Full length genome sequencing and phylogenetic analysis of two strains of Getah virus / 中国热带医学

@#Abstract: Objective　The main aim of the study is to sequence the complete genome of two Getah virus strains (GS11-155 and HNDZ1712-1) isolated in Gansu Province and Hainan Province in 2011 and 2017 respectively and analyze the molecular and genetic evolution of the two strains compared with M1, which was first isolated in 1964 in Hainan Province, China. Methods　Genome of two newly isolated Getah viruses were sequenced by virus gene amplification technique, and the genomic database of Getah viruses was established. The molecular characteristics and genetic evolution of the viruses were analyzed by bioinformatics software. Results　The genome length of two new isolated Getah virus strains (GS11-155 and HNDZ1712-1) was 11 690 nt and 11 621 nt, respectively. Both strains had the structural characteristics of Alphavirus genome. Although the nucleotide sequence lengths of structural genes, non-structural genes and non-coding junction regions of the two strains were identical, the nucleotide sequence lengths of the 5' and 3' non-coding regions of the viral genomes were a few different. The 3'UTR repeats elements in the genomes of the two virus strains did not change. It was 97.7% and 98.1% different of nucleotide and amino acid homology between both strains of Getah virus, HNDZ1712-1 isolated in 2017 and M1 isolated in 1964 in Hainan Province. Interesting, Gansu 2011 cluster and Hainan 2017 cluster were emerged leading by both strains GS11-155 and HNDZ1712-1 respectively, those two clusters totally independent with M1 virus isolated from Hainan in 1964 in whole genome phylogenetic analysis first. Conclusions　Although the HNDZ1712-1 was also isolated from mosquito samples in Hainan Province, it was in a completely different evolutionary branch from the M1 isolated from Hainan Island in 1964, and was closely related to the strain isolated from Gansu Province (GS11-155) thousands of kilometers away. It is suggested that the two new strains of Getah virus are different from the Getah virus isolated in 1964.

Antihypertensive drug-related genes polymorphisms in hypertensive patients at a certain hospital / 中国热带医学

@#Abstract: Objective　By analyzing the frequency distribution of antihypertensive drug-related genotypes in hypertensionpatients treated in our hospital, so as to provide a clinical basis for individualized treatment of hypertension patients. Methods　A total of 72 hypertensive patients treated in Hainan Hospital of PLA General Hospital from June 2021 to April 2022 were collected. PCR-melting curve method was used to detect CYP2D6*10 (c.100 C>T), CYP2C9*3 (c.1075 A>C), ADRB1 (c.1165 G>C), AGTR1 (c.1166 A>C), ACE (I/D), NPPA (T2238C) and CYP3A5*3 (A6986G), and the relationship between different genotypes and biochemical indexes was analyzed. Results　According to the statistics of the gene and genotype frequency of each point in 72 patients, the gene frequencies of 7 sites all conformed to Hardy Weinberg equilibrium. There were gender differences in ADRB1 genotypes (χ2 = 5.878, P<0.05). There were statistical differences in triglycerides [AA: 1.4 (1.0, 2.0)mmol/L; AC: 2.2 (1.5, 2.5)mmol/L; P=0.038], total cholesterol [AA: 4.0 (3.1, 4.9) mmol/L; AC: 4.8 (4.0, 5.3) mmol/L; P=0.040] and low-density lipoprotein cholesterol [(AA: 2.4 (1.8, 3.3) mmol/L; AC: 3.2 (2.5, 3.5) mmol/L; P=0.035] among patients with different genotypes of AGTR1 locus. The patients with different genotypes of CYP2C9 locus had significant differences in their alanine transferase (ALT) [AA:16.9 (11.4,30.2) mmol/L; AC:10.4 (9.4, 18.2) mmol/L; P=0.040]. Aftergene-directed individualized therapy, different genotypes of CYP3A5 andAGTR1 affected the heart rate [CYP3A5: AA: (79.3±7.0) beats/min; AG： (69.8±6.8) beats/min; GG： (68.8±7.3) beats/min; P=0.010], systolic blood pressure [AGTR1: AA: (131.3±16.7) mmHg; AC: (140.6±11.8) mmHg; P=0.014] and diastolic blood pressure [CYP3A5: AA: (90.0±8.3) mmHg; AG: (78.7±10.8) mmHg; GG: (74.9±10.7) mmHg; P=0.025; AGTR1: AA: (75.3±10.2) mmHg; AC: (86.3±10.6) mmHg; P=0.001] of patients. Conclusions　The related gene loci of antihypertensive drugs are an important basis for guiding the diversification and individualization of clinical medication. Clinicians need to consider the impact of related genes on drug efficacy and adverse reactions when prescribing.

Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

Specific Detection of Acanthamoeba species using Polyclonal Peptide Antibody Targeting the Periplasmic Binding Protein of A. castellanii

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.

An in vitro model study of computer-guided endodontic microsurgery / 中华口腔医学杂志

Objective: To assess and compare the accuracies and operating time of endodontic microsurgery performed by operators with different levels of experience in endodontics using computer-guided techniques including dynamic and static navigations in a surgical simulation model. Methods: Six pairs of three dimensional (3D)-printed models of upper and lower jaws were set up on dental manikins. A total of 120 teeth (10 teeth each jaw) were included in the models. Microsurgeries of osteotomy and root-resection were performed on the models by two operators with different experience, namely novices and experts, under of free hand (FH)(n=20), dynamic navigation (DN)(n=20), and static navigation (SN)(n=20) conditions, respectively. The duration of each operation was recorded. Cone-beam CT was taken for 3D-printed models before and after the operation. The path of preoperative surgery planning was simulated. The linear deviations at the entry and the end point and the angular deviation of the access path between the simulated and the actual operation were compared by the software. Results: Significant difference of the entry deviation was observed between the novices and the experts in the FH group [(1.44±0.49) and (1.02±0.58) mm] (q=4.67, P=0.020). There were no significant differences between the novices and the experts in the end point and angular deviations (P>0.05). For the novices, the entry deviations in both DN and SN groups [(0.76±0.32) and (0.66±0.20) mm] were significantly lower than those in FH group (q=7.58, P<0.001; q=8.66, P<0.001). The angular deviations in the abovementioned two groups (5.0°±3.5°, 3.9°±2.1°) were significantly lower than that in FH group (10.9°±6.1°) (q=7.38, P<0.001; q=8.70, P<0.001). For the experts, significant differences were found only in the angular deviations among DN, SN and FH groups (3.6°±1.9°, 3.2°±1.7° and 8.2°±3.9°) (q=5.74, P=0.001; q=6.29, P<0.001). The operation durations were significantly shortened for both the novices [(4.80±2.15), (1.09±0.48) min] (q=14.60, P<0.001; q=20.10, P<0.001) and the experts [(3.40±1.96),(1.02±0.34) min] (q=5.86, P<0.001; q=9.37, P<0.001) by using DN and SN techniques. Regarding the differences between tooth types, in FH group, the operating time on the anterior teeth was significantly shorter than that on the posterior teeth (q=8.14, P<0.001; q=5.20, P=0.007), while in DN and SN groups, there were no significant differences in the operating time between two tooth types (P>0.05). No significant differences were discovered in the accuracies on the anterior and posterior teeth among three techniques or between two kinds of operators (P>0.05). Conclusions: Dynamic and static navigation techniques could assist the clinicians, especially the novices, to improve the accuracies and shorten the operating time of osteotomy and root resection microsurgeries.

Differentially Expressed Gene Profile of Acanthamoeba castellanii Induced by an Endosymbiont Legionella pneumophila

Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term ‘integral component of membrane’ were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.

Detection of Toxoplasma gondii Infections using Virus-Like Particles Displaying T. gondii ROP4 Antigen

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.

