Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess

Background

Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba''s pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties.

Methodology/Principal Findings

A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene.

Conclusions/Significance

Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.     

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates

Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia^® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.     

Glutathione disulfide liposomes – A research tool for the study of glutathione disulfide associated functions and dysfunctions

Glutathione disulfide (GSSG) is the oxidized form of glutathione (GSH). GSH is a tripeptide present in the biological system in mM concentration and is the major antioxidant in the body. An increase in GSSG reflects an increase in intracellular oxidative stress and is associated with disease sates. The increase has also been demonstrated to lead to an increase in protein S-glutathionylation that can affect the structure and function of proteins. Protein S-glutathionylation serves as a regulatory mechanism during cellular oxidative stress. Though GSSG is commercially available, its roles in various GSSG-associated normal/abnormal physiological functions have not been fully delineated due to the reason that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed cationic liposomes that can effectively deliver GSSG into cells. Various concentrations of GSSG liposomes can be conveniently prepared. At 1 mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds (n=3) in 4 h and led to a significant increase in protein S-glutathionylation confirming that the increased GSSG is functionally effective. The Trypan blue assay demonstrated that GSSG liposomes were not cytotoxic; the cell viability was greater than 95% after cells were treated with the GSSG liposomes for 4 h. A stability study showed that the dry form of the GSSG liposomes were stable for at least 70 days when stored at ?80 °C. Our data demonstrate that the GSSG liposomes can be a valuable tool in studying GSSG-associated physiological/pathological functions.     

Mechanism investigation for poloxamer 188 raw material variation in cell culture

Variability in poloxamer 188 (P188) raw material, which is routinely used in cell culture media to protect cells from hydrodynamic forces, plays an important role in the process performance. Even though tremendous efforts have been spent to understand the mechanism of poloxamer's protection, the root cause for lot‐to‐lot variation was not clear. A recent study reported that the low performance was not due to toxicity but inefficiency to protect cells (Peng et al., Biotechnol Prog. 2014;30:1411–1418). In this study, it was demonstrated for the first time that the addition of other surfactants even at a very low level can interfere with P188 resulting in a loss of efficiency. It was also found that the performance of P188 lots correlated well with its foam stability. Foam generated from low performing lots in baffled shaker flask lasts longer, which suggests that the components in the foam layers are different. The spiking of foam generated from a low performing lot into the media containing a high performance lot resulted in cell damage and low growth. Analytical studies using size exclusion chromatography (SEC) identified differences in high molecular weight (HMW) species present in the P188 lots. These differences are much clearer when comparing the HMW region of the SEC chromatogram of foam vs. bulk liquid samples. This study shows that low performing lots have enriched HMW species in foam samples due to high hydrophobicity, which can be potentially used as a screening assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:767–775, 2016     

Initial Application of Diffusional Kurtosis Imaging in Evaluating Brain Development of Healthy Preterm Infants

ObjectiveTo explore the parametric characteristics of diffusional kurtosis imaging (DKI) in the brain development of healthy preterm infants.ResultsMK and FA values were positively correlated with PMA in most selected WM regions, such as the posterior limbs of the internal capsule (PLIC) and the splenium of the corpus callosum (SCC). The positive correlation between MK value and PMA in the deep GM region was higher than that between FA and PMA. The MK value gradually decreased from the PLIC to the cerebral lobe. In addition, DKI parameters exhibited subtle differences in the parietal WM between the preterm and term control groups.ConclusionsMK may serve as a more reliable imaging marker of the normal myelination process and provide a more robust characterization of deep GM maturation.     

Fine Mapping and Candidate Gene Analysis of the Leaf-Color Gene ygl-1 in Maize

A novel yellow-green leaf mutant yellow-green leaf-1 (ygl-1) was isolated in self-pollinated progenies from the cross of maize inbred lines Ye478 and Yuanwu02. The mutant spontaneously showed yellow-green character throughout the lifespan. Meanwhile, the mutant reduced contents of chlorophyll and Car, arrested chloroplast development and lowered the capacity of photosynthesis compared with the wild-type Lx7226. Genetic analysis revealed that the mutant phenotype was controlled by a recessive nuclear gene. The ygl-1 locus was initially mapped to an interval of about 0.86 Mb in bin 1.01 on the short arm of chromosome 1 using 231 yellow-green leaf individuals of an F₂ segregating population from ygl-1/Lx7226. Utilizing four new polymorphic SSR markers, the ygl-1 locus was narrowed down to a region of about 48 kb using 2930 and 2247 individuals of F₂ and F₃ mapping populations, respectively. Among the three predicted genes annotated within this 48 kb region, GRMZM2G007441, which was predicted to encode a cpSRP43 protein, had a 1-bp nucleotide deletion in the coding region of ygl-1 resulting in a frame shift mutation. Semi-quantitative RT-PCR analysis revealed that YGL-1 was constitutively expressed in all tested tissues and its expression level was not significantly affected in the ygl-1 mutant from early to mature stages, while light intensity regulated its expression both in the ygl-1 mutant and wild type seedlings. Furthermore, the mRNA levels of some genes involved in chloroplast development were affected in the six-week old ygl-1 plants. These findings suggested that YGL-1 plays an important role in chloroplast development of maize.     

