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BACKGROUND: Parasitoid venom is composed of a complex mixture of various active substances with different biological functions and is injected in the host during the parasitoid oviposition. Anastatus japonicus (Hymenoptera: Eupelmidae) is an egg parasite of Tessaratoma papillosa (Hemiptera: Tessaratomidae). Although the venom of this egg parasitoid plays an important role in the parasitic process, relatively little work has been done to address the mechanism. RESULTS: In the present study, proteomic analysis was performed to identify the proteins that play an important role in the parasitic process of A. japonicus. A total of 2084 proteins were identified, including 81 putative venom proteins, most of which were identified as Hexamerin, Chitinase 2, Calreticulin, Heat shock protein 83‐like, Serine protease, Arginine kinase, Phosphoserine aminotransferase and Actin protein. Together the before (Be) and after (Af) parasitization venom contains 1628 proteins, including 212 DEPs with 181 and 31 significantly up‐regulated and down‐regulated respectively. In addition, 10 differentially expressed proteins (DEPs) with fold change ≥8.71 were subjected to RT‐qPCR to validate the proteomic data. The differential expression analysis revealed that nine proteins were specifically present in the pre‐parasitic venom, whereas 26 proteins were specific to the post‐parasitic treatments. Results of RT‐qPCR analysis showed high expression of the selected DEPs which further validated our proteomics data. CONCLUSION: These new proteomic data greatly enrich our current knowledge about key venom proteins associated with parasitic process in A. japonicus and contribute to better understanding of the parasitic mechanisms leading to the development of new biological control strategies. © 2020 Society of Chemical Industry

Background and aims Atherosclerosis is characterized by lipid deposition, oxidative stress, and inflammation in the arterial intima. Ganoderma lucidum triterpenoids (GLTs) and polysaccharides (GLPs) are traditional Chinese medicines with potential cardiovascular benefits. We aimed to comprehensively evaluate the effect of GLTs and GLPs on atherosclerosis and the associated underlying mechanisms in vivo and in vitro. Methods and results Japanese big-ear white rabbits were randomly divided into three groups of blank, model, and treatment, and the treatment group was fed with GLSO and GLSP (0.3 g/kg body-weight/day) for 4 months. Serum levels of triglyceride (TG), total (TC), and low density lipoprotein cholesterol (LDL-C) in GL treatment group were significantly lower than those in the model group. The area of aortic plaques was significantly reduced in the treatment group. Further, GL administration in oxidized low-density lipoprotein (ox-LDL) stimulated human umbilical vein endothelial cells (HUVECs) reduced the generation of reactive oxygen species (ROS) and malondialdehyde (MDA) by inhibiting the upregulation of the nuclear transcription factor (NF)-κB p65 and the relative receptor LOX-1. In THP-1 cells treated with phorbol myristate acetate, GL inhibited the inflammatory polarization of macrophages (as evidenced by reduced TNF-α levels) via regulation of Notch1 and DLL4 pathways. Ox-LDL-stimulated THP-1 cells treated with GL showed an increase in the apoptosis of foam cells. Conclusions GLTs and GLPs attenuated the progression of atherosclerosis by alleviating endothelial dysfunction and inflammatory polarization of macrophages, thus promoting apoptosis of foam cells.

Summary5-AZA-2′-deoxycytidine (5-AZA-CdR) is a demethylating, teratogenic agent and a mutagen, which causes defects in the developing mouse and rat after implantation. Our previous data indicated that 5-AZA-CdR (0.2 and 1.0 μM) inhibited the development of mouse preimplantation embryos. Pronuclear embryos exposed to 5-AZA-CdR at the pronuclear stage were unable to form 8-cell embryos, while 2-cell-stage embryos exposed to 5-AZA-CdR only developed into uncompacted 8-cell-stage embryos. And there was no formation of blastocysts when 4-cell embryos cultured in 5-AZA-CdR. In our present study, we detected Dnmt1o protein and some developmental gene expression in order to find the reasons for the developmental arrest. Dnmt1o could not traffic to 8-cell nuclei as control when embryos were exposed to 5-AZA-CdR. Dnmt1o was in cytoplasm at 2-cell and 4-cell stages before and after treated with 5-AZA-CdR. Gene expression changes were also detected in this research. Our data indicated that connexin 31 (Cx31), connexin 43 (Cx43), connexin 45 (Cx45), E-cadherin (Cdh1) and β-catenin (Ctnnb1) were all downregulated by 5-AZA-CdR. Cx31, Cx43 and Cx45 are members of connexins family, which have a central role in gap junctions. Cdh1 and Ctnnb1 are necessary for the foundation of tight junctions. Therefore, developmental arrest induced by 5-AZA-CdR may be caused by the failure of Dnmt1o cytoplasmic–nuclear traffic and the down-regulation of developmental gene expression. Normal compaction and blastocoel cavitation need Dnmt1o traffic to 8-cell nuclei and the right gene expression, especially the correlative genes in gap junctions and tight junctions.