Effect of hypothalamic nerve growth factor precursor on field potential of paraventrucular nucleus during rat cardiopulmonary bypass / 临床麻醉学杂志

Objective To evaluate the effect of cardiopulmonary bypass (CPB) on the expression of hypothalamic nerve growth factor precursor (proNGF) and the influence of hypothalamic proNGF on the sympathetic output of paraventrucular nucleus. Methods Forty-two male SD rats, aged 3-4 months, weighing 350-500 g, were divided into control group, CPB group and ischemia reperfusion (IR) group. At the end of CPB for 110 min, hypothalamus and dorsal root ganglion (DRG) were taken to measure the levels of proNGF mRNA and hypothalamic proNGF protein. Mini pipe was put into bilateral paraventrucular nucleus (PVN) and human recombination proNGF protein was injected into PVN for 7 d before the local field potentials (LFP) of RVLM was recoreded. Human recombination proNGF protein was administrated into lateral ventricle, the prior-administration-LFP of PVN and post-administration-LFP were recorded and compared. At the end of the experiment, hypothalamus was taken to measure the levels of glutamate and gammer amino butyric acid (GABA). Results Hypothalamic proNGF protein in CPB group and IR group was higher than that in the control group (P ＜ 0.05); NGF mRNA of hypothalamus and DRG in CPB and IR group were higher than those of control group (P ＜ 0.05). In PVN and RVLM, after the administration of proNGF protein, the power of delta band significantly decreased and other bands increased (P ＜ 0.05). The hypothalamic GABA level decreased (P ＜ 0.05) with no change of hypothalamic glutamate after proNGF was injected into lateral ventricle. Conclusion CPB increases the expression of proNGF in the hypothalamus contributing to the changes of hypothalamic sympathetic output.

Serum levels of miR‐21 and miR‐155 in T2DM + CHD patients and their relationship with lipid me‐tabolism／ / 心血管康复医学杂志

To explore serum levels of miR‐21 and miR‐155 in patients with T2DM complicated CHD and their relationship with lipid metabolism .Methods : A total of 134 T2DM patients treated in our hospital from 2016 to 2017 , were divided into T2DM + CHD group (n＝60) and pure T2DM group (n＝74).Blood glucose and blood lipid levels and serum miR‐21 and miR‐155 levels were measured and were compared between two groups .Results :There were no significant difference in general data , blood pressure , body mass index (BMI) , glycosylated hemo‐globin A1c (HbA1c) , plasma glucose and total cholesterol (TC) between two groups , P＞0. 05 all.Compared with pure T2DM group , there were significant rise in levels of triglyceride (TG) [ (1. 89 ± 0.92) mmol／L vs.(2. 75 ± 1.61) mmol／L] , LDL‐C [ (2.83 ± 0.79) mmol／L vs.(3. 52 ± 1.24) mmol／L] and serum miR‐21 [ (0. 93 ± 0. 15) vs.(1. 86 ± 0.24 )] , and significant reductions in levels of HDL‐C [ (1.35 ± 0. 34 ) mmol／L vs.(0. 94 ± 0.31 ) mmol／L] and serum miR‐155 [ (0. 95 ± 0.19) vs.(0. 27 ± 0. 10)] in T2DM + CHD group , P＝0.001 all.Multiva‐riate Logistic regression analysis indicated that TG , LDL‐C and miR‐21 were independent risk factors for T2DM +CHD (OR＝2. 800～4. 986 , P＜0.05 all) , while HDL‐C and miR‐155 were its independent protective factors (OR＝0.314 , 0.327 , P＜ 0.05 both).Pearson correlation analysis indicated that serum miR‐21 level was significant positively correlated with TG and LDL‐C levels ( r＝0. 415 , 0.506 , P＜0.05 or ＜0. 01) , and serum miR‐155 level was significant inversely correlated with TG and LDL‐C levels ( r＝ －0. 397 ,－0. 526 , P＜0.05 or ＜0. 01 ).Con‐clusion : Serum miR‐21 level was significant positively correlated with TG and LDL‐C levels , but serum miR‐155 level was significant inversely correlated with TG and LDL‐C levels ,

Influence of ticagrelor on myocardial injury in patients with acute myocardial infarction after emergen-cy PCI / 心血管康复医学杂志

