Tracking the Primary Sources of Fecal Pollution in a Tropical Watershed in a One-Year Study

A study was conducted to determine the primary sources of fecal pollution in a subtropical watershed using host-specific assays developed in temperate regions. Water samples (n = 534) from 10 different sites along the Rio Grande de Arecibo (RGA) watershed were collected mostly on a weekly basis (54 sampling events) during 13 months. DNA extracts from water samples were used in PCR assays to determine the occurrence of fecal bacteria (Bacteroidales, Clostridium coccoides, and enterococci) and human-, cattle-, swine-, and chicken-specific fecal sources. Feces from 12 different animals (n = 340) and wastewater treatment samples (n = 16) were analyzed to determine the specificity and distribution of host-specific assays. The human-specific assay (HF183) was found to be highly specific, as it did not cross-react with nontarget samples. The cattle marker (CF128) cross-reacted to some extent with swine, chicken, and turkeys and was present in 64% of the cattle samples tested. The swine assays showed poor host specificity, while the three chicken assays showed poor host distribution. Differences in the detection of host-specific markers were noted per site. While human and cattle assays showed moderate average detection rates throughout the watershed, areas impacted by wastewater treatment plants and cattle exhibited the highest prevalence of these markers. When conditional probability for positive signals was determined for each of the markers, the results indicated higher confidence levels for the human assay and lower levels for all the other assays. Overall, the results from this study suggest that additional assays are needed, particularly to track cattle, chicken, and swine fecal pollution sources in the RGA watershed. The results also suggest that the geographic stability of genetic markers needs to be determined prior to conducting applied source tracking studies in tropical settings.     

Sequence polymorphisms in Pvs48/45 and Pvs47 gametocyte and gamete surface proteins in Plasmodium vivax isolated in Korea

Nucleotide sequence analyses of the Pvs48/45 and Pvs47 genes were conducted in 46 malaria patients from the Republic of Korea (ROK) (n = 40) and returning travellers from India (n = 3) and Indonesia (n = 3). The domain structures, which were based on cysteine residue position and secondary protein structure, were similar between Plasmodium vivax (Pvs48/45 and Pvs47) and Plasmodium falciparum (Pfs48/45 and Pfs47). In comparison to the Sal-1 reference strain (Pvs48/45, PVX_083235 and Pvs47, PVX_083240), Korean isolates revealed seven polymorphisms (E35K, H211N, K250N, D335Y, A376T, I380T and K418R) in Pvs48/45. These isolates could be divided into five haplotypes with the two major types having frequencies of 47.5% and 20%, respectivelfy. In Pvs47, 10 polymorphisms (F22L, F24L, K27E, D31N, V230I, M233I, E240D, I262T, I273M and A373V) were found and they could be divided into four haplotypes with one major type having a frequency of 75%. The Pvs48/45 isolates from India showed a unique amino acid substitution site (K26R). Compared to the Sal-1 and ROK isolates, the Pvs47 isolates from travellers returning from India and Indonesia had amino acid substitutions (S57T and I262K). The current data may contribute to the development of the malaria transmission-blocking vaccine in future clinical trials.     

Lineage and Genogroup-Defining Single Nucleotide Polymorphisms of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic human pathogen for which cattle are an important reservoir host. Using both previously published and new sequencing data, a 48-locus single nucleotide polymorphism (SNP)-based typing panel was developed that redundantly identified 11 genogroups that span six of the eight lineages recently described for E. coli O157:H7 (J. L. Bono, T. P. Smith, J. E. Keen, G. P. Harhay, T. G. McDaneld, R. E. Mandrell, W. K. Jung, T. E. Besser, P. Gerner-Smidt, M. Bielaszewska, H. Karch, M. L. Clawson, Mol. Biol. Evol. 29:2047–2062, 2012) and additionally defined subgroups within four of those lineages. This assay was applied to 530 isolates from human and bovine sources. The SNP-based lineage groups were concordant with previously identified E. coli O157:H7 genotypes identified by other methods and were strongly associated with carriage of specific Stx genes. Two SNP lineages (Ia and Vb) were disproportionately represented among cattle isolates, and three others (IIa, Ib, and IIb) were disproportionately represented among human clinical isolates. This 48-plex SNP assay efficiently and economically identifies biologically relevant lineages within E. coli O157:H7.     

