Combined therapy of CAR-IL-15/IL-15Rα-T cells and GLIPR1 knockdown in cancer cells enhanced anti-tumor effect against gastric cancer.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has shown remarkable responses in hematological malignancies with several approved products, but not in solid tumors. Patients suffer from limited response and tumor relapse due to low efficacy of CAR-T cells in the complicated and immunosuppressive tumor microenvironment. This clinical challenge has called for better CAR designs and combined strategies to improve CAR-T cell therapy against tumor changes. METHODS: In this study, IL-15/IL-15Rα was inserted into the extracellular region of CAR targeting mesothelin. In-vitro cytotoxicity and cytokine production were detected by bioluminescence-based killing and ELISA respectively. In-vivo xenograft mice model was used to evaluate the anti-tumor effect of CAR-T cells. RNA-sequencing and online database analysis were used to identify new targets in residual gastric cancer cells after cytotoxicity assay. CAR-T cell functions were detected in vitro and in vivo after GLI Pathogenesis Related 1 (GLIPR1) knockdown in gastric cancer cells. Cell proliferation and migration of gastric cancer cells were detected by CCK-8 and scratch assay respectively after GLIPR1 were overexpressed or down-regulated. RESULTS: CAR-T cells constructed with IL-15/IL-15Rα (CAR-ss-T) showed significantly improved CAR-T cell expansion, cytokine production and cytotoxicity, and resulted in superior tumor control compared to conventional CAR-T cells in gastric cancer. GLIPR1 was up-regulated after CAR-T treatment and survival was decreased in gastric cancer patients with high GLIPR1 expression. Overexpression of GLIPR1 inhibited cytotoxicity of conventional CAR-T but not CAR-ss-T cells. CAR-T treatment combined with GLIPR1 knockdown increased anti-tumor efficacy in vitro and in vivo. CONCLUSIONS: Our data demonstrated for the first time that this CAR structure design combined with GLIPR1 knockdown in gastric cancer improved CAR-T cell-mediated anti-tumor response.

Using Self-Reported Sexual Positioning as an Indicator for Rectal Chlamydia and Gonorrhea Screening Among Men Who Have Sex With Men in China: A Missed Opportunity.

BACKGROUND: Rectal Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections among men who have sex with men (MSM) are escalating public health concerns. This study aimed to explore (1) the reliability of self-reported sexual positioning as an indicator for rectal CT and NG screening, and (2) factors associated with rectal CT and NG infections in Shenzhen, China. METHODS: A cross-sectional study was conducted in 2 settings in Shenzhen, China, from April 1, 2021, to March 31, 2022. Data on sociodemographic characteristics, sexual behaviors, and basic CT knowledge were collected. Urine and self-collected rectal swabs were collected for CT and NG testing. RESULTS: In total, 195 MSM participated in the study, and 5.1% tested positive for urogenital CT, 29.2% for rectal CT, 1.0% for urogenital NG, and 8.2% for rectal NG. Among those who reported exclusively insertive anal sex, 69.2% of CT infections and 85.7% of NG infections would have remained undetected with urine testing alone. Risk factors for rectal CT infection included engaging in both insertive and receptive anal sex, with a significant association found for coinfection with rectal NG. CONCLUSIONS: Self-reported sexual positioning was found to be an unreliable indicator for CT and NG screening, as a substantial proportion of infections would have remained undetected. The findings suggest that CT and NG screening in China should be offered to all MSM regardless of self-reported sexual positioning, and that the dual CT/NG testing is recommended.

Galectin from Trichinella spiralis alleviates DSS-induced colitis in mice by regulating the intestinal microbiota.