Objective :To explore influence of ticagrelor on myocardial injury ,serum levels of N terminal pro brain natriuretic peptide (NT-proBNP) ,tumor necrosis factor (TNF)-α and endothelin (ET) in patients with acute myo-cardial infarction (AMI) after emergency percutaneous coronary intervention (PCI).Methods : A total of 94 AMI patients undergoing emergency PCI were selected ,and were divided into clopidogrel group (n＝ 45 ) and ticagrelor group (n＝ 49 ) ,two groups received postoperative dual-antiplatelet therapy of aspirin combined clopidogrel or ti-cagrelor respectively .After one-month treatment ,index of microcirculatory resistance (IMR ) ,coronary flow re-serve (CFR ) , serum levels of NT-proBNP , TNF-α , ET , and incidence of major adverse cardiovascular events (MACE) within one year were compared between two groups .Results : Compared with clopidogrel group after 12- month treatment ,there were significant reductions in IMR [21-24 ± 4-07 ) vs.(15-33 ± 4-82)] ,serum levels of NT-proBNP [(123-17 ± 16-25) ng／L vs.(63-46 ± 12-13) ng／L] ,TNF-α [(4-04 ± 0-84) mg／L vs.(3-07 ± 0-52) mg／L] ,ET-1 [ (48-71 ± 6-53) ng／L vs.(38-04 ± 5-89) ng／L] ,and significant rise in CFR [ (1-73 ± 0-34) vs. (2-36 ± 0-42)] in ticagrelor group , P＝ 0-001 all .Incidence rate of MACE in ticagrelor group was significantly lower than that of clopidogrel group (6-12% vs .24-44%, P＝ 0-018 ).Conclusion : Compared with clopidogrel group ,ticagrelor group possesses significant therapeutic effect in AMI patients after emergency PCI .And it′s safe .

Respiratory Syncytial Virus Fusion Protein-encoding DNA Vaccine Is Less Effective in Conferring Protection against Inflammatory Disease than a Virus-like Particle Platform

Formalin-inactivated respiratory syncytial virus (RSV) vaccination causes vaccine-enhanced disease (VED) after RSV infection. It is considered that vaccine platforms enabling endogenous synthesis of RSV immunogens would induce favorable immune responses than non-replicating subunit vaccines in avoiding VED. Here, we investigated the immunogenicity, protection, and disease in mice after vaccination with RSV fusion protein (F) encoding plasmid DNA (F-DNA) or virus-like particles presenting RSV F (F-VLP). F-DNA vaccination induced CD8 T cells and RSV neutralizing Abs, whereas F-VLP elicited higher levels of IgG2a isotype and neutralizing Abs, and germinal center B cells, contributing to protection by controlling lung viral loads after RSV challenge. However, mice that were immunized with F-DNA displayed weight loss and pulmonary histopathology, and induced F specific CD8 T cell responses and recruitment of monocytes and plasmacytoid dendritic cells into the lungs. These innate immune parameters, RSV disease, and pulmonary histopathology were lower in mice that were immunized with F-VLP after challenge. This study provides important insight into developing effective and safe RSV vaccines.

Parasite Infiltration and Apoptosis in Spleen upon Toxoplasma gondii Infection

Toxoplasma gondii infection induces parasite infiltration and apoptosis in the spleen. However, dose-dependent parasite infiltration, apoptosis, body weight alternations and survival in mice remain largely unknown. In this study, mice were intraperitoneally infected with 10, 30 or 100 tachyzoites of T. gondii, respectively. Parasite infiltration and apoptosis in the spleen were analyzed on days 3, 7, and 9 post-infection by immunohistochemistry and flow cytometry. Significantly higher levels of T. gondii infiltration and apoptosis in the spleen were found in 30 and 100 tachyzoites infected mice compared to 10 tachyzoites infected mice on days 7 and 9 post-infection. Although 30 and 100 tachyzoites infected mice showed significant body weight loss compared to 10 tachyzoites infected mice, all of the 100, 30, and 10 tachyzoites infected mice died by days 12, 15, and 17, each respectively. Interestingly, T. gondii infiltration in 10 tachyzoites infected mice were limited to capsule area of the spleen on day 9 post-infection. Several areas of parasite infiltrations were found in the 30 tachyzoites infected mice, where noticeable levels of splenic capsule de-adhesion occurred. These results indicated that parasite infiltration and apoptosis in the spleen, as well as body weight loss (survival) are closely correlated with infection dosage. The level of T. gondii infiltration and apoptosis in the spleen and splenic de-adhesion were dependent on the parasite dose.