Molecular Detection of Campylobacter spp. and Fecal Indicator Bacteria during the Northern Migration of Sandhill Cranes (Grus canadensis) at the Central Platte River

The risk to human health of the annual sandhill crane (Grus canadensis) migration through Nebraska, which is thought to be a major source of fecal pollution of the central Platte River, is unknown. To better understand potential risks, the presence of Campylobacter species and three fecal indicator bacterial groups (Enterococcus spp., Escherichia coli, and Bacteroidetes) was assayed by PCR from crane excreta and water samples collected during their stopover at the Platte River, Nebraska, in 2010. Genus-specific PCR assays and sequence analyses identified Campylobacter jejuni as the predominant Campylobacter species in sandhill crane excreta. Campylobacter spp. were detected in 48% of crane excreta, 24% of water samples, and 11% of sediment samples. The estimated densities of Enterococcus spp. were highest in excreta samples (mean, 4.6 × 10⁸ cell equivalents [CE]/g), while water samples contained higher levels of Bacteroidetes (mean, 5.1 × 10⁵ CE/100 ml). Enterococcus spp., E. coli, and Campylobacter spp. were significantly increased in river water and sediments during the crane migration period, with Enterococcus sp. densities (∼3.3 × 10⁵ CE/g) 2 to 4 orders of magnitude higher than those of Bacteroidetes (4.9 × 10³ CE/g), E. coli (2.2 × 10³ CE/g), and Campylobacter spp. (37 CE/g). Sequencing data for the 16S rRNA gene and Campylobacter species-specific PCR assays indicated that C. jejuni was the major Campylobacter species present in water, sediments, and crane excreta. Overall, migration appeared to result in a significant, but temporary, change in water quality in spring, when there may be a C. jejuni health hazard associated with water and crops visited by the migrating birds.     

PEGylated human catalase elicits potent therapeutic effects on H1N1 influenza-induced pneumonia in mice

Therapeutic recombinant human catalase (rhCAT) can quench infection-induced reactive oxygen species (ROS), thereby alleviating the associated tissue damage. Although the intranasal route is efficient to deliver native rhCAT to the lung, the therapeutic effect is limited by rapid elimination from the blood. In this study, we modified rhCAT with the active polymer, polyethylene glycol monomethyl ether (PEG)-5000, and analyzed the pharmacokinetics of PEGylated rhCAT in mice. The high tetra-PEGylation ratio was about 60 %, and PEGylation prolonged the half-life of rhCAT in serum (75 vs. 13.5 min for native rhCAT). The protective effects of PEG-rhCAT were investigated in a mouse model of influenza virus A (H1N1)-associated pneumonia. PEG-rhCAT was more effectively delivered than native rhCAT and was associated with higher survival ratio, less extensive lung injuries, reduced ROS levels, and lower viral replication. Collectively, these findings indicate that PEGylation can enhance the therapeutic efficacy of native rhCAT and suggest that PEGylated rhCAT may represent a novel complement therapy for H1N1 influenza-induced pneumonia.     

δ-catenin overexpression promotes angiogenic potential of CWR22Rv-1 prostate cancer cells via HIF-1α and VEGF

This study revealed that CWR22Rv-1 cells overexpressing δ-catenin display bigger tumor formation and higher angiogenic potentials than their matched control cells in the CAM assay. In addition, δ-catenin overexpression in CWR22Rv-1 cells results in increased hypoxia-inducible factor 1-alpha (HIF-1α and vascular endothelial growth factor (VEGF) expression. Furthermore, δ-catenin overexpression was found to enhance nuclear distribution of both β-catenin and HIF-1α in hypoxic condition, which is diminished by knockdown of δ-catenin. Our current study adds novel evidence regarding contribution of δ-catenin to the progression of prostate cancer.     

SHORT Syndrome with Partial Lipodystrophy Due to Impaired Phosphatidylinositol 3 Kinase Signaling

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.