According to numerous reports, Trichinella spiralis (T. spiralis) and its antigens can reduce intestinal inflammation by modulating regulatory immunological responses in the host to maintain immune homeostasis. Galectin has been identified as a protein that is produced by T. spiralis, and its characterization revealed this protein has possible immune regulatory activity. However, whether recombinant T. spiralis galectin (rTs-gal) can cure dextran sulfate sodium (DSS)-induced colitis remains unknown. Here, the ability of rTs-gal to ameliorate experimental colitis in mice with inflammatory bowel disease (IBD) as well as the potential underlying mechanism were investigated. The disease activity index (DAI), colon shortening, inflammatory cell infiltration, and histological damage were used as indicators to monitor clinical symptoms of colitis. The results revealed that the administration of rTs-gal ameliorated these symptoms. According to Western blotting and ELISA results, rTs-gal may suppress the excessive inflammatory response-mediated induction of TLR4, MyD88, and NF-κB expression in the colon. Mice with colitis exhibit disruptions in the gut flora, including an increase in gram-negative bacteria, which in turn can result in increased lipopolysaccharide (LPS) production. However, injection of rTs-gal may inhibit changes in the gut microbiota, for example, by reducing the prevalence of Helicobacter and Bacteroides, which produce LPS. The findings of the present study revealed that rTs-gal may inhibit signalling pathways that involve enteric bacteria-derived LPS, TLR4, and NF-κB in mice with DSS-induced colitis and attenuate DSS-induced colitis in animals by modulating the gut microbiota. These findings shed additional light on the immunological processes underlying the beneficial effects of helminth-derived proteins in medicine.

Bacterial cellulose production by a strain of Komagataeibacter rhaeticus isolated from residual loquat.

In this study, loquat extract was selected as a promising substrate for bacterial cellulose (BC) production. A new BC-producing bacterial strain was isolated from residual loquat and identified as Komagataeibacter rhaeticus. BC production with different carbon sources and with loquat extract was investigated. Among all tested carbon sources, glucose was demonstrated to be the best substrate for BC production by K. rhaeticus, with up to 7.89 g/L dry BC obtained under the optimal initial pH (5.5) and temperature (28 °C) with 10 days of fermentation. The total sugar and individual sugars were investigated in different loquat extracts, in which fructose, glucose, and sucrose were the three main sugars. When loquat extract was prepared with a solid‒liquid (S-L) ratio of 2:1, the concentrations of glucose, fructose, and sucrose were 7.91 g/L, 9.31 g/L, and 2.84 g/L, respectively. The BC production obtained from loquat extract was higher than that of other carbon sources except glucose, and 6.69 g/L dry BC was obtained from loquat extract with an S-L ratio of 2:1. After BC production, all sugars substantially decreased, with the utilization of glucose, fructose, and sucrose reaching 93.9%, 87.9%, and 100%, respectively. These results suggested that the different sugars in loquat extract were all carbon sources participating in BC production by K. rhaeticus. Structural and physicochemical properties were investigated by SEM, TGA, XRD, and FT-IR spectroscopy. The results showed that the structural, chemical group, and water holding capacity of BC obtained from loquat extract were similar to those of BC obtained from glucose, but the crystallinity and thermal stability of BC were higher than those of BC from mannose and lactose but lower than those of BC from glucose and fructose. KEY POINTS: • A new BC-producing strain was isolated and identified as Komagataeibacter rhaeticus. • Loquat extract is an alternative substrate for BC production. • The BC obtained from loquat extract owns advanced physicochemical properties.

Chlamydia-related knowledge, opinion to opportunistic testing, and practices of providers among different sexually transmitted infections related departments in hospitals in Shenzhen city, China.

BACKGROUND: Chlamydia trachomatis (CT) infection could lead to seriously adverse outcomes if left untreated. This study aimed to determine CT-related knowledge, opinion to testing, and practices of providers among different sexually transmitted infections (STI) related departments in hospitals in Shenzhen city, China, and also to explore the differences in these responses. MATERIALS AND METHODS: From 1st April 2018 to 15th April 2018, a cross-sectional study was conducted in Shenzhen and 64 of 66 hospitals agreed to participate in this study. In the hospital sites, all the providers from the department of obstetrics and gynecology, department of dermatology and venereology, department of urology, and anorectal surgical department were recruited. A structured paper-based questionnaire was used to obtain data on CT-related information. RESULTS: A total of 355 providers from 64 hospitals participated in the current study. Compared to providers from the department of dermatology and venereology, those from the department of obstetrics and gynecology (OR = 0.31, 95% CI 0.16-0.62), department of urology (OR = 0.32, 95% CI 0.16-0.65), and anorectal surgical department (OR = 0.25, 95% CI 0.09-0.71) were less likely to identify that "Be in a long-term mutually monogamous relationship with a partner who has been tested and has negative STI test results." is an appropriate way for a sexually active person to reduce risk of getting CT. Also, those from the department of obstetrics and gynecology (OR = 0.45, 95% CI 0.23-0.87) were less likely to identify that "Use latex condoms the right way every time you have sex" is another appropriate way. A high proportion of providers agreed that all sexually active patients attending to their department should be screened regularly (77.1%), and they are willing to offer opportunistic CT screening (96.0%). Only 11.4% of respondents correctly identified that the appropriate time frame of the CT retesting is three months. CONCLUSIONS: Providers among STI-related departments in hospitals showed a very high willingness to offer opportunistic CT screening. However, this study showed important gaps in providers' knowledge and practices in China, targeted training in CT-related knowledge and practice is urgently needed.

