Symmetric array of the urothelium surface controlled by the lipid lattice composition

Proteins on the luminal surface of the mammalian membrane urothelium form an hexagonal two-dimensional (2D) lattice of approximately 12nm particles embedded in a lipid matrix. The establishment and/or maintenance of this two-dimensional lattice have not yet been elucidated. To understand the lipid involvement in these effects, which may have a potential role on the permeability barrier, the lattice structural changes induced by different dietary fatty acids were analyzed. Image analysis of the uranyl formate stained urothelial membrane from mice fed with a commercial diet (control) or a formula containing 5% (w/w) corn oil (CO) or olein (O) and our previous data of fluorescence anisotropy indicated that the olein diet profoundly alters the two-dimensional particle network. Besides the increase from 15nm (control) to 17nm center-to-center particle, a statistically significant increase of the particle size was induced by the olein diet. The same parameters on urothelial membranes from animals fed with a corn diet differed on the long-range super-array from the control values. A specific structural change of the urothelial plaque particle organization as a response to changes of lipid composition is described for the first time. These results are a starting point for further research of the lipid implications on the permeability barrier.     

Centromere binding and evolution of chromosomal partition systems in the Burkholderiales

How split genomes arise and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2, and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3, and pBC share many features, suggesting that they arose within an extended family. Database searching revealed seven subfamilies of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of the Burkholderiales, which reinforces the proposal of an extended family. The subfamily of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using electrophoretic mobility shift assay (EMSA), we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. In contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome. These observations suggest that we are witnessing a plasmid-chromosome interaction from which a third chromosome will emerge in a two-chromosome species.     

Heat shock protein production and immunity and altered fetal development in diabetic pregnant rats

We evaluated associations between the concentrations of heat shock proteins (hsp60 and hsp70) and their respective antibodies, alterations in maternal reproductive performance, and fetal malformations in pregnant rats with hyperglycemia. Mild diabetes (MD) or severe diabetes (SD) was induced in Sprague-Dawley rats prior to mating; non-treated non-diabetic rats (ND) served as controls. On day 21 of pregnancy, maternal blood was analyzed for hsp60 and hsp70 and their antibodies; and fetuses were weighed and analyzed for congenital malformations. Hsp and anti-hsp levels were correlated with blood glucose levels during gestation. There was a positive correlation between hsp60 and hsp70 levels and the total number of malformations (R = 0.5908, P = 0.0024; R = 0.4877, P = 0.0134, respectively) and the number of malformations per fetus (R = 0.6103, P = 0.0015; R = 0.4875, P = 0.0134, respectively). The anti-hsp60 IgG concentration was correlated with the number of malformations per fetus (R = 0.3887, P = 0.0451) and the anti-hsp70 IgG level correlated with the total number of malformations (R = 0.3999, P = 0.0387). Moreover, both hsp and anti-hsp antibodies showed negative correlations with fetal weight. The results suggest that there is a relationship between hsp60 and hsp70 levels and their respective antibodies and alterations in maternal reproductive performance and impaired fetal development and growth in pregnancies associated with diabetes.     

The 12th International Workshops on Opportunistic Protists (IWOP‐12)

The 12th International Workshops on Opportunistic Protists (IWOP‐12) was held in August 2012 in Tarrytown, New York. The objectives of the IWOP meetings are to: (1) serve as a forum for exchange of new information among active researchers concerning the basic biology, molecular genetics, immunology, biochemistry, pathogenesis, drug development, therapy, and epidemiology of these immunodeficiency‐associated pathogenic eukaryotic microorganisms that are seen in patients with AIDS and (2) foster the entry of new and young investigators into these underserved research areas. The IWOP meeting focuses on opportunistic protists, e.g. the free‐living amoebae, Pneumocystis, Cryptosporidium, Toxoplasma, the Microsporidia, and kinetoplastid flagellates. This conference represents the major conference that brings together research groups working on these opportunistic pathogens. Slow but steady progress is being achieved on understanding the biology of these pathogenic organisms, their involvement in disease causation in both immune‐deficient and immune‐competent hosts, and is providing critical insights into these emerging and reemerging pathogens. This IWOP meeting demonstrated the importance of newly developed genomic level information for many of these pathogens and how analysis of such large data sets is providing key insights into the basic biology of these organisms. A great concern is the loss of scientific expertise and diversity in the research community due to the ongoing decline in research funding. This loss of researchers is due to the small size of many of these research communities and a lack of appreciation by the larger scientific community concerning the state of art and challenges faced by researchers working on these organisms.     

Novel Camelid Antibody Fragments Targeting Recombinant Nucleoprotein of Araucaria hantavirus: A Prototype for an Early Diagnosis of Hantavirus Pulmonary Syndrome

In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ₈₅ by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone {"type":"entrez-nucleotide","attrs":{"text":"KC329705","term_id":"468362168","term_text":"KC329705"}}KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus infections.     

Non-invasive cytology brush PCR diagnostic testing in mucosal leishmaniasis: superior performance to conventional biopsy with histopathology

Background

Traditional methods of diagnosing mucosal leishmaniasis (ML), such as biopsy with histopathology, are insensitive and require collection of an invasive diagnostic specimen.

Methods

We compared standard invasive procedures including biopsy histopathology, biopsy PCR, and leishmanin skin test (LST) to a novel, non-invasive, cytology-brush based PCR for the diagnosis of ML in Lima, Peru. Consensus reference standard was 2/4 tests positive, and outcome measures were sensitivity and specificity. Leishmania species identification was performed by PCR-based assays of positive specimens.

Results

Twenty-eight patients were enrolled, 23 of whom fulfilled criteria for a diagnosis of ML. Sensitivity and specificity of biopsy with histopathology were 21.7% [95% CI 4.9–38.5%] and 100%; 69.6% [95% CI 50.8–88.4%] and 100% for LST; 95.7% [95% CI 87.4–100%] and 100% for biopsy PCR; and 95.7% [95% CI 87.4–100%] and 90% [95% CI 71.4–100%] for cytology brush PCR using both Cervisoft® and Histobrush® cervical cytology brushes. Represented species identified by PCR-RFLP included: L. (V). braziliensis (n = 4), and L. (V). peruviana (n = 3).

Conclusions

Use of commercial grade cytology brush PCR for diagnosis of ML is sensitive, rapid, well tolerated, and carries none of the risks of invasive diagnostic procedures such as biopsy. Further optimization is required for adequate species identification. Further evaluation of this method in field and other settings is warranted.     

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment ^1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded ². This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances ^{4, 5}. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation ^{4, 6}. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) ^7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators ^10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al⁷. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere ^{14, 15}. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. ^{16, 17}. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin ¹⁸. This approach proved to be sufficiently sensitive and reproducible. Download video file.^{(77M, mov)}     

Parasites and malignancies, a review, with emphasis on digestive cancer induced by Cryptosporidium parvum (Alveolata: Apicomplexa)

The International Agency for Research on Cancer (IARC) identifies ten infectious agents (viruses, bacteria, parasites) able to induce cancer disease in humans. Among parasites, a carcinogenic role is currently recognized to the digenetic trematodes Schistosoma haematobium, leading to bladder cancer, and to Clonorchis sinensis or Opisthorchis viverrini, which cause cholangiocarcinoma. Furthermore, several reports suspected the potential association of other parasitic infections (due to Protozoan or Metazoan parasites) with the development of neoplastic changes in the host tissues. The present work shortly reviewed available data on the involvement of parasites in neoplastic processes in humans or animals, and especially focused on the carcinogenic power of Cryptosporidium parvum infection. On the whole, infection seems to play a crucial role in the etiology of cancer.