Application of enzyme-linked immunosorbent assay for mass examination of strongyloidiasis in okinawa, Japan

The enzyme-linked immunosorbent assay was tested to evaluate whether it could be applicable in screening for mass examination of strongyloidiasis. A total of 2906 inhabitants in three areas (858 in Gushikawa Village, 849 in Nakazato Village and 1199 in Sashiki Town) were screened by the enzymatic assay and approximately 11–30% (11.8% in Gushikawa, 17.0% in Nakazato and 27.7% in Sashiki) were considered to be antibody positive. In the parasitological follow-up examinations of those who were antibody positive, actual infection was found in more than half (51%) the subjects. The overall infection rates estimated from the results reached 5.8% in Gushikawa, 9.1% in Nakazato and 14.0% in Sashiki (mean = 10.4%). The infection rates were significantly higher than those in previous surveys conducted in the same areas. The ELISA technique was found to be useful for strongyloidiasis screening and for seroepidemiological purposes in Okinawa.     

Evaluating long-term cellular effects of the arsenic species thio-DMAV: qPCR-based gene expression as screening tool

Thio-dimethylarsinic acid (thio-DMA^V) is a human urinary metabolite of the class 1 human carcinogen inorganic arsenic as well as of arsenosugars. Thio-DMA^V exerts strong cellular toxicity, whereas its toxic modes of action are not fully understood. For the first time, this study characterises the impact of a long-term (21 days) in vitro incubation of thio-DMA^V on the expression of selected genes related to cell death, stress response, epigenetics and DNA repair. The observed upregulation of DNMT1 might be a cellular compensation to counterregulate the in a very recent study observed massive global DNA hypomethylation after chronic thio-DMA^V incubation. Moreover, our data suggest that chronic exposure towards subcytotoxic, pico- to nanomolar concentrations of thio-DMA^V causes a stress response in human urothelial cells. The upregulation of genes encoding for proteins of DNA repair (Apex1, Lig1, XRCC1, DDB2, XPG, ATR) as well as damage response (GADD45A, GADD45G, Trp53) indicate a potential genotoxic risk emanating from thio-DMA^V after long-term incubation.     

Ancient Cytokines,the Role of Astakines as Hematopoietic Growth Factors

Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. Vertebrate prokineticins (PKs) are known to take part in this process, as are the invertebrate prokineticin domain proteins, astakines. In Pacifastacus leniusculus, astakine 1 is essential for the release of new hemocytes into the open circulatory system of these animals. In addition to astakine 1, we have now cloned a homologue of astakine 1 with an insert of 13 amino acids, named as astakine 2. Both crustacean astakines lack the N-terminal AVIT motif, which is present in vertebrate PKs, and hence receptor binding differs from that of vertebrate PKs. We have found astakine-like sequences in 19 different invertebrate species, and the sequences show that some motifs are conserved among invertebrate groups. Previously we showed that astakine 1 is directly involved in hematopoiesis, and now we show that astakine 1 and astakine 2 have different roles in hemocyte lineage differentiation. Astakine 1 can stimulate proliferation of hematopoietic tissue (Hpt) cells (precursor of hemocytes) as well as specifically induce differentiation of Hpt cells along the semigranular cell lineage, whereas astakine 2 plays a role in granular cell differentiation. Moreover, we discuss the impact of the putative structures of different astakines in comparison with the vertebrate prokineticins.     

Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

Parkinson’s disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by α-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z′ factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD.     

In silico and pharmacological screenings identify novel serine racemase inhibitors

d-Serine is a coagonist of the N-methyl-d-aspartate (NMDA)-type glutamate receptor and its biosynthesis is catalyzed by serine racemase (SR). The overactivation of the NMDA receptor has been implicated in the development of neurodegenerative diseases, strokes, and epileptic seizures, thus, the inhibitors of SR have potential against these pathological states. Here, we have developed novel inhibitors of SR by in silico screening and in vitro enzyme assay. The newly developed inhibitors have lower IC₅₀ value comparing with that of malonate, one of the standard SR inhibitor. The structural features of novel inhibitors suggest the importance of central amide structure having a phenoxy substituent in their structure for the SR inhibitory activity. The present findings suggest the importance and rational development of new drugs for diseases of NMDAR overactivation.     

