Immunization with Live-Attenuated RHΔhad2a Strain Confers Partial Protective Immunity against Acute and Chronic Infection of Toxoplasma gondii in Mice.

Toxoplasmosis caused by Toxoplasma gondii is an important zoonosis of human and animal health significance. Current chemical therapeutics have side effects, and no commercially available vaccine is licensed for the prevention of toxoplasmosis in humans and most animals. Developing a safe and effective vaccine with long-term protection against T. gondii infection is necessary to control toxoplasmosis. HAD2a is a key member of the haloacid dehalogenase (HAD) phosphatase family, which is essential for T. gondii daughter budding. However, the role of HAD2a in T. gondii virulence remains unknown. In this study, we successfully constructed the had2a gene knockout strain in the T. gondii-type I RH strain (RHΔhad2a) and determined its role in virulence and vaccination. These results demonstrate that HAD2a played an important role in parasite daughter budding and in vitro replication. Knockout of the had2a gene attenuated the virulence of the T. gondii-type I RH strain. Vaccination with RHΔhad2a tachyzoites induced a Th1-biased immune response, provided partial protection against acute T. gondii infection in mice by highly virulent tachyzoites of RH and PYS (ToxoDB#9, Chinese I) strains, and conferred strong protection against challenge infection by cysts and oocysts of the less virulent type II Pru strain. These results demonstrate that T. gondii had2a is important for its in vitro proliferation and virulence in mice and that RHΔhad2a may be used as a candidate strain to generate a multiple gene knockout live-attenuated strain or be collaboratively applied with other live-attenuated strains to confer more effective protection against T. gondii infection.

Effect of deletion of gra17 and gra23 genes on the growth, virulence, and immunogenicity of type II Toxoplasma gondii.

The protozoan parasite Toxoplasma gondii secretes a number of dense granule proteins (GRAs) from the dense granule organelle to manipulate the host cell. Two of these effector proteins (GRA17 and GRA23) are involved in the trafficking of molecules between the parasitophorous vacuole (PV) and the host cell cytoplasm. However, their roles in establishing chronic infection remain obscured. In this study, CRISPR-Cas9 was used to delete gra17 or gra23 gene in T. gondii Pru strain (type II). The growth, the virulence, the ability to establish chronic infection, and the immunogenicity of the constructed mutant strains were investigated in Kunming mice. Pru:Δgra17 and Pru:Δgra23 mutants developed PVs with abnormal morphology and exhibited reduced growth rate, compared with the wild-type Pru strain. Deletion of gra17 abrogated acute infection and blocked cyst formation. Although the deletion of gra23 caused slight attenuation of the parasite virulence in mice, it caused a significant reduction in cyst formation. Immunization with Pru:Δgra17 induced high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (interleukin-2 [IL-2], IL-10, IL-12, and interferon gamma [IFN-γ]), which conferred significant protection in mice challenged with virulent type I (RH), ToxoDB#9 (PYS) strains, or less virulent type II (Pru) strain of T. gondii. These findings show that GRA17 and GRA23 play important roles in T. gondii chronic infection and that irreversible deletion of gra17 in T. gondii type II Pru strain can be a viable option for stimulating protective immunity to T. gondii infection.

Prevalence and Genotype Distribution of Giardia duodenalis in Rabbits in Shandong Province, Eastern China.

Giardia duodenalis is a zoonotic enteric parasite that can infect humans and a number of animal species including rabbits with a worldwide distribution. Infection with G. duodenalis can cause serious public health problems and significant economic losses to animal husbandry. So accurate understanding of the prevalence and genotype distribution of G. duodenalis in rabbits is necessary. In the present study, a total of 616 fecal samples were collected from rabbits in Shandong province, eastern China, and examined in G. duodenalis prevalence and genotypes by nested PCR amplification of ß-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) gene loci of G. duodenalis. Sixty-nine (11.2%) of the examined rabbit fecal samples were G. duodenalis-positive. Of them, the prevalence of G. duodenalis is 8.4% (41/490) in Rizhao city and 22.2% (28/126) in Weihai city. Breeds, region, and feeding modes were highly correlated with G. duodenalis infection in rabbits. Moreover, three genotypes (assemblages A, B, and E) were identified in rabbits at three gene loci, and the assemblage E was the dominant genotype, while the assemblage A was reported in rabbits in China for the first time. It is noticeable that two rabbits were found to be infected with two different G. duodenalis assemblages (assemblages A and E, assemblages B and E, respectively). These findings enrich the genotype distribution of G. duodenalis in rabbits and provide baseline data for preventing and controlling G. duodenalis infection in rabbits in eastern China.

