Anti-Sporothrix Antibody Detection in Domestic Cats as an Indicator of a Possible New Occurrence Area for Sporotrichosis in North Brazil

Mycopathologia - Feline sporotrichosis has emerged as an important public health issue in some countries, especially Brazil. Currently, zoonotic transmission of Sporothrix brasiliensis by domestic...     

Use of F1 progeny of HolsteinxZebu cross cattle as oocyte donors for in vitro embryo production and gene microinjection

This study was designed to determine the possibility of using F1 crossbreed cattle (Holstein x Zebu) as donors of oocytes for in vitro fertilization (IVF) and for pronuclear gene microinjection into in vitro-produced embryos. In the first part of the experiment oocytes from Bos taurus (Holstein), Bos indicus (Zebu) and F1 crossbred Bos taurus x Bos indicus (Holstein x Zebu) genotypes were inseminated with Bos taurus (Holstein) semen and were allocated for in vitro embryo production using conventional IVF procedures. No differences were observed on the in vitro maturation (IVM) rates between breeds (Holstein x Holstein:85%, Zebu x Holstein:84% and Zebu x Holstein x Holstein:88%). Holstein cows yielded the highest number of cumulus oocyte complexes (6.8 per ovary) for in vitro maturation, differing (P<0.05) from Zebu x Holstein and Zebu x Holstein x Holstein F1 by 5.1 and 5.8, respectively. However, the Holstein breed also yielded the lowest percentage of cleavage (45.1 vs 71.9% for Zebu x Holstein and 65.1% for Zebu x Holstein x Holstein). Of the 3 genotypes, the hybrid F1 breed was the most efficient source of oocytes for the production of embryos capable of reaching morulae and blastocyst stages (76 250 ; P< 0.001). In the second part of the study, 599 oocytes from the F1 breed were fertilized in vitro, 1 group of 150 oocytes was used for the determination of the optimal pronuclear visualization period. The highest number of oocytes with 2 pronuclei was observed between 24 to 28 h after IVF (27 to 42%). The remaining 399 oocytes were microinjected with a gene construct bearing the bacterial lacZ gene as the reporter for gene expression. Survival of embryos to microinjection was 73.8%, and 45.5% of them (50 110 ) cleaved in culture. Of the microinjected embryos, 1 out of 50 showed beta-galactosidase activity. These findings indicate that a tropical crossbreed of cattle (Zebu x Holstein x Holstein) can be used as a source of oocytes for IVF programs and gene microinjection studies.     

Conserved central domains control the quaternary structure of type I and type II Hsp40 molecular chaperones

Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed.     

Germline mutation in RNASEL predicts increased risk of head and neck, uterine cervix and breast cancer

THE BACKGROUND: Ribonuclease L (RNASEL), encoding the 2'-5'-oligoadenylate (2-5A)-dependent RNase L, is a key enzyme in the interferon induced antiviral and anti-proliferate pathway. Mutations in RNASEL segregate with the disease in prostate cancer families and specific genotypes are associated with an increased risk of prostate cancer. Infection by human papillomavirus (HPV) is the major risk factor for uterine cervix cancer and for a subset of head and neck squamous cell carcinomas (HNSCC). HPV, Epstein Barr virus (EBV) and sequences from mouse mammary tumor virus (MMTV) have been detected in breast tumors, and the presence of integrated SV40 T/t antigen in breast carcinomas correlates with an aggressive phenotype and poor prognosis. A genetic predisposition could explain why some viral infections persist and induce cancer, while others disappear spontaneously. This points at RNASEL as a strong susceptibility gene. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the implication of an abnormal activity of RNase L in the onset and development of viral induced cancers, the study was initiated by searching for germline mutations in patients diagnosed with uterine cervix cancer. The rationale behind is that close to 100% of the cervix cancer patients have a persistent HPV infection, and if a defective RNase L were responsible for the lack of ability to clear the HPV infection, we would expect to find a wide spectrum of mutations in these patients, leading to a decreased RNase L activity. The HPV genotype was established in tumor DNA from 42 patients diagnosed with carcinoma of the uterine cervix and somatic tissue from these patients was analyzed for mutations by direct sequencing of all coding and regulatory regions of RNASEL. Fifteen mutations, including still uncharacterized, were identified. The genotype frequencies of selected single nucleotide polymorphisms (SNPs) established in the cervix cancer patients were compared between 382 patients with head and neck squamous cell carcinomas (HNSCC), 199 patients with primary unilateral breast cancer and 502 healthy Danish control individuals. We found that the genotype frequencies of only one of the 15 mutations, the yet uncharacterized 5'UTR mutation rs3738579 differed significantly between cancer patients and control individuals (P-value: 4.43x10(-5)). CONCLUSION/SIGNIFICANCE: In conclusion, we have discovered an increased risk, a heterozygous advantage and thereby a protective effect linked to the RNASEL SNP rs3738579. This effect is found for patients diagnosed with carcinoma of the uterine cervix, HNSCC, and breast cancer thus pointing at RNASEL as a general marker for cancer risk and not restricted to familial prostate cancer.     