Attempts to develop gossypol and steroidal hormones alone as a male contraceptive have been tested for many years; however, both caused undesirable side effects that have prevented their acceptance. In this study, we formulated a regimen of combined gossypol at a low dose of 12 mg/kg or a high dose of 50 mg/kg plus methyltestosterone 20 mg/kg and ethinylestradiol 100 g/kg daily (12 mg G+H and 50 mg G+H) administered for 6 weeks in adult rats. The possible roles of germ cell apoptosis and related genes expression were studied by techniques of TdT-mediated dUTP nick end-labeling (TUNEL), agarose gel electrophoresis of low-molecular–weight DNA, in situ hybridization and reverse transcription-polymerase chain reaction detection. Results showed that germ cell apoptosis and related genes expression were significantly induced after combined drug administration. The apoptosis index increased 3.86- and 9.65-fold in the 12-mg and 50-mg G+H-treated groups, respectively, as compared to the control group. DNA ladder formation on the agarose gel further validated the findings of TUNEL-stained apoptotic cells. The apoptosis-related genes fas mRNA expression levels increased 0.44- and 1.39-fold, bax mRNA 0.74- and 2.56-fold, caspase-3 mRNA 0.60- and 1.29-fold, and caspase-9 mRNA 2.50- and 4.08-fold, respectively, in the 12-mg and 50-mg G+H-treated groups vs. the control group. These results indicated that our drug regimen applied as a contraceptive could induce rat germ cell apoptosis. The apoptotic process involved fas system, bax and caspase family genes and the apoptotic extent and cell types were gossypol dose-dependent.

An effective immune response against human immunodeficiency virus or simian immunodeficiency virus (SIV) is critical in achieving control of viral replication. Here, we show in SIV-infected rhesus monkeys that an effective and durable SIV-specific cellular and humoral immunity is elicited by a vaccination with chemically inactivated SIV-pulsed dendritic cells. After three immunizations made at two-week intervals, the animals exhibited a 50-fold decrease of SIV DNA and a 1,000-fold decrease of SIV RNA in peripheral blood. Such reduced viral load levels were maintained over the remaining 34 weeks of the study. Molecular and cellular analyses of axillary and inguinal node lymphocytes of vaccinated monkeys revealed a correlation between decreased SIV DNA and RNA levels and increased SIV-specific T-cell responses. Neutralizing antibody responses were augmented and remained elevated. Inactivated whole virus-pulsed dendritic cell vaccines are promising means to control diseases caused by immuno- deficiency viruses.

