Diversity of trypanosomes in humans and cattle in the HAT foci Mandoul and Maro,Southern Chad—A matter of concern for zoonotic potential?

BackgroundAfrican trypanosomes are parasites mainly transmitted by tsetse flies. They cause trypanosomiasis in humans (HAT) and animals (AAT). In Chad, HAT/AAT are endemic. This study investigates the diversity and distribution of trypanosomes in Mandoul, an isolated area where a tsetse control campaign is ongoing, and Maro, an area bordering the Central African Republic (CAR) where the control had not started.Methods717 human and 540 cattle blood samples were collected, and 177 tsetse flies were caught. Trypanosomal DNA was detected using PCR targeting internal transcribed spacer 1 (ITS1) and glycosomal glyceraldehyde-3 phosphate dehydrogenase (gGAPDH), followed by amplicon sequencing.ResultsTrypanosomal DNA was identified in 14 human samples, 227 cattle samples, and in tsetse. Besides T. b. gambiense, T. congolense was detected in human in Maro. In Mandoul, DNA from an unknown Trypanosoma sp.-129-H was detected in a human with a history of a cured HAT infection and persisting symptoms. In cattle and tsetse samples from Maro, T. godfreyi and T. grayi were detected besides the known animal pathogens, in addition to T. theileri (in cattle) and T. simiae (in tsetse). Furthermore, in Maro, evidence for additional unknown trypanosomes was obtained in tsetse. In contrast, in the Mandoul area, only T. theileri, T. simiae, and T. vivax DNA was identified in cattle. Genetic diversity was most prominent in T. vivax and T. theileri.ConclusionTsetse control activities in Mandoul reduced the tsetse population and thus the pathogenic parasites. Nevertheless, T. theileri, T. vivax, and T. simiae are frequent in cattle suggesting transmission by other insect vectors. In contrast, in Maro, transhumance to/from Central African Republic and no tsetse control may have led to the high diversity and frequency of trypanosomes observed including HAT/AAT pathogenic species. Active HAT infections stress the need to enforce monitoring and control campaigns. Additionally, the diverse trypanosome species in humans and cattle indicate the necessity to investigate the infectivity of the unknown trypanosomes regarding their zoonotic potential. Finally, this study should be widened to other trypanosome hosts to capture the whole diversity of circulating trypanosomes.     

一株油脂降解菌的筛选鉴定及降解效果分析

为了筛选分离得到一株具有油脂降解能力的菌株,同时探究菌株的特性和降解能力。以屠宰场污染土作为菌源,通过梯度驯化法最终筛选分离得到能够将橄榄油作为单一碳源生长的降解菌。随后通过形态特征观察、Biolog生理生化测试以及16S rRNA基因序列比对分析鉴定,实验菌株为革兰氏阴性菌,属于无色杆菌属（Achromobacter sp.）,在构建的系统发育树上与Achromobacter pulmonis聚为一支。综合运用紫外分光光度法和高效液相色谱法检测,测得实验菌株培养4~5 d时对橄榄油的降解率可以达到90%,同时测得菌株降解油脂的最适pH和最适温度分别为7.5和35 ℃,该菌株在盐浓度低于40 g·L-1环境中降解率较高。此外实验结果表明,实验菌株对各类型油脂均具有较高的降解效率,具有广泛的应用前景。     

肠出血性大肠杆菌O157∶H7 EspA和EspB蛋白的重组表达及其免疫效果

目的：通过DNA重组技术表达肠出血性大肠杆菌（EHEC）0157：H7的EspA和EspB蛋白,并分析它们的免疫保护性。方法：采用PCR技术从EHEC0157：H7基因组中扩增espA和espB基因,连接至pET-22b（4-）载体上,转化至宿主细胞大肠杆菌BL21（DE3）,经IPTG诱导表达,用亲和层析纯化目的蛋白,SDS-PAGE测定其相对分子质量,免疫小鼠分析其免疫保护性。结果：重组espA和espB基因片段的测序结果与GenBank中的相应基因序列完全一致,一致性均为100％;得到了纯度为95％以上的重组EspA和EspB蛋白,免疫小鼠所得到的抗体效价均为10^6。结论：重组EspA和EspB蛋白获得了可溶性表达,表达的蛋白具有良好的免疫保护性,为进一步制备疫苗奠定了基础。     

Combination of Ascorbate and α-Tocopherol as a Preventive Therapy against Structural and Functional Defects of Erythrocytes in Visceral Leishmaniasis