New Insights into the in situ Microscopic Visualization and Quantification of Inorganic Polyphosphate Stores by 4’,6-Diamidino-2-Phenylindole (DAPI)-Staining

Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4’,6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multiphoton microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.Key words: DAPI, polyphosphate, fluorescence, fluorimetry     

Simplifier: a web tool to eliminate redundant NGS contigs

Modern genomic sequencing technologies produce a large amount of data with reduced cost per base; however, this data consists of short reads. This reduction in the size of the reads, compared to those obtained with previous methodologies, presents new challenges, including a need for efficient algorithms for the assembly of genomes from short reads and for resolving repetitions. Additionally after abinitio assembly, curation of the hundreds or thousands of contigs generated by assemblers demands considerable time and computational resources. We developed Simplifier, a stand-alone software that selectively eliminates redundant sequences from the collection of contigs generated by ab initio assembly of genomes. Application of Simplifier to data generated by assembly of the genome of Corynebacterium pseudotuberculosis strain 258 reduced the number of contigs generated by ab initio methods from 8,004 to 5,272, a reduction of 34.14%; in addition, N50 increased from 1 kb to 1.5 kb. Processing the contigs of Escherichia coli DH10B with Simplifier reduced the mate-paired library 17.47% and the fragment library 23.91%. Simplifier removed redundant sequences from datasets produced by assemblers, thereby reducing the effort required for finalization of genome assembly in tests with data from Prokaryotic organisms.

Availability

Simplifier is available at http://www.genoma.ufpa.br/rramos/softwares/simplifier.xhtmlIt requires Sun jdk 6 or higher.     

Deficient IFN Signaling by Myeloid Cells Leads to MAVS-Dependent Virus-Induced Sepsis

The type I interferon (IFN) signaling response limits infection of many RNA and DNA viruses. To define key cell types that require type I IFN signaling to orchestrate immunity against West Nile virus (WNV), we infected mice with conditional deletions of the type I IFN receptor (IFNAR) gene. Deletion of the Ifnar gene in subsets of myeloid cells resulted in uncontrolled WNV replication, vasoactive cytokine production, sepsis, organ damage, and death that were remarkably similar to infection of Ifnar ^−/− mice completely lacking type I IFN signaling. In Mavs^−/−×Ifnar^−/− myeloid cells and mice lacking both Ifnar and the RIG-I-like receptor adaptor gene Mavs, cytokine production was muted despite high levels of WNV infection. Thus, in myeloid cells, viral infection triggers signaling through MAVS to induce proinflammatory cytokines that can result in sepsis and organ damage. Viral pathogenesis was caused in part by massive complement activation, as liver damage was minimized in animals lacking complement components C3 or factor B or treated with neutralizing anti-C5 antibodies. Disease in Ifnar ^−/− and CD11c Cre⁺ Ifnar ^f/f mice also was facilitated by the proinflammatory cytokine TNF-α, as blocking antibodies diminished complement activation and prolonged survival without altering viral burden. Collectively, our findings establish the dominant role of type I IFN signaling in myeloid cells in restricting virus infection and controlling pathological inflammation and tissue injury.     

Genomic organization of gypsy chromatin insulators in Drosophila melanogaster

Chromatin insulators have been implicated in the regulation of higher-order chromatin structure and may function to compartmentalize the eukaryotic genome into independent domains of gene expression. To test this possibility, we used biochemical and computational approaches to identify gypsy-like genomic-binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein, a component of the gypsy insulator. EMSA and FISH analyses suggest that these are genuine Su(Hw)-binding sites. In addition, functional tests indicate that genomic Su(Hw)-binding sites can inhibit enhancer-promoter interactions and thus function as bona fide insulators. The insulator strength is dependent on the genomic location of the transgene and the number of Su(Hw)-binding sites, with clusters of two to three sites showing a stronger effect than individual sites. These clusters of Su(Hw)-binding sites are located mostly in intergenic regions or in introns of large genes, an arrangement that fits well with their proposed role in the formation of chromatin domains. Taken together, these data suggest that genomic gypsy-like insulators may provide a means for the compartmentalization of the genome within the nucleus.