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system.

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)+) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS.

Geographic and position-based variations in phyllospheric bacterial communities present on flue-cured tobacco.

Although tobacco leaves (TLs) contain abundant bacteria, how the geography and leaf position of TLs affect these bacteria is unclear. Here, TLs at different positions from Henan (HN, strong flavor style) and Yunnan (YN, fresh flavor style) provinces were collected, and the bacteria were characterized by Illumina sequencing at harvest and 1 year of storage. Bacterial communities were very different between TLs originating from different geographical areas and positions, and beta diversity analysis showed that leaf position was the most important factor for phyllospheric bacterial communities, followed by geographical area and storage time. At the genus level, Subdoligranulum, Thermus, and Acinetobacter were obviously more abundant in HN than in YN, while Blautia and Ruminococcus were significantly more abundant in YN. These differences in bacterial communities decreased after 1 year of storage, indicating that the microbiota tends to become similar during tobacco processing. Storage time also affected the phyllospheric bacteria of TLs, as the bacterial communities shifted significantly on both HN and YN TLs after 1 year of storage. Significant differences in the predicted genes were also observed between the different geographic locations and leaf positions. Potential human pathogens, including Acinetobacter, Methylobacterium, and Escherichia-Shigella, were greatly different between TLs originating from different areas and positions. These data suggested that geographic variations and positions were associated with phyllospheric bacterial communities on TLs, which may be related to not only the flavor style and quality of TLs but also the potential health risks to humans. KEY POINTS: • Tobacco leaf position and tobacco growth location affected bacterial communities. • Microbial communities of TLs shifted significantly after one year of storage. • Potential human pathogens differed at different leaf positions and growth locations.

Group B Streptococcus DNA Copy Numbers Measured by Digital PCR Correlates with Perinatal Outcomes.

Group B Streptococcus (GBS) is a one of the main causes of perinatal disease, yet the method for GBS detection, broth-enriched culture, is time-consuming and has low sensitivity and accuracy. We aimed to develop a GBS digital PCR (GBS-dPCR) assay for detecting GBS colonization. More rapid and accurate detection of GBS colonization could increase GBS diagnosis and treatment closer to delivery. A single-center, retrospective, case-controlled study was performed. A total of 182 rectovaginal swabs from pregnant women, who were undergoing prenatal screening by broth-enriched culture, were evaluated using GBS-dPCR targeting the cfb gene of GBS. Pregnant women with GBS colonization were followed up for correlation analysis between GBS DNA copy numbers and perinatal outcomes. The results of the GBS-dPCR assay were compared to those from the broth-enriched culture, which is the gold standard for GBS detection. The sensitivity and specificity of GBS-dPCR were 98% and 92.5%, respectively. By discrepant result analysis, the specificity of GBS-dPCR was raised to 97.4%. The incidence of premature rupture of membrane (PROM) and neonatal infection were statistically significantly positively correlated with GBS DNA copy numbers. GBS-dPCR has the advantage of directly detecting GBS colonization from swabs with high specificity and sensitivity, while reducing turnaround time (<4 h). Analysis of clinical samples with GBS-dPCR shows that GBS DNA copy numbers are positively correlated with the incidence of PROM and neonatal infection, suggesting that dPCR is a promising method for detection of GBS colonization during pregnancy.

Higher notch expression implies poor survival in pancreatic ductal adenocarcinoma: A systematic review and meta-analysis.