The redox unbalance in erythrocytes has been found to contribute significantly in the development of anemia in visceral leishmaniasis (VL). The present study revealed enhanced production of reactive oxygen species (ROS) and gradual depletion of α-tocopherol and ascorbate in the erythrocytes of infected animals. The response of erythrocytes to chronic treatment with antioxidants was studied in hamsters during leishmanial infection. Treatment with a combination of α-tocopherol and ascorbate proved to be the most effective preventive for the proteolytic degradation of erythrocyte membrane. Erythrocytes from infected animals were thermally more sensitive compared to the control ones. Combination of both antioxidants was most successful in resisting heat induced structural defects in the cells. Cross-linking of membrane proteins subsequent to oxidative damage in the red cells was accompanied by the formation of high molecular weight protein band at the top of the resolving gel in the presence of the cross-linking agent dimethyladepimidate (DMA). Marked inhibition of cross-linking was observed with combination of both antioxidants. Treatment with α-tocopherol and ascorbate together could withstand osmotic lysis of erythrocytes in the infected animals very efficiently. Decreased hemoglobin (Hb) level was successfully replenished and was coupled with significant increase in the life span of red cells after treating the animals with both antioxidants. Results indicate better efficacy of the combination therapy with α-tocopherol and ascorbate in protecting the erythrocytes from structural and functional damages during leishmanial infection.     

Dickkopf-1 Is Oncogenic and Involved in Invasive Growth in Non Small Cell Lung Cancer

Dickkopf-1 (DKK1) is an inhibitor of the Wnt/β-catenin signaling pathway. However, the role of DKK1 in the progression of non small cell lung cancer (NSCLC) is not fully understood. In this study, RT-PCR and Western blot were used to examine the expression of DKK1 in a panel of ten human NSCLC cell lines and NSCLC tissues. DKK1 expression was highly transactivated in the great majority of these cancer lines. The expression of DKK1 was upregulated on both mRNA and protein levels in NSCLC tissues compared with the adjacent normal lung tissues. Immunohistochemistry and immunofluoresence revealed that DKK1 was mainly distributed in the cytoplasm in both carcinoma tissues and cell lines. DKK1 protein expression was also evaluated in paraffin sections from 102 patients with NSCLC by immunohistochemistry, and 65(63.73%)tumors were DKK1 positive. Relative analysis showed a significant relationship between DKK1 positive expression and lymph node metastasis(P<0.05). Patients with DKK1-positive tumors had poorer DFS than those with negative ESCC (5-year DFS; 15.4% versus 27%, P = 0.007). To further explore the biological effects of DKK1 in NSCLC cells, we over-expressed DKK1 in NSCLC 95C cell using eukaryotic expression vector pCMV-Tab-2b and performed a knockdown of DKK1 in LTEP-a-2 cell using a short hairpin RNA expression vector pSilencer 5.1. DKK1 did not have any effect on proliferation, but seemed to play a role in migration and invasion capability. Overexpression of DKK1 promotes migratory and invasive activity of 95C, while DKK1 knockdown resulted in the suppression of migration and invasion potentials of LTEP-a-2 cell. Taken together, these results indicate that DKK1 may be a crucial regulator in the progression of NSCLC. DKK1 might be a potential therapeutic target in NSCLC.     

MicroRNA-138 Regulates Hypoxia-Induced Endothelial Cell Dysfunction By Targeting S100A1

The Ca²⁺ sensor S100A1 is essential for proper endothelial cell (EC) nitric oxide (NO) synthase (eNOS) activation. S100A1 levels are greatly reduced in primary human microvascular ECs subjected to hypoxia, rendering them dysfunctional. However mechanisms that regulate S100A1 levels in ECs are unknown. Here we show that ECs transfected with a S100A1–3′ untranslated region (UTR) luciferase reporter construct display significantly reduced gene expression when subjected to low oxygen levels or chemical hypoxia. Bioinformatic analysis suggested that microRNA -138 (MiR-138) could target the 3′UTR of S100A1. Patients with critical limb ischemia (CLI) or mice subjected to femoral artery resection (FAR) displayed increased MiR-138 levels and decreased S100A1 protein expression. Consistent with this finding, hypoxia greatly increased MiR-138 levels in ECs, but not in skeletal muscle C2C12 myoblasts or differentiated myotubes or primary human vascular smooth muscle cells. Transfection of a MiR-138 mimic into ECs reduced S100A1–3 ‘UTR reporter gene expression, while transfection of an anti MiR-138 prevented the hypoxia-induced downregulation of the reporter gene. Deletion of the 22 nucleotide putative MiR-138 target site abolished the hypoxia-induced loss of reporter gene expression. Knockdown of Hif1-α mediated by siRNA prevented loss of hypoxia-induced reporter gene expression. Conversely, specific activation of Hif1-α by a selective prolyl-hydroxylase inhibitor (IOX2) reduced reporter gene expression even in the absence of hypoxia. Finally, primary ECs transfected with a MiR-138 mimic displayed reduced tube formation when plated onto Matrigel matrix and expressed less NO when stimulated with VEGF. These effects were reversed by gene transfer of S100A1 using recombinant adenovirus. We conclude that hypoxia-induced MiR-138 is an essential mediator of EC dysfunction via its ability to target the 3′UTR of S100A1.     