Functional Characterization of Two Thioredoxin Proteins of Toxoplasma gondii Using the CRISPR-Cas9 System.

Toxoplasmosis caused by infection with Toxoplasma gondii is an important parasitic zoonosis with a worldwide distribution. In this study, we examined the functions of two thioredoxins (namely CTrp26 and CTrx1) of T. gondii tachyzoites by generation of HA tag strains or gene deficient parasites in Type I RH strain (ToxoDB#10). Immunofluorescence analysis (IFA) was used to investigate the subcellular localization of the thioredoxins (Trxs). Results of IFA showed that both CTrp26 and CTrx1 were located in the cytoplasm of T. gondii. Functional characterizations of CTrp26 and CTrx1-deficient parasites were performed by plaque assay, intracellular replication, egress, H2O2 resistance, detection of reactive oxygen species (ROS) level and total antioxidant capacity (T-AOC) assays in vitro, as well as mouse infection in vivo. Our results showed that deletion of CTrp26 or CTrx1 did not influence the ability of T. gondii RH strain to replicate, egress, form plaque, resist H2O2 exposure, maintain the ROS level, and T-AOC, and also did not serve as virulence factors in Kunming mice. Taken together, these results provide new properties of the two Trxs. Although they are not essential for RH strain, they may have roles in other strains of this parasite due to their different expression patterns, which warrants future research.

Composition, Distribution, and Antioxidant Activity of Phenolic Compounds in 18 Soybean Cultivars.

Natural phenols are an important functional compound widely distributed in plants with benefits that promote human health. The content of total phenols, flavonoids, and anthocyanins and their composition distribution in 18 soybean cultivars was investigated. There are four phenolic acid distribution forms in these soybean cultivars, namely free, esterified, glycosided, and insoluble-bound. Total phenols, flavonoids, and anthocyanins from 6 black soybean cultivars were found in higher numbers than those from 12 other yellow soybean cultivars. Free and esterified phenolic acids were the main phenolic acid form in all 18 soybean samples. Chlorogenic acid and caffeic acid were the dominant phenolic acids in eight detected phenolic acids, and daidzin and genistin were the abundant isoflavones in five detected isoflavones. Furthermore, the antioxidant activities of total phenols from the 6 black soybean cultivars were greater than those from the 12 yellow soybean cultivars, and there was a significant positive correlation between antioxidant activity and total phenolic content. Black soybeans could be a potential resource for developing natural antioxidants that may play a crucial role in human health protection.

Enhancing the enzymatic saccharification of bamboo shoot shell by sequential biological pretreatment with Galactomyces sp. CCZU11-1 and deep eutectic solvent extraction.

In this study, sequential biological pretreatment (BP) with Galactomyces sp. CCZU11-1 at 30 °C for 3 days and deep eutectic solvent (DES) choline chloride: oxalic acid (ChCl:OA, 1 mol/2 mol) extraction at 120 °C for 1.5 h was used for pretreating BSS. It was found that combination pretreatment could effectively remove xylan and lignin for enhancing enzymatic saccharification. The reducing sugars and glucose from the hydrolysis of 100 g/L pretreated BSS with complexed cellulases of Galactomyces sp. CCZU11-1 were obtained in the yields of 81.0% and 74.1%, respectively. The BSS-hydrolyzates had no inhibitory effects on the lipid-accumulating microorganism Bacillus sp. CCZU11-1, and the cell mass and TAG accumulation were 4.8 g CDW/L and 2.2 g TAG/L, respectively. Fatty acids including palmitic acid (C16:0; 25.3%), palmitoleic acid (C16:1; 24.4%), stearic acid (C18:0; 15.1%), and oleic acid (C18:1; 21.6%) were accumulated in cells. Clearly, this combination pretreatment has high potential application in future.

Effective Biotransformation of Ethyl 4-Chloro-3-Oxobutanoate into Ethyl (S)-4-Chloro-3-Hydroxybutanoate by Recombinant E. coli CCZU-T15 Whole Cells in [ChCl][Gly]-Water Media.