Our previous studies suggested that low-dose gossypol combined with steroid hormones has a reversible antifertility role in adult male rats, and the course of treatment was shorter than that of either gossypol or steroid hormones alone. This result suggested that low-dose gossypol and steroid hormones have a drug synergistic effect on antifertility. The aim of the study was to find the target organs of the antifertility synergistic effect of the combined regimen.Thirty-two adult male rats were divided into four groups randomly: group GH, rats were fed orally with gossypol acetic acid (GA, 12.5 mg×kg(-1)×d(-1)) and desogestrel (DSG, 0.125 mg×kg(-1)×d(-1))/ethinylestradiol (EE, 0.025 mg×kg(-1)×d(-1))/testosterone undecanoate (TU, 100 mg×kg(-1)×d(-1)); group G, a single dose of GA (12.5 mg×kg(-1)×d(-1)) was given; group H, the same dosage of DSG/EE/TU as in group GH were administered; group C, rats were treated with vehicle (1% methyl cellulose) as control. Testes and epididymis were removed at 8 weeks post-treatment for evaluating their weight, volumes, volume fraction, and total volume of testicular tissue structures and the seminiferous tubule diameter using stereological assay. Sperm cell numbers and the motility of epididymal sperm were quantitated by flow cytometry and morphological methods.Compared with group C, spermatogenesis was normal in group G and suppressed in groups H and GH. Similar changes of testicular tissue structures and sperm number were found in groups H and GH. The decreases of epididymal sperm number and motility in group GH were greater than that of the low-dose gossypol or steroid hormones alone group.The suppression of spermatogenesis was induced by steroid hormones in the combined regimen, and the epididymis was the target organ of low-dose gossypol. Combined use of low-dose gossypol and steroid hormones played a comprehensive antifertility role in their synergistic effect on reducing the number and motility of epididymal sperm.

To explore the effectiveness of the traditional Chinese herbal medicine Lingzhi (Ganoderma lucidum) preparation in treating simian acquired immune deficiency syndrome (SAIDS).Five female adult Chinese rhesus monkeys were inoculated rectally with SIVmac239, and were all diagnosed as SAIDS by laboratory and clinical examinations 17 months later. Of these 5 monkeys, 3 (#393, #374, and #381; treatment group) were orally administered with Ganoderma lucidum (2 spores powder capsules plus 2 spores oil capsules on a daily basis), and the remaining other two monkeys (#348 and #361) served as control and did not receive treatment.Animal #393 (treatment group), #361 (control group) and #348 (control group) died of SAIDS (opportunity infection) 3.5 months, 6 months, and 11 months later, respectively. Two animals (#374 and #381) survived. The necropsy revealed depletion and/or exhaustion of their lymphoid tissue. In the monkey #374, the peripheral CD4(+) T lymphocyte increased by 30% in the 6(th) month compared with the baseline level and then fluctuated. The plasma viral load gradually fell and reached about 1 log(10) in the treatment group, but remained stable in the control group. As shown by pathological examinations, the lymph node and spleen of monkeys #374 (treatment group) and #381 (treatment group) showed rehabilitation and reconstruction in the lymphatic tissue, thymus, nerve tissue of gyrus hippocampi, pituitary gland, pineal body, thyroid gland, adrenal gland, and ovary. In the control group, however, animals experienced depletion of lymph nodes, atrophy of spleen, disappearance of thymus, and other disorders in endocrine organs.Ganoderma lucidum preparation may have certain protective effect on the immune system, nervous system, and endocrine system of monkeys with SAIDS.

Cryptorchidism is associated with male infertility due to germ cell loss in response to elevated temperature. However, there is a great deal of contradictory information prevalent on the status of germ cells and their process of removal in the cryptorchid testis. In the present study, we investigate the cell removal from cryptorchid rat testis by the methods of morphology and stereology. The testis weight is reduced according to previous reports after surgical induction of cryptorchidism. Interestingly, the epididymal weight is significantly increased in 7 days after surgery, and the caput epididymis tubules show filling with countless round germ cells. We found that the elongating spermatids (steps 10-13), newborn spermatids (step 1) and the dividing spermatocytes are the most susceptible cells to elevated temperature, and are the first disappeared cells from the seminiferous tubules after surgery. Germ cell removal followed the order, starting first with elongating spermatids and newborn spermatids, followed by round spermatids and elongated spermatids and later extending to spermatocytes.