Cis-regulatory characterization of sequence conservation surrounding the Hox4 genes

Hox genes are key regulators of anterior-posterior axis patterning and have a major role in hindbrain development. The zebrafish Hox4 paralogs have strong overlapping activities in hindbrain rhombomeres 7 and 8, in the spinal cord and in the pharyngeal arches. With the aim to predict enhancers that act on the hoxa4a, hoxb4a, hoxc4a and hoxd4a genes, we used sequence conservation around the Hox4 genes to analyze all fish:human conserved non-coding sequences by reporter assays in stable zebrafish transgenesis. Thirty-four elements were functionally tested in GFP reporter gene constructs and more than 100 F1 lines were analyzed to establish a correlation between sequence conservation and cis-regulatory function, constituting a catalog of Hox4 CNEs. Sixteen tissue-specific enhancers could be identified. Multiple alignments of the CNEs revealed paralogous cis-regulatory sequences, however, the CNE sequence similarities were found not to correlate with tissue specificity. To identify ancestral enhancers that direct Hox4 gene activity, genome sequence alignments of mammals, teleosts, horn shark and the cephalochordate amphioxus, which is the most basal extant chordate possessing a single prototypical Hox cluster, were performed. Three elements were identified and two of them exhibited regulatory activity in transgenic zebrafish, however revealing no specificity. Our data show that the approach to identify cis-regulatory sequences by genome sequence alignments and subsequent testing in zebrafish transgenesis can be used to define enhancers within the Hox clusters and that these have significantly diverged in their function during evolution.     

Isolation of polyhydroxyalkanoate-producing bacteria from an integrated-farming pond and palm-oil mill effluent ponds

Bacterial isolates from two environments, an integrated-farming pond in the university and palm-oil mill effluent (POME) ponds at a local palm-oil-processing factory, were screened for polyhydroxyalkanoates (PHAs). Initially Sudan Black B staining was performed to detect lipid cellular inclusions. Lipid-positive isolates were then grown in a nitrogen-limiting medium containing 2% (w/v) glucose to promote accumulation of PHA before the subsequent Nile Blue A staining. The PHA extracted from positive isolates was confirmed by nuclear magnetic resonance (NMR) spectroscopy. The proportion of PHA-positive bacterial isolates was higher in the POME ponds compared to the integrated-farming pond.     

A screening campaign in sea urchin egg homogenate as a platform for discovering modulators of NAADP-dependent Ca2+ signaling in human cells

The Ca²⁺ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. Targeting NAADP-evoked Ca²⁺ signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. To aid discovery of novel scaffolds that modulate NAADP-evoked Ca²⁺ signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign. Known pharmacological inhibitors of NAADP-evoked Ca²⁺ release (but not cADPR- or IP₃-evoked Ca²⁺ release) in this invertebrate system strongly correlated with inhibition of MERS-pseudovirus infectivity in a human cell line. A primary screen of 1534 compounds yielded eighteen ‘hits’ exhibiting >80% inhibition of NAADP-evoked Ca²⁺ release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca²⁺ release without depleting acidic Ca²⁺ stores in a human cell line. These candidates displayed a similar penetrance of inhibition in both the sea urchin system and the human cell line, and the extent of inhibition of NAADP-evoked Ca²⁺ signals correlated well with observed inhibition of infectivity of a Middle East Respiratory syndrome coronavirus (MERS-CoV) pseudovirus. These experiments support the potential of this simple, homogenate system for screening campaigns to discover modulators of NAADP, cADPR and IP₃-dependent Ca²⁺ signaling with potential therapeutic value.     

The Drosophila Platelet-derived Growth Factor and Vascular Endothelial Growth Factor-Receptor Related (Pvr) Protein Ligands Pvf2 and Pvf3 Control Hemocyte Viability and Invasive Migration

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) family members are essential and evolutionary conserved determinants of blood cell development and dispersal. In addition, VEGFs are integral to vascular growth and permeability with detrimental contributions to ischemic diseases and metastatic cancers. The PDGF/VEGF-receptor related (Pvr) protein is implicated in the migration and trophic maintenance of macrophage-like hemocytes in Drosophila melanogaster embryos. pvr mutants have a depleted hemocyte population and a breakdown in hemocyte distribution. Previous studies suggested redundant functions for the Pvr ligands, Pvf2 and Pvf3 in the regulation of hemocyte migration, proliferation, and size. However, the precise roles that Pvf2 and Pvf3 play in hematopoiesis remain unclear due to the lack of available mutants. To determine Pvf2 and Pvf3 functions in vivo, we generated a genomic deletion that simultaneously disrupts Pvf2 and Pvf3. From our studies, we identified contributions of Pvf2 and Pvf3 to the Pvr trophic maintenance of hemocytes. Furthermore, we uncovered a novel role for Pvfs in invasive migrations. We showed that Pvf2 and Pvf3 are not required for the directed migration of hemocytes, but act locally in epithelial cells to coordinate trans-epithelial migration of hemocytes. Our findings redefine Pvf roles in hemocyte migration and highlight novel Pvf roles in hemocyte invasive migration. These new parallels between the Pvr and PDGF/VEGF pathways extend the utility of the Drosophila embryonic system to dissect physiological and pathological roles of PDGF/VEGF-like growth factors.