To increase the biocatalytic activity of Escherichia coli CCZU-T15 whole cells, choline chloride/glycerol ([ChCl][Gly]) was firstly used as biocompatible solvent for the effective biotransformation of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE]. Furthermore, L-glutamine (150 mM) was added into [ChCl][Gly]-water ([ChCl][Gly] 12.5 vol%, pH 6.5) media instead of NAD+ for increasing the biocatalytic efficiency. To further improve the biosynthesis of (S)-CHBE (>99 % e.e.) by E. coli CCZU-T15 whole cells, Tween-80 (7.5 mM) was also added into this reaction media, and (S)-CHBE (>9 % e.e.) could be effectively synthesized from 2000 and 3000 mM COBE in the yields of 100 and 93.0 % by whole cells of recombinant E. coli CCZU-T15, respectively. TEM image indicated that the cell membrane was permeabilized and lost its integrity and when the cell was exposed to [ChCl][Gly]-water media with Tween-80. Clearly, this bioprocess has high potential for the effective biosynthesis of (S)-CHBE (>99 % e.e.).

Mitofusin-2 prevents skeletal muscle wasting in cancer cachexia.

Cancer cachexia remains a leading cause of morbidity and mortality worldwide, despite extensive research and clinical trials. The prominent clinical feature of cancer cachexia is the continuous loss of skeletal muscle that cannot be fully reversed by conventional nutritional support, and that leads to progressive functional impairment. The mechanism underlying muscle loss in patients with cachexia is poorly understood. The present study analyzed 21 cancer patients with or without cachexia, and demonstrated that mitofusin-2 (Mfn2) was downregulated in the rectus abdominis of patients with cachexia, which was associated with body weight loss. In vitro cell experiments indicated that loss of Mfn2 was associated with atrophy of the C2C12 mouse myoblast cell line. Furthermore, in vivo animal experiments demonstrated that cachexia decreased gastrocnemius muscle mass and Mfn2 expression, and overexpression of Mfn2 in gastrocnemius muscle was able to partially attenuate cachexia-induced gastrocnemius muscle loss. The results of the present study suggested that Mfn2 is involved in cachexia-induced muscle loss and may serve as a potential target for therapy of cachexia.

miR-346 promotes migration and invasion of nasopharyngeal carcinoma cells via targeting BRMS1.

The aim of this study is to determine the expression and roles of miR-346 in nasopharyngeal carcinoma (NPC). We showed that miR-346 was upregulated in NPC tissues compared with adjacent non-tumorous nasopharyngeal tissues. Inhibition of miR-346 significantly attenuated the migration and invasion of NPC cells. Luciferase reporter assay showed that miR-346 targeted the 3'-untranslated region (3'-UTR) of breast cancer metastasis suppressor 1 (BRMS1). Overexpression of miR-346 suppressed the endogenous expression of BRMS1 in NPC cells. There was a significant negative correlation between miR-346 and BRMS1 protein expression in NPC tissues (r = -0.372, P = 0.008). Rescue experiments demonstrated that overexpression of BRMS1 lacking the 3'-UTR impaired the invasiveness of NPC cells transfected with miR-346 mimic. Taken together, miR-346 shows the ability to promote the migration and invasion of nasopharyngeal cancer cells via targeting BRMS1 and represents a potential therapeutic target for NPC.

PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function.

Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.

The Endoplasmic Reticulum Stress Sensor IRE1α in Intestinal Epithelial Cells Is Essential for Protecting against Colitis.

Intestinal epithelial cells (IECs) have critical roles in maintaining homeostasis of intestinal epithelium. Endoplasmic reticulum (ER) stress is implicated in intestinal epithelium homeostasis and inflammatory bowel disease; however, it remains elusive whether IRE1α, a major sensor of ER stress, is directly involved in these processes. We demonstrate here that genetic ablation of Ire1α in IECs leads to spontaneous colitis in mice. Deletion of IRE1α in IECs results in loss of goblet cells and failure of intestinal epithelial barrier function. IRE1α deficiency induces cell apoptosis through induction of CHOP, the pro-apoptotic protein, and sensitizes cells to lipopolysaccharide, an endotoxin from bacteria. IRE1α deficiency confers upon mice higher susceptibility to chemical-induced colitis. These results suggest that IRE1α functions to maintain the intestinal epithelial homeostasis and plays an important role in defending against inflammation bowel diseases.

[Impact in response control, attention and hyperactivity behavior on children with obstructive sleep apnea hypopnea syndrome by integrated visual and auditory continuous performance test].

