Binding site on human C-reactive Protein (CRP) recognized by the Leukocyte CRP-receptor

C-reactive protein (CRP), the prototypical inflammatory acute phase reactant in humans, interacts with monocytes and neutrophils via a specific receptor. To map the site on CRP recognized by the CRP receptor (CRP-R), synthetic peptides corresponding to the surface region on each of the five identical subunits were tested as competitors vs. [¹²⁵l]-CRP for cell binding. A peptide of residues 27–38 (TKPLKAFTVCLH) efficiently inhibited CRP binding when compared to other nonoverlapping peptides. This peptide was termed the cell-binding peptide (CB-Pep). The F(ab′)₂ of an IgG Ab to the CB-Pep specifically inhibited CRP binding upon reacting with the ligand. Competitive binding studies with synthetic peptides truncated from either the NH₂- or COOH-terminus of the CB-Pep revealed that the minimum length recognized by the CRP-R consisted of residues 31–36: KAFTVC. Conservative substitutions of residues within the CB-Pep indicated that the four residues AFTV were critical for CRP-R binding. The CB-Pep also inhibited induced superoxide generation by HL-60 granulocytes. The minimum length required for the inhibition was also KAFTVC; however, only Phe-33 and Leu-37 were critical residues in this assay. Anti-CB-Pep IgG Ab reacted more extensively with heat-modified CRP, suggesting that an altered conformation of CRP is preferentially recognized by the CRP-R. The results suggest that this contiguous sequence on a β-strand on one face of each of five subunits of the CRP pentamer serves as a unique recognition motif for inflammatory leukocytes. J. Cell. Biochem. 64:140–151. © 1997 Wiley-Liss, Inc.     

Hourly Activity and Natural Infection of Sandflies (Diptera: Psychodidae) Captured from the Aphotic Zone of a Cave,Minas Gerais State,Brazil

Sandflies are holometabolous insects that are of great epidemiological importance in the neotropical region as vectors of leishmaniases. Caves are ecotopes that significantly differ from external environments and, among the insects that live or visit their internal area and adjacent environment, sandflies are commonly found. Based on this context, the objective of this work was to examine the period of activity of sandflies in the cave environment in the aphotic zone. Thus, four sandfly captures were conducted, one in each season of the year, in a cave where studies on the bioecological aspects of sandfly fauna have been conducted since 2008. In this same study, we have also noticed the presence of flagellates in some captured females. Catches were carried out for 24 hours using a Shannon trap, light bait, and cave walls were actively searched. We collected a total of 638 sandflies, representing 11 species. The most abundant species and with more intense period of activity were, in descending order: Lu. cavernicola (62%), Ev. spelunca (16%) and Ev. sallesi (14%). A total of 69 females were dissected to check for natural infection, and in five specimens we found living flagellated forms: two Ev. spelunca, two Ev. sallesi and one Sc. sordellii. This study shows that the activity of some species caught in the aphotic zone of the cave, especially Lu. cavernicola, differs from what has already been reported in previous sandfly captures, which are almost always conducted at night and during twilight. The existence of sandflies that were naturally infected with flagellates and the lack of awareness regarding the behaviour of sandflies in cave environments are strong indicators of the need for further study on this group of insects in this ecotope, as a safety measure to protect the visitors of such environment.     

Surveillance of fish species composition using environmental DNA

Prompt and accurate methods for assessing the species composition of given areas are indispensable in addressing the rapid loss of biodiversity. Here, we propose a method for the surveillance of fish species composition in freshwater using environmental DNA as species markers. First, the applicability of the method was demonstrated through aquarium experiments. DNA was extracted from 120?ml aquarium water, and the degenerated primers targeting the fish mitochondrial cytochrome b gene were used for amplification. PCR-amplified fragments were analysed by random cloning, and all species reared in the aquarium were detected. Next, this method was applied to natural freshwater environments. Water samples were collected from three sites in the Yura River, Japan; DNA was concentrated from 2?l of environmental water, and then amplified and cloned. Up to four species of fish were detected by sequencing 47 randomly selected clones from a single water sample. Overall, the results were consistent with previous knowledge of fish habitat utilisation. Using this method, the surveillance of fish species composition can be conducted less laboriously than with traditional methods.     

FAD mutants unable to increase neurotoxic Abeta 42 suggest that mutation effects on neurodegeneration may be independent of effects on Abeta

Strong support for a primary causative role of the Abeta peptides in the development of Alzheimer's disease (AD) neurodegeneration derives from reports that presenilin familial AD (FAD) mutants alter amyloid precursor protein processing, thus increasing production of neurotoxic Abeta 1-42 (Abeta 42). This effect of FAD mutants is also reflected in an increased ratio of peptides Abeta 42 over Abeta 1-40 (Abeta 40). In the present study, we show that several presenilin 1 FAD mutants failed to increase production of Abeta 42 or the Abeta 42/40 ratio. Our data suggest that the mechanism by which FAD mutations promote neurodegeneration and AD may be independent of their effects on Abeta production.     

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

Background

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.

Results

The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.

Conclusion

This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.     

Identification of Ebola virus microRNAs and their putative pathological function

Ebola virus(EBOV),a member of the filovirus family,is an enveloped negative-sense RNA virus that causes lethal infections in humans and primates.Recently,more than 1000 people have been killed by the Ebola virus disease in Africa,yet no specific treatment or diagnostic tests for EBOV are available.In this study,we identified two putative viral microRNA precursors(pre-miRNAs)and three putative mature microRNAs(miRNAs)derived from the EBOV genome.The production of the EBOV miRNAs was further validated in HEK293T cells transfected with a pcDNA6.2-GW/EmGFP-EBOV-pre-miRNA plasmid,indicating that EBOV miRNAs can be produced through the cellular miRNA processing machinery.We also predicted the potential target genes of these EBOV miRNAs and their possible biological functions.Overall,this study reports for the first time that EBOV may produce miRNAs,which could serve as non-invasive biomarkers for the diagnosis and prognosis of EBOV infection and as therapeutic targets for Ebola viral infection treatment.