Surgery-induced cryptorchidism, in which the testes are prevented from descending into the scrotal sac, results in testicular germ cell death, and it is commonly used as an experimental tool in the study of spermatogenesis. However, the molecular events underlying the activation of germ cell death remain poorly understood. In the present study, we investigate selective cell loss from cryptorchid rat testis by using DNA flow cytometry and by determining protein and mRNA expression of Hsf1, Hsf2, and Phlda1. The hypo-haploid cell fraction is significantly decreased as early as 3 days after surgical induction of cryptorchidism (from 42.01 ± 5.74% to 15.98 ± 3.88%), followed by a significant decrease in the haploid cell fraction at Day 7. At the latter time point, an apoptotic peak of spermatocytes appears in DNA histograms just before the tetraploid peak; the percentage of aneuploid cells between diploid and tetraploid rises as high as 14.05 ± 2.98% of the total cells in 7-day cryptorchid testis, suggesting that a large number of spermatocytes are undergoing apoptosis. The expression of Phlda1 mRNA is significantly elevated 3 days after induction of cryptorchidism. After 7 days of cryptorchidism, Hsf1 and Phlda1 are strongly expressed in the nucleus and cytoplasm, respectively, of primary spermatocytes. Numerous apoptotic spermatocytes are also observed at this time point. These results suggest that the Hsf1/Phlda1 pathway plays an important role in the apoptosis of primary spermatocytes in cryptorchid testis. We present evidence suggesting that Hsf2 is also involved in germ cell removal in cryptorchid testis.

The morphological measurements of seminiferous tubules are important in the studies of testis tissues. The purpose of this study was to evaluate the feasibility of using a stereological method to measure the geometric parameters of seminiferous tubule and to optimize the method.A stereological image processing program was developed with Delphi for the stereological measurement. Fields of view were obtained from 15 healthy Wistar rats' testis tissues (n = 247). The diameter, area and volume of seminiferous tubule were estimated with the image processing program by two individual observers. The area results were compared with those obtained by the standard morphometric method of planimetry.Diameter measurements showed the diameters of different seminiferous tubules were almost the same and the mean value of about 50 tubules could be a good representation of the whole structure. Area measurements indicated there was no significant difference between stereology and planimetry (P0.05). But the stereological method required about 45% less time. Volume measurement showed the inter-observer variability was small (P0.05) and the reproducibility of the stereological method was good.The stereological technique was practical and efficient in the quantitative measurement of the rat's seminiferous tubule.

To investigate the biological and clinical features of Chinese rhesus monkeys after intravenous (IV) and intrarectal (IR) challenge with SIVmac239 in rhesus monkeys of Chinese origin, and compare the differences between the routes of infection.Rhesus monkeys of Chinese origin were inoculated with SIVmac239 either by IV (n = 19) or IR (n = 6) routes. Simian immunodeficiency virus (SIV)-specific antibody titer, CD4 + T cell counting, plasma SIV load, lymph node pathology, and clinical manifestations were compared between these two groups 232 or 168 days after challenging.All SIVmac239-inoculated animals became seropositive for SIV-specific antibodies. SIV-specific IgM was detected in IV groups as from day 10 but was not detected in IR for all the time points. Although SIV-specific IgG was detected as from day 30 in both groups, the IgG titers were ten-fold higher in IV group than in IR group after day 168. CD4 + T-cell counting decreased progressively in IV group but remained stable in IR group over time. Plasma SIV RNA loads peaked in all animals between day 10 and day 14 (10(7) copies/ml), then declined to "setpoint" (10(3) - 10(6) copies/ml) about 2 months later. Most inoculated animals manifested lymphadenopathy. Two animals in IV group and one in IR group died of simian AIDS between day 150 and day 210, as evidenced by the autopsies showing the depletion of lymph tissues, Pneumocystis carinii pneumonia and other opportunity infections. Conclusion IV or IR inoculation of SIVmac239 in Chinese rhesus monkeys will result in chronic SIV infection with a similar clinical feature of natural HIV infection, which provides an excellent experimental animal model for AIDS.

To study the relationship between simian acquired immunodeficiency syndromn (SAIDS) and autoimmunity in simian immunodeficiency virus (SIV)-infected monkeys.Indirect immunofluorescence assays were performed to detect plasma or serum autoantibodies in SIV-infected monkeys. The heart, liver, spleen, lung, kidney, and lymph node of BALB/c mice, a strain of endothelial cell ECV304, and granulocytes were used as target antigens. These results were compared with HE stained slides of SIV-infected monkeys.The levels of various autoantibodies, including anti-lymphocyte autoantibodies, anti-endothelial cell autoantibodies, and anti-granulocyte antibodies, increased after SIV infection in monkeys. Moreover, pathological examinations showed injuries in the lymphoid tissue and vascular pathological changes in cerebral cortex, submucosa of gastrointestinal tract, interstitial capillaries of myocardium, nephron of the kidney, and sinusoid cell of liver.The increased autoantibodies and the pathological changes of tissues and organs confirm the existence of autoimmunity in SIV-infected monkeys.