Virus-like Particle Vaccine Containing Toxoplasma gondii Rhoptry Protein 13 Induces Protection against T. gondii ME49 Infection in Mice

Toxoplasma gondii can infect humans worldwide, causing serious diseases in pregnant women and immunocompromised individuals. T. gondii rhoptry protein 13 (ROP13) is known as one of the key proteins involved in host cell invasion. In this study, we generated virus-like particles (VLPs) vaccine expressing T. gondii rhoptry ROP13 and investigated VLPs vaccine efficacy in mice. Mice immunized with ROP13 VLPs vaccine elicited significantly higher levels of T. gondii-specific IgG, IgG1, IgG2a, and IgA antibody responses following boost immunization and challenge infection, whereas antibody inductions were insignificant upon prime immunization. Differing immunization routes resulted in differing antibody induction, as intranasal immunization (IN) induced greater antibody responses than intramuscular immunization (IM) after boost and challenge infection. IN immunization induced significantly higher levels of IgG and IgA antibody responses from feces, antibody-secreting cells (ASCs), CD4⁺ T, CD8⁺ T cells and germinal center B cell responses in the spleen compared to IM immunization. Compared to IM immunization, IN immunization resulted in significantly reduced cyst counts in the brain as well as lesser body weight loss, which contributed to better protection. All of the mice immunized through either route survived, whereas all naïve control mice perished. These results indicate that the ROP13 VLPs vaccine could be a potential vaccine candidate against T. gondii infection.

Low Levels of Polymorphisms and Negative Selection in Plasmodum knowlesi Merozoite Surface Protein 8 in Malaysian Isolates

Human infections due to the monkey malaria parasite Plasmodium knowlesi is increasingly being reported from most Southeast Asian countries specifically Malaysia. The parasite causes severe and fatal malaria thus there is a need for urgent measures for its control. In this study, the level of polymorphisms, haplotypes and natural selection of full-length pkmsp8 in 37 clinical samples from Malaysian Borneo along with 6 lab-adapted strains were investigated. Low levels of polymorphism were observed across the full-length gene, the double epidermal growth factor (EGF) domains were mostly conserved, and non-synonymous substitutions were absent. Evidence of strong negative selection pressure in the non-EGF regions were found indicating functional constrains acting at different domains. Phylogenetic haplotype network analysis identified shared haplotypes and indicated geographical clustering of samples originating from Peninsular Malaysia and Malaysian Borneo. This is the first study to genetically characterize the full-length msp8 gene from clinical isolates of P. knowlesi from Malaysia; however, further functional characterization would be useful for future rational vaccine design.

Previous Infection with Plasmodium berghei Confers Resistance to Toxoplasma gondii Infection in Mice

Both Plasmodium spp. and Toxoplasma gondii are important apicomplexan parasites, which infect humans worldwide. Genetic analyses have revealed that 33% of amino acid sequences of inner membrane complex from the malaria parasite Plasmodium berghei is similar to that of Toxoplasma gondii. Inner membrane complex is known to be involved in cell invasion and replication. In this study, we investigated the resistance against T. gondii (ME49) infection induced by previously infected P. berghei (ANKA) in mice. Levels of T. gondii-specific IgG, IgG1, IgG2a, and IgG2b antibody responses, CD4+ and CD8+ T cell populations were found higher in the mice infected with P. berghei (ANKA) and challenged with T. gondii (ME49) compared to that in control mice infected with T. gondii alone (ME49). P. berghei (ANKA) + T. gondii (ME49) group showed significantly reduced the number and size of T. gondii (ME49) cysts in the brains of mice, resulting in lower body weight loss compared to ME49 control group. These results indicate that previous exposure to P. berghei (ANKA) induce resistance to subsequent T. gondii (ME49) infection.