OBJECTIVE: To study the impact in response control, attention and hyperactivity behavior on children with obstructive sleep apnea hypopnea syndrome (OSAHS) by the integrated visual and auditory continuous performance test (IVA-CPT). METHODS: Fifty-one children aged between 5 and 12 years were diagnosed as OSAHS by polysomnography (PSG), and received adenotonsillectomy and adenoidectomy or only adenoidectomy. Then received IVA-CPT and PSG before surgery, 3 months after surgery and 6 months after surgery (named as first, second and third time point). These children were divided into two groups according to the disease course (group A: course of disease < 5 years; group B: course of disease ≥ 5 years). The SPSS 19.0 was used for statistical analysis. RESULTS: By balanced test, there were no differences in gender, body mass index (BMI) and disease severity among the two groups before surgery (P > 0.05). The numbers of children with abnormal psychological behavior at three time points were 32 (62.7%), 25 (49.0%) and 8 (15.7%). The abnormal rate did not show statistical difference between the first and second time point (χ(2) = 1.49, P = 0.163), but did show statistical difference between the second and third time point (χ(2) = 12.95, P < 0.001). Repetitive measurement and analysis of variance showed that there were statistical differences in means of FRCQ, FAQ and HYP between three time points in two groups (F were 342.15, 263.12, 380.57, P < 0.001), and all the means improved with time. It also showed that there were statistical differences in means of FRCQ, FAQ and HYP between two groups at every time point (F were 167.05, 126.47, 117.683, P < 0.001). FRCQ and HYP all showed interation effect between two groups (P < 0.001). Means of apnea hypopnea index (AHI) and lowest arterial (LaSO2) were compared between three time points in two groups and all showed statistical differences (F were 99.057, 70.742, P < 0.001). Means of AHI and LaSO2 were compared between two groups at every time point. AHI and LaSO2 did not show statistical difference (P > 0.05). AHI and LaSO2 did not exist interation effect of disease course and time. CONCLUSIONS: OSAHS obviously affect the children's response control, attention and hyperactivity behavior, but can recover gradually after adenotonsillectomy and adenoidectomy or only adenoidectomy. Therefore, Children with OSAHS should receive treatment as early as possible so as to reduce the influence on psychology.

PAX2 protein induces expression of cyclin D1 through activating AP-1 protein and promotes proliferation of colon cancer cells.

Paired box (PAX) 2, a transcription factor, plays a critical role in embryogenesis. When aberrantly expressed in adult tissues, it generally exhibits oncogenic properties. However, the underlying mechanisms remain unclear. We reported previously that the expression of PAX2 was up-regulated in human colon cancers. However, the role of PAX2 in colon cancer cells has yet to be determined. The aim of this study is to determine the function of PAX2 in colon cancer cells and to investigate the possible mechanisms underlain. We find that knockdown of PAX2 inhibits proliferation and xenograft growth of colon cancer cells. Inhibition of PAX2 results in a decreased expression of cyclin D1. Expression of cyclin D1 is found increased in human primary colon malignant tumors, and its expression is associated with that of PAX2. These data indicate that PAX2 is a positive regulator of expression of cyclin D1. We find that knockdown of PAX2 inhibits the activity of AP-1, a transcription factor that induces cyclin D1 expression, implying that PAX2 induces cyclin D1 through AP-1. PAX2 has little effect on expression of AP-1 members including c-Jun, c-Fos, and JunB. Our data show that PAX2 prevents JunB from binding c-Jun and enhances phosphorylation of c-Jun, which may elevate the activity of AP-1. Taken together, these results suggest that PAX2 promotes proliferation of colon cancer cells through AP-1.

Prolyl hydroxylase domain protein 3 targets Pax2 for destruction.

Prolyl hydroxylase domain proteins (PHDs) hydroxylate HIFα in the presence of oxygen, leading to HIFα proteasomal destruction. The PHDs family comprises PHD1, 2, and 3. Recent studies indicate that, in addition to HIFα, PHDs have other substrates. Paired box (Pax) 2, a transcription factor, was found aberrantly expressed in a variety of cancers. However, the underlying mechanisms remain unknown. Here we demonstrate that PHD3 is a negative regulator of expression of Pax2. We found that PHD3 bound to Pax2 and mediated Pax2 destruction directly. Inhibition of PHD3 hydroxylase activity led to upregulation of Pax2 protein but not mRNA level. We found that Pax2 protein was increased and PHD3 protein was decreased in colorectal cancer, and the increased Pax2 was associated with decreased PHD3. Our results suggest that PHD3 targets Pax2 for destruction. The findings may disclose a mechanism for the regulation of Pax2 expression in cancer cells.