Impacts of temperature on giant panda habitat in the north Minshan Mountains

Understanding the impacts of meteorological factors on giant pandas is necessary for future conservation measures in response to global climate change. We integrated temperature data with three main habitat parameters (elevation, vegetation type, and bamboo species) to evaluate the influence of climate change on giant panda habitat in the northern Minshan Mountains using a habitat assessment model. Our study shows that temperature (relative importance = 25.1%) was the second most important variable influencing giant panda habitat excepting the elevation. There was a significant negative correlation between temperature and panda presence (ρ = −0.133, P < 0.05), and the temperature range preferred by giant pandas within the study area was 18–21°C, followed by 15–17°C and 22–24°C. The overall suitability of giant panda habitats will increase by 2.7%, however, it showed a opposite variation patterns between the eastern and northwestern region of the study area. Suitable and subsuitable habitats in the northwestern region of the study area, which is characterized by higher elevation and latitude, will increase by 18007.8 hm² (9.8% habitat suitability), while the eastern region will suffer a decrease of 9543.5 hm² (7.1% habitat suitability). Our results suggest that increasing areas of suitable giant panda habitat will support future giant panda expansion, and food shortage and insufficient living space will not arise as problems in the northwest Minshan Mountains, which means that giant pandas can adapt to climate change, and therefore may be resilient to climate change. Thus, for the safety and survival of giant pandas in the Baishuijiang Reserve, we propose strengthening the giant panda monitoring program in the west and improving the integrity of habitats to promote population dispersal with adjacent populations in the east.     

The Effects of Intra-Aortic Balloon Pumps on Mortality in Patients Undergoing High-Risk Coronary Revascularization: A Meta-Analysis of Randomized Controlled Trials of Coronary Artery Bypass Grafting and Stenting Era

Background

Intra-aortic balloon pumps (IABP) have generally been used for patients undergoing high-risk mechanical coronary revascularization. However, there is still insufficient evidence to determine whether they can improve outcomes in reperfusion therapy patients, mainly by percutaneous coronary intervention (PCI) with stenting or coronary artery bypass graft (CABG). This study was designed to determine the difference between high-risk mechanical coronary revascularization with and without IABPs on mortality, by performing a meta-analysis on randomized controlled trials of the current era.

Methods

Pubmed and Embase databases were searched from inception to May 2015. Unpublished data were obtained from the investigators. Randomized clinical trials of IABP and non-IABP in high-risk coronary revascularization procedures (PCI or CABG) were included. In the case of PCI procedures, stents should be used in more than 80% of patients. Numbers of events at the short-term and long-term follow-up were extracted.

Results

A total of 12 randomized trials enrolling 2155 patients were included. IABPs did not significantly decrease short-term mortality (relative risk (RR) 0.66; 95% CI, 0.42–1.01), or long-term mortality (RR 0.79; 95% CI, 0.47–1.35), with low heterogeneity across the studies. The findings remained stable in patients with acute myocardial infarction with or without cardiogenic shock. But in high-risk CABG patients, IABP was associated with reduced mortality (71 events in 846 patients; RR 0.40; 95%CI 0.25–0.67).

Conclusion

In patients undergoing high-risk coronary revascularization, IABP did not significantly decrease mortality. But high-risk CABG patients may be benefit from IABP. Rigorous criteria should be applied to the use of IABPs.     

疏花水柏枝幼苗生物量与构件对模拟土壤地下水位变化的响应

通过模拟不同地下水位的方法,对疏花水柏枝（Myricaria laxiflora（Franch.）P.Y.Zhang et Y.J.Zhang）一年生幼苗在不同条件下地上与地下部生物量及构件的变化进行测定,分析幼苗生长对地下水位变化的响应。结果显示：随着地下水位的降低,疏花水柏枝幼苗的生长特征指标均呈先增加后减少的趋势,其中地上、地下部分生物量的最高值分别为0.0438、0.0100 g,最低值分别为0.0177、0.0026 g。幼苗地上部生物量在-10 cm处理水平最高;地下部生物量在-15 cm处理水平最高。幼苗直径、根表面积、株高、主根长度、根体积、一级枝数、二级枝数等指标也分别在-10 cm或-15 cm处理水平达到最高值。疏花水柏枝幼苗主要构件的生长与地下水位的变化存在显著相关性。主成分分析结果表明,幼苗的地下部分更容易受到土壤地下水位变化的影响,幼苗性状症候群随地下水位的变化而发生移动,说明该物种幼苗在不同地下水位时的生长投资策略具有较大差异。