BACKGROUND: At present, pancreatic ductal adenocarcinoma (PDAC) is a fetal disease lack of effective prognostic and therapeutic methods resulting in high mortality. The Notch signaling has been demonstrated being up- or down-regulated in many cancers, but the effects in pancreatic ductal adenocarcinoma are still controversial. Moreover, the available cases in an individual study are of small samples. Therefore, it is essential to define the effect of Notch signaling in pancreatic ductal adenocarcinoma with larger samples. METHODS: Conducted from 6 eligible studies and 463 pancreatic ductal adenocarcinoma patients, this was the first meta-analysis to analyze the correlation between the Notch signal pathway and pancreatic ductal adenocarcinoma. All data were sourced from The National Center for Biotechnology Information, Web of Science and Cochrane. The articles which matched the inclusion criteria were included. All included data were analyzed and performed by Review Manager 5.3. RESULTS: The results indicated that high expression of Notch signaling proteins was associated with poor overall survival of pancreatic ductal adenocarcinoma patients (pooled hazard ratio>2.00; P < 0.001). Moreover, poor survival was related to high expression of Notch3 (pooled hazard ratio: 2.05; confidence interval: 1.49-2.82; P < 0.001) and DLL4 (pooled hazard ratio: 2.13; confidence interval: 1.37-3.32; P < 0.001). CONCLUSIONS: This meta-analysis supports that Notch signaling proteins may be available as prognostic factors for pancreatic ductal adenocarcinoma progression and patient survival. Higher expression of Notch signaling proteins indicated poor survival of pancreatic ductal adenocarcinoma patients. Targeting Notch signaling components, especially Notch3 protein, would be beneficial for therapies.

The effects of threshing and redrying on bacterial communities that inhabit the surface of tobacco leaves.

Before being subjected to the aging process, raw tobacco leaves (TLs) must be threshed and redried. We propose that threshing and redrying affect the bacterial communities that inhabit the TL surface, thereby influencing the aging process. However, these effects remain unclear. In this study, Illumina sequencing was applied to analyze the bacterial communities on both raw and redried TLs. Shannon's diversity value decreased from 3.38 to 2.52 after the threshing and redrying processes, indicating a large reduction in TL bacterial diversity. The bacterial communities also largely differed between raw TLs and redried TLs. On unaged raw TLs, Proteobacteria was the most dominant phylum (56.15%), followed by Firmicutes (38.99%). In contrast, on unaged redried TLs, Firmicutes (76.49%) was the most dominant phylum, followed by Proteobacteria (21.30%). Thus, the dominant genus Proteobacteria, which includes Sphingomonas, Stenotrophomonas, and Pantoea, decreased after the threshing and redrying processes, while the dominant genus Firmicutes, which includes Bacillus and Lactococcus, increased. Changes in the bacterial communities between raw and redried TLs were also noted after 1 year of aging. The relative abundance of dominant Proteobacteria taxa on raw TLs decreased from 56.15 to 16.92%, while the relative abundance of Firmicutes taxa increased from 38.99 to 79.10%. However, small changes were observed on redried TLs after 1 year of aging, with a slight decrease in Proteobacteria (21.30 to 17.64%) and a small increase in Firmicutes (76.49 to 79.10%). Based on these results, Firmicutes taxa may have a higher tolerance for extreme environments (such as high temperature or low moisture) than Proteobacteria bacteria. This study is the first report to examine the effects of threshing and redrying on bacterial communities that inhabit TLs.

Degradation of phytosterols in tobacco waste extract by a novel Paenibacillus sp.

Phytosterols have been demonstrated to be precursors of polycyclic aromatic hydrocarbons (PAHs) formed during biomass pyrolysis. Here, a novel Paenibacillus sp. was evaluated for its ability to degrade phytosterols in tobacco waste extract (TWE). The optimal conditions for cell growth and stigmasterol (a representative of phytosterols) degradation were 37 °C, pH 7.0, 1.0 g/L yeast extract, and 6.0 g/L glucose. Paenibacillus sp. could degrade stigmasterol under high concentrations of glucose (up to 130 g/L) and tolerate wide pH (5.0-9.0) and temperature (25-42 °C) ranges. The new strain could degrade stigmasterol completely into CO2 and H2 O, and no intermediate steroids were detected during the degradation process. Phytosterol degradation in TWE was demonstrated by high-performance liquid chromatography-tandem mass spectrometry. Under optimal conditions (37 °C, pH 7.0, with the exponential-phase cells), the total degradation ratio of phytosterols reached 38.5% in TWE, including 45.2% of stigmasterol, 37.4% of ß-sitosterol, 27.3% of campesterol, and 28.7% of cholesterol. These results showed that Paenibacillus sp. is a candidate for phytosterol degradation in TWE and other biomass and is potentially useful in reducing the PAHs generated from biomass pyrolysis.