Background Myocardial fibrosis is a major component of ventricular remodeling after myocardial infarction (MI). The aim of the present study was to determine the outcome of transplantation into ischemic myocardium of bone marrow derived stem cell (MSC) on left ventricular (LV) function and remodeling, and to look closely at extracellular matrix gene expression. Methods and Results MI was induced by direct ligation of the left anterior descending coronary artery in rats, followed by MSC transplantation into the ischemic myocardium. Hemodynamic evaluations were performed at 7 and 28 days after coronary ligation. Changes in the mRNA expressions of collagen type I and type III, matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and transforming growth factor (TGF)-β1 were investigated using reverse transcription polymerase chain reaction and in situ hybridization after MI. Heart/body weight ratio in the MI + MSC group decreased after coronary ligation. However, LV systolic pressure and LV peak velocities of contraction and relaxation significantly increased compared with the MI group (p

The present study was to determine the effects of mesenchymal stem cell (MSC) transplantation into ischemic myocardium on left ventricular (LV) function and remodeling, especially the changes in extracellular matrix (ECM) and in transforming growth factor (TGF)-beta1 after myocardial infarction (MI).In this study, we investigated the changes in ECM involving collagen type I, collagen type III, matrix metalloproteinase (MMP)-1, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and in TGF-beta1 mRNA expressions by using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization after MSC transplantation into infarcted myocardium in rats. Protein expressions were detected by immunohistochemistry and western blot. Seven and 28 days after coronary artery ligation, hemodynamic evaluations were performed.Heart : body weight ratio in the MI+MSC group decreased after MSC transplantation into infarcted myocardium compared with the MI group. However, LV systolic pressure (LVSP) and LV peak velocities of contraction and relaxation (+dP/dtmax and -dP/dtmax) significantly increased compared with the MI group (P0.01). There was marked up-regulation of mRNA transcription for ECM and TGF-beta1 proteins in MI rats. Immunohistochemical and western blot studies confirmed that the expressions of these ECM and TGF-beta1 proteins were significantly increased in the MI group. The mRNA and protein expressions of collagen type I, collagen type III, TIMP-1 and TGF-beta1 were all decreased by MSC transplantation in our study, although no significant difference was found for the mRNA and protein expressions of MMP-1 between the MI group and the MI+MSC group.Our results demonstrated that MSC transplantation could inhibit LV remodeling and improve heart function.

To investigate the effects of excessive intake of fluoride on the expression of type II collagen gene and types and morphological change of collagen fiber in the bone tissues of rats.A rat model with fluorosis was established by adding 221 mg/L of sodium fluoride (NaF) to drinking water for the rats for 15 days, 30 days and two months, respectively. Type II collagen alpha1 (II) cDNA probe was prepared, and cDNA-mRNA in-situ hybridization was employed to detect change in expression of type II collagen mRNA in the bone tissues of rats with excessive intake of fluoride (221 mg/L NaF). Picrosirius-polarization method was used to observe types of collagen and morphology of collagen fiber in the bone tissues.Chondroblasts were found in the femur and other bone tissues of the rats after exposure to fluoride. cDNA-mRNA in-situ hybridization showed that expression of type II collagen gene could be observed in the cytoplasm of chondrocytic lacuna and chondrified bone tissues. mRNA in collagen of chondrocytes of the rib cartilage reached the peak level 15 days after exposure to fluoride, and decreased gradually one month and two months after exposure. Polychromatic type II collagen, breakage of collagen fiber, disorder array and reduced content of type II collagen could be found in the bone tissues with picrosirius-polarization method.Excessive intake of fluoride could lead to changes in types and structure of collagen (cross-linkage) of bone tissues, which caused expression of type II collagen gene in the chondrified bone tissues and enhanced its expression in the rib cartilage tissues.