Comparison of Proteins Secreted into Extracellular Space of Pathogenic and Non-pathogenic Acanthamoeba castellanii

Pathogenic Acanthamoeba spp. cause granulomatous amoebic encephalitis and keratitis. Acanthamoeba keratitis (AK) is a rare but serious ocular infection that can result in permanent visual impairment or blindness. However, pathogenic factors of AK remain unclear and treatment for AK is arduous. Expression levels of proteins secreted into extracellular space were compared between A. castellanii pathogenic (ACP) and non-pathogenic strains. Two-dimensional polyacrylamide gel electrophoresis revealed 123 differentially expressed proteins, including 34 increased proteins, 7 qualitative increased proteins, 65 decreased proteins, and 17 qualitative decreased proteins in ACP strain. Twenty protein spots with greater than 5-fold increase in ACP strain were analyzed by liquid chromatography triple quadrupole mass spectrometry. These proteins showed similarity each to inosine-uridine preferring nucleoside hydrolase, carboxylesterase, oxygen-dependent choline dehydrogenase, periplasmic-binding protein proteinases and hypothetical proteins. These proteins expressed higher in ACP may provide some information to understand pathogenicity of Acanthamoeba.

Virus-Like Particles Expressing Toxoplasma gondii Rhoptry Protein 18 Induces Better Protection Than Rhoptry Protein 4 against T. gondii Infection

Toxoplasma gondii is a ubiquitous protozoan parasite responsible for causing toxoplasmosis. Preventive measures for toxoplasmosis are currently lacking and as such, development of novel vaccines are of urgent need. In this study, we generated 2 virus-like particles (VLPs) vaccines expressing T. gondii rhoptry protein 4 (ROP4) or rhoptry protein 18 (ROP18) using influenza matrix protein (M1) as a core protein. Mice were intranasally immunized with VLPs vaccines and after the last immunization, mice were challenged with ME49 cysts. Protective efficacy was assessed and compared by determining serum antibody responses, body weight changes and the reduction of cyst counts in the brain. ROP18 VLPs-immunized mice induced greater levels of IgG and IgA antibody responses than those immunized with ROP4 VLPs. ROP18 VLPs immunization significantly reduced body weight loss and the number of brain cysts in mice compared to ROP4 VLPs post-challenge. These results indicate that T. gondii ROP18 VLPs elicited better protective efficacy than ROP4 VLPs, providing important insight into vaccine design strategy.

Effect of 2, 6-Dichlorobenzonitrile on Amoebicidal Activity of Multipurpose Contact Lens Disinfecting Solutions

Multipurpose contact lens disinfecting solutions (MPDS) are widely used to cleanse and disinfect microorganisms. However, disinfection efficacy of these MPDS against Acanthamoeba cyst remain insufficient. 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, is capable of increasing the amoebical effect against Acanthamoeba by inhibiting its encystation. In this study, we investigated the possibility of DCB as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba cyst. Eight commercial MPDS (from a to h) were assessed, all of which displayed insufficient amoebicidal activity against the mature cysts. Solution e, f, and h showed strong amoebicidal effect on the immature cysts. Amoebicidal efficacy against mature cysts remained inadequate even when the 8 MPDS were combined with 100 μM DCB. However, 4 kinds of MPDS (solution d, e, f, and h) including 100 μM DCB demonstrated strong amoebicidal activity against the immature cysts. The amoebicidal activity of solution d was increased by addition of DCB. Cytotoxicity was absent in human corneal epithelial cells treated with either DCB or mixture of DCB with MPDS. These results suggested that DCB can enhance the amoebicical activity of MPDS against Acanthamoeba immature cyst in vitro.

Identification and Characterization of Protein Arginine Methyltransferase 1 in Acanthamoeba castellanii

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.

DNA Methylation of Gene Expression in Acanthamoeba castellanii Encystation

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1–3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.