Bioconversion of lutein by Enterobacter hormaechei to form a new compound, 8-methyl-α-ionone.

OBJECTIVES: To investigate the final product of the bioconversion of lutein by a novel lutein-degrading bacterium, Enterobacter hormaechei A20, and the kinetics of the process. RESULTS: A new product, 8-methyl-α-ionone, was resolved by GC-MS. The compound was further identified by NMR. A conversion yield of 90% was achieved by E. hormaechei in 36 h with 10 g lutein l-1. CONCLUSIONS: This is the first report of the bioconversion of lutein to form 8-methyl-α-ionone. A degradation pathway is proposed.

Zoonotic Enterocytozoon bieneusi in raw wastewater in Zhengzhou, China.

Contamination of Enterocytozoon bieneusi Desportes, Charpentier, Galian, Bernard, Cochand-Priollet, Laverne, Ravisse, et Modigliani, 1985 in water sources may cause outbreaks of microsporidiosis. To examine the occurrence of E. bieneusi, 108 raw wastewater samples were collected from three wastewater treated plants in Zhengzhou, China. In total, 46 samples were PCR positive for E. bieneusi. A total of 15 ITS genotypes was identified, including ten known genotypes (D, BEB6, I, J, PigEbIX, PigEBITS5, EbpA, Peru6, Peru8, Type IV) and five novel genotypes (HNWW1, HNWW2, HNWW3, HNWW4, HNWW5). Nine genotypes belonged to a known zoonotic group (group 1) and the other genotypes belonged to potential zoonotic group (group 2). Most of the genotypes had been identified in wildlife or domestic animals in former reports in Zhengzhou. The occurrence of E. bieneusi in wastewater was probably related to the rainfall day before sampling. Of 36 sampling days, 20 days had rainfall on the previous day and 16 days had none. As many as 43 of 60 samples were found to be E. bieneusi-positive in the 20 days which had rainfall on the previous day. Only three of 48 samples were found to be E. bieneusi-positive in the 16 days without rainfall the day before. The significant difference of the occurrence of E. bieneusi was observed between wet days and dry days by t-test (43/60 vs 3/48, p < 0.01). This indicates that the occurrence of E. bieneusi in wastewater in Zhengzhou mainly originated from animals and was probably related to rainfall the day before sample collection. Given the zoonotic genotypes detected in wastewater, animal faeces should be treated appropriately before being drained into the water source.

Antibody Production and Th1-biased Response Induced by an Epitope Vaccine Composed of Cholera Toxin B Unit and Helicobacter pylori Lpp20 Epitopes.

BACKGROUND: The epitope vaccine is an attractive potential for prophylactic and therapeutic vaccination against Helicobacter pylori (H. pylori) infection. Lpp20 is one of major protective antigens which trigger immune response after H. pylori invades host and has been considered as an excellent vaccine candidate for the control of H. pylori infection. In our previous study, one B-cell epitope and two CD4(+) T-cell epitopes of Lpp20 were identified. OBJECTIVE: In this study, an epitope vaccine composed of mucosal adjuvant cholera toxin B subunit (CTB) and these three identified Lpp20 epitopes were constructed to investigate the efficacy of this epitope vaccine in mice. METHODS: The epitope vaccine including CTB, one B-cell, and two CD4(+) T-cell epitopes of Lpp20 was constructed and named CTB-Lpp20, which was then expressed in Escherichia coli and used for intraperitoneal immunization in BALB/c mice. The immunogenicity, specificity, and ability to induce antibodies against Lpp20 and cytokine secretion were evaluated. After that, CTB-Lpp20 was intragastrically immunized to investigate the prophylactic and therapeutic efficacy in infected mice. RESULTS: The results indicated that the epitope vaccine CTB-Lpp20 possessed good immunogenicity and immunoreactivity and could elicit specific high level of antibodies against Lpp20 and the cytokine of IFN-γ and IL-17. Additionally, CTB-Lpp20 significantly decreased H. pylori colonization in H. pylori challenging mice, and the protection was correlated with IgG, IgA, and sIgA antibody and Th1-type cytokines. CONCLUSION: This study will be better for understanding the protective immunity of epitope vaccine, and CTB-Lpp20 may be an alternative strategy for combating H. pylori invasion.

Cryptosporidium species and Cryptosporidium parvum subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey.

