen, Vibrio parahaemolyticus continueo be a major health issue worldwide. The pathogenic mechanisms of V. parahaemolyticus are still not fully understood. One of the most abundant and widely distributed groups of helix-turn-helix transcription factors is the GntR family of regulators, which are involved in the regulation of various biological processes in bacteria, but little is known about their functions in V. parahaemolyticus. Here, we identified a gene designated as hutC in V. parahaemolyticus SH112 that encodes a member belongs to the HutC subfamily of the large GntR transcriptional regulator family. Compared to the wild type, the hutC mutant strain was significantly more sensitive to acid, bile salt, Triton X-100, and sodium dodecyl sulfate stresses. Our results showed that HutC is required for optimal swimming motility but not necessary for the swarming of V. parahaemolyticus. In addition, inactivation of hutC in V. parahaemolyticus SH112 led to decreased biofilm formation, reduced cytotoxicity in Coca-2 cells, and defective virulence in vivo compared to the wild-type strain. Furthermore, transcriptome sequencing (RNA-Seq) analysis and real-time PCR indicated 4 upregulated and 14 downregulated genes in the hutC mutant strain. Functional analysis revealed that 4 upregulated genes were related to the histidine metabolism pathway. The 14 downregulated genes were mostly related to the cellular metabolic process, binding, and membrane part. This study presents evidence that HutC is involved in bacterial survival under conditions of stress, swimming motility, biofilm formation, cytotoxicity, virulence, and gene regulation of V. parahaemolyticus during infection.

Seroprevalence and the Risk Factor of Toxoplasma gondii Infection to Slaughter Pigs in Chongqing, China.

Toxoplasma gondii can infect all mammals, including humans, and can cause serious public health problems and economic losses. Pork is considered an important source of infection for humans, and seroepidemiological surveys are used to assess the level of infection in pig herds. To understand the current seroprevalence and potential risk factors of T. gondii in pigs in Chongqing, a total of 1221 serum samples collected from seven slaughterhouses in five districts from 2015 to 2019 were analyzed for antibodies against the protozoan by enzyme-linked immunosorbent assay. The overall seropositive rate for T. gondii antibodies in Chongqing was 11.1%. The rate varied among the different districts (9.8-15.2%), slaughterhouses (8.0-18.4%), seasons (8.1-14.6%), and years (6.3-14.7%). These results suggest that the season and year were potential risk factors for T. gondii infection in pigs of Chongqing. The study provides prevalence and risk factor data that may help manage livestock and human infections in downstream areas. The Clinical Trial Registration number was SV-20150605-01.

Avian Pathogenic Escherichia coli through Pfs Affects the Tran-Scription of Membrane Proteins to Resist ß-Lactam Antibiotics.

Avian pathogenic Escherichia coli (APEC) is a causative agent of colibacillosis, one of the principal causes of morbidity and mortality in poultry worldwide. Nowadays, antibiotics are mainly used to prevent and control poultry colibacillosis, but the situation of drug resistance is serious. 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) is involved in methylation reactions, polyamine synthesis, vitamin synthesis, and quorum sensing (QS) pathways. In this study, compared with the APEC wild-type strain DE17, the pfs deletion strain DE17Δpfs was more susceptible to ß-lactam antibiotics (amoxicillin, ceftazidime, cefuroxime) by drug sensitivity test and minimum inhibitory concentration (MIC), and the MIC of the DE17Δpfs was half that of the DE17. Quorum sensing signal molecule AI-2 is involved in antibiotic resistance. In the case of pfs inactivation, the DE17Δpfs cannot synthesize AI-2, so it is necessary to add AI-2 to study whether it affects APEC resistance. When the exogenous AI-2 was added, the MIC of all APEC did not change. Transcriptome sequencing indicated that the transcription levels of a lot of outer membrane protein genes and metabolic genes had changed due to the deletion of pfs. Moreover, the transcription levels of the efflux pump gene tolC and penicillin binding protein (fstI and mrcA) were significantly reduced (p < 0.05), while the transcription levels of the porin protein genes (ompF, ompC, and ompD) were significantly increased (p < 0.05). In addition, it was also found that the outer membrane permeability of the DE17Δpfs was significantly increased (p < 0.05). The results indicated that pfs does not affect APEC strain DE17 resistance to ß-lactam antibiotics through AI-2, but pfs affects the sensitivity of APEC to ß-lactam antibiotics by affecting antibiotic-related genes. This study can provide a reference for screening new drug targets.