The Protein Kinase Double-Stranded RNA-Dependent (PKR) Enhances Protection against Disease Cause by a Non-Viral Pathogen

PKR is well characterized for its function in antiviral immunity. Using Toxoplasma gondii, we examined if PKR promotes resistance to disease caused by a non-viral pathogen. PKR^−/− mice infected with T. gondii exhibited higher parasite load and worsened histopathology in the eye and brain compared to wild-type controls. Susceptibility to toxoplasmosis was not due to defective expression of IFN-γ, TNF-α, NOS2 or IL-6 in the retina and brain, differences in IL-10 expression in these organs or to impaired induction of T. gondii-reactive T cells. While macrophages/microglia with defective PKR signaling exhibited unimpaired anti-T. gondii activity in response to IFN-γ/TNF-α, these cells were unable to kill the parasite in response to CD40 stimulation. The TRAF6 binding site of CD40, but not the TRAF2,3 binding sites, was required for PKR phosphorylation in response to CD40 ligation in macrophages. TRAF6 co-immunoprecipitated with PKR upon CD40 ligation. TRAF6-PKR interaction appeared to be indirect, since TRAF6 co-immunoprecipitated with TRAF2 and TRAF2 co-immunoprecipitated with PKR, and deficiency of TRAF2 inhibited TRAF6-PKR co-immunoprecipitation as well as PKR phosphorylation induced by CD40 ligation. PKR was required for stimulation of autophagy, accumulation the autophagy molecule LC3 around the parasite, vacuole-lysosomal fusion and killing of T. gondii in CD40-activated macrophages and microglia. Thus, our findings identified PKR as a mediator of anti-microbial activity and promoter of protection against disease caused by a non-viral pathogen, revealed that PKR is activated by CD40 via TRAF6 and TRAF2, and positioned PKR as a link between CD40-TRAF signaling and stimulation of the autophagy pathway.     

A Study of the Mechanism of the Chaperone-like Function of an scFv of Human Creatine Kinase by Computer Simulation

A new application of antibodies is to use them as macromolecular chaperones. Protein antigens usually have multiple epitopes, thus, there may be a plurality of antibodies binding to one antigen. However, not all antibodies that bind to one antigen could act as a chaperone. Experiments show that some screened anti-human creatine kinase single chain antibodies (scFV) could assist in the folding and stabilizing of the enzyme, while others could not. We built the model of the single chain antibody (scFv-A4) that increased the stability of human creatine kinase (HCK) by the homology modeling method. Epitopes of human creatine kinase were predicted by computer and then the binding of scFv-A4 and HCK was modeled with computer. The calculation results were further combined with the peptide array membrane experiment results to obtain reliable models for the scFv-A4-HCK complex. Based on the above study we gave an explanation about how scFv-A4 could act as a macromolecular chaperone assisting the folding of HCK. This study provides an approach for predicting antigen-antibody binding mode and also a useful theoretical guidance for the study of antibodies'' chaperone-like function.     

Valproic Acid Treatment Inhibits Hypoxia-Inducible Factor 1α Accumulation and Protects against Burn-Induced Gut Barrier Dysfunction in a Rodent Model

Objective

Burn-induced gut dysfunction plays an important role in the development of sepsis and multiple organ dysfunction. Emerging evidence suggests that hypoxia-inducible factor-1α (HIF-1α) is critical in paracelluar barrier functions via regulating vascular endothelial growth factor (VEGF) and myosin light chain kinase (MLCK) expression. Previous studies have also demonstrated that histone deacetylase inhibitors (HDACIs) can repress HIF-1α. This study aims to examine whether valproic acid (VPA), a HDACI, protects against burn-induced gut barrier dysfunction via repressing HIF-1α-dependent upregulation of VEGF and MLCK expression.

Methods

Rats were subjected to third degree 55% TBSA burns and treated with/ without VPA (300mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and histologic evaluation. Histone acetylation, tight junction protein zonula occludens 1 (ZO-1), VEGF, MLCK and HIF-1α were measured. In addition, CaCO₂ cells were transfected with siRNA directed against HIF-1α and were stimulated with CoCl2 (1mM) for 24 hours with/without VPA (2mM) followed by analysis of HIF-1α, MLCK, VEGF and ZO-1.

Results

Burn insults resulted in a significant increase in intestinal permeability and mucosal damage, accompanied by a significant reduction in histone acetylation, ZO-1, upregulation of VEGF, MLCK expression, and an increase in HIF-1α accumulation. VPA significantly attenuated the increase in intestinal permeability, mucosa damage, histone deacetylation and changes in ZO-1 expression. VPA also attenuated the increased VEGF, MLCK and HIF-1α protein levels. VPA reduced HIF-1α, MLCK and VEGF production and prevented ZO-1 loss in CoCl2-stimulated Caco-2 cells. Moreover, transfection of siRNA directed against HIF-1α led to inhibition of MLCK and VEGF production, accompanied by upregulation of ZO-1.

Conclusions

These results indicate that VPA can protect against burn-induced gut barrier dysfunction. These protective effects may be due to its inhibitory action on HIF-1α, leading to a reduction in intestinal VEGF and MLCK expression and minimizing ZO-1 degradation.