Molecular characterizations of Cryptosporidium spp. in ruminants reared under traditional animal management systems are scarce and studies conducted thus far have revealed largely an absence of the pathogenic and zoonotic species Cryptosporidium parvum in pre-weaned animals. In this study, we examined Cryptosporidium species and subtype distribution in free-range pre-weaned dairy calves and goat kids with diarrhea. Cryptosporidium-positive specimens from pre-weaned calves on 10 farms and goat kids on 4 farms in Ankara, Balikesir, Corum, Kirikkale, and Kirsehir Provinces, Turkey were genotyped by PCR-restriction length polymorphism analysis of the small subunit rRNA gene, which identified C. parvum in 27 calves and 9 goat kids and Cryptosporidium ryanae in 1 calf. Among the C. parvum isolates successfully subtyped by DNA sequence analysis of the 60 kDa glycoprotein gene, three subtypes were detected in calves, including IIaA13G2R1 (20/23), IIdA18G1 (2/23), and IIdA20G1b (1/23), and four subtypes were detected in goat kids, including IIaA13G2R1 (3/8), IIaA15G1R1 (2/8), IIdA22G1 (2/8), and IIdA18G1 (1/8). Data of the study suggest that dairy calves reared in a traditional cow-calf system in Turkey are mainly infected with a C. parvum subtype rarely seen elsewhere, whereas goat kids are infected with diverse subtypes. As all five C. parvum subtypes found in this study are known human pathogens, pre-weaned farm animals could play a potential role in the transmission of human cryptosporidiosis.

Identification and morphologic and molecular characterization of Cyclospora macacae n. sp. from rhesus monkeys in China.

Cyclospora spp. in nonhuman primates are most closely related to Cyclospora cayetanensis, an emerging human pathogen causing outbreaks of cyclosporiasis in North America. Studies thus far indicate the possible existence of host specificity in Cyclospora spp. In this study, 411 fecal specimens from free-range rhesus monkeys (Macaca mulatta) were collected and examined for Cyclospora by sequence analysis of the small subunit rRNA gene. A novel Cyclospora species was identified in 28 (6.8%) specimens and named Cyclospora macacae based on morphologic and molecular characterizations. The oocyst of C. macacae is spherical and measures 8.49 ± 0.55 × 8.49 ± 0.49 µm in diameter. Phylogenetic analysis grouped this species together with the other four Cyclospora species infecting primates, including C. cayetanensis in humans, forming a monophyletic group closely related to avian Eimeria species. In addition, C. cayetanensis was detected in one specimen, although whether rhesus monkeys can serve as a natural reservoir host of C. cayetanensis needs further investigation.

Prevalence and characterization of Cryptosporidium spp. in dairy cattle in Nile River delta provinces, Egypt.

Molecular characterizations of Cryptosporidium spp. in dairy cattle in industrialized nations have mostly shown a dominance of Cryptosporidium parvum, especially its IIa subtypes in pre-weaned calves. Few studies, however, have been conducted on the distribution of Cryptosporidium species and C. parvum subtypes in various age groups of dairy cattle in developing countries. In this study, we examined the prevalence and molecular characteristics of Cryptosporidium in dairy cattle in four Nile River delta provinces in Egypt. Modified Ziehl-Neelsen acid-fast microscopy was used to screen for Cryptosporidium oocysts in 1974 fecal specimens from animals of different ages on 12 farms. Positive fecal specimens were identified from all studied farms with an overall prevalence of 13.6%. By age group, the infection rates were 12.5% in pre-weaned calves, 10.4% in post-weaned calves, 22.1% in heifers, and 10.7% in adults. PCR-RFLP and DNA sequence analyses of microscopy-positive fecal specimens revealed the presence of four major Cryptosporidium species. In pre-weaned calves, C. parvum was most common (30/69 or 43.5%), but Cryptosporidium ryanae (13/69 or 18.8%), Cryptosporidium bovis (7/69 or 10.2%), and Cryptosporidium andersoni (7/69 or 10.2%) were also present at much higher frequencies seen in most industrialized nations. Mixed infections were seen in 12/69 (17.4%) of genotyped specimens. In contrast, C. andersoni was the dominant species (193/195 or 99.0%) in post-weaned calves and older animals. Subtyping of C. parvum based on sequence analysis of the 60kDa glycoprotein gene showed the presence of subtypes IIdA20G1 in nine specimens, IIaA15G1R1 in 27 specimens, and a rare subtype IIaA14G1R1r1b in one specimen. The common occurrence of non-C. parvum species and IId subtypes in pre-weaned calves is a distinct feature of cryptosporidiosis transmission in dairy cattle in Egypt. The finding of the same two dominant IIa and IId C. parvum subtypes recently found in humans in Egypt suggests calves can be potential reservoirs of zoonotic cryptosporidiosis.

Alteration of the oral and gut microbiota in patients with Kawasaki disease.

Background: Kawasaki disease (KD) is a multi-systemic vasculitis that primarily affects children and has an unknown cause. Although an increasing number of studies linking the gut microbiota with KD, the unchallengeable etiology of KD is not available. Methods: Here, we obtained fecal and oral samples from KD patients and healthy controls, and then we use high-throughput sequencing to examine the diversity and composition of microbiota. Results: Results showed that both in the gut and oral microbiota, the diversity of KD patients was significantly lower than that of the healthy controls. In the gut microbiota, a higher abundance of Enterococcus (40.12% vs less than 0.1%), Bifidobacterium (20.71% vs 3.06%), Escherichia-Shigella (17.56% vs 0.61%), Streptococcus (5.97% vs 0.11%) and Blautia (4.69% vs 0.1%) was observed in the KD patients, and enrichment of Enterococcus in the patients was observed. In terms of oral microbiota, the prevalence of Streptococcus (21.99% vs 0.1%), Rothia (3.02% vs 0.1%), and Escherichia-Shigella (0.68% vs 0.0%) were significantly higher in the KD patients, with the enrichment of Streptococcus and Escherichia-Shigella. Additionally, significant differences in microbial community function between KD patients and healthy controls in the fecal samples were also observed, which will affect the colonization and reproduction of gut microbiota. Conclusions: These results suggested that the dysbiosis of gut and oral microbiota are both related to KD pathogenesis, of which, the prevalence of Enterococcus in the gut and higher abundance of Streptococcus and Escherichia-Shigella in the oral cavity will be a potential biomarker of the KD. Overall, this study not only confirms that the disturbance of gut microbiota is a causative trigger of KD but also provides new insight into the oral microbiota involved in KD pathogenesis.

Anthroponotic enteric parasites in monkeys in public park, China.

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi were detected in 45, 35, and 116 of 411 free-range rhesus monkeys, respectively, in a popular public park in the People's Republic of China. Most genotypes and subtypes detected were anthroponotic, indicating these animals might be reservoirs for human cryptosporidiosis, giardiasis, and microsporidiosis.

Elevated DLL3 in stomach cancer by tumor-associated macrophages enhances cancer-cell proliferation and cytokine secretion of macrophages.

Background: The notch signal pathway is important in the development of both tumor-associated macrophages (TAMs) and stomach cancer, but how Notch signaling affects TAMs in stomach cancer is barely understood. Methods: The expressions of Notch1, Notch2, Notch3, Notch4, hes family bHLH transcription factor 1 (Hes1), and delta-like canonical Notch ligand 3 (DLL3) were detected by Western blot and the expressions of interleukin (IL)-10, IL-12, and IL1-ß were detected using enzyme-linked immunosorbent assay after the co-culture of macrophages and stomach-cancer cells. The proliferation and migration of cancer cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scratch assay, respectively, and the cell cycle was detected using Annexin V/propidium iodide assay. The protein interactions with DLL3 were detected using co-immunoprecipitation and mass spectrometry. Results: The co-culture of macrophages and stomach-cancer cells MKN45 and BGC823 could enhance cell proliferation accompanied by the activation of Notch1/Notch2 signaling and upregulation of DLL3. Notch signaling gamma-secretase inhibitor (DAPT) blocked this process. The overexpression of DLL3 in stomach-cancer cells could promote the proliferation of cancer cells, enhance the activation of Notch1/Notch2 signaling, induce the expression of IL-33, lead to the degradation of galectin-3-binding protein (LG3BP) and heat shock cognate 71 kDa protein (HSPA8), and result in elevated IL-1ß, IL-12, and IL-10 secretion by macrophages. Higher expression of DLL3 or IL-33 could lead to a lower survival rate based on University of California, Santa Cruz Xena Functional Genomics Explorer and The Cancer Genome Atlas data set. Conclusions: This is evidence that DLL3 regulates macrophages in stomach cancer, suggesting that DLL3 may be a novel and potential target for stomach-cancer therapy.