Antibody raised against extracellular proteinases ofSporothrix schenckii inS. schenkii inoculated hairless mice

Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin, while proteinase II is an aspartic proteinase, inhibited by pepstatin. Studies on substrate specificity and the effect of proteinase inhibitors on cell growth suggest an important role for these proteinases in terms of fungal invasion and growth. There has, however, been no evidence presented demonstrating thatS. schenckii produces 2 extracellular proteinases in vivo. In order to substantiate the in vivo production of proteinases and to attempt a preliminary serodiagnosis of sporotrichosis, serum antibodies against 2 proteinases were assayed usingS. schenckii inoculated hairless mice. Subsequent to an intracutaneous injection ofS. schenckii to the mouse skin, nodules spontaneously formed and disappeared for a period of 4 weeks. Histopathological examination results were in accordance with the microscopic observations. Micro-organisms disappeared during the fourth week. Serum antibody titers against purified proteinases I and II were measured weekly, using enzyme-linked immunosorbent assay (EIA). As a result, the time course of the antibody titers to both proteinases I and II were parallel to that of macroscopic and microscopic observations in an experimental mouse sporotrichosis model. These results suggest thatS. schenckii produces both proteinases I and II in vivo. Moreover, the detection of antibodies against these proteinases can contribute to a serodiagnosis of sporotrichosis.     

Prospective characterization of full-length hepatitis C virus NS5A quasispecies during induction and combination antiviral therapy

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR(237-276)) sequence associated with IFN resistance was not found, although the presence of Ala(245) within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P<0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P<0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P<0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.     

TNF-α-induced exosomal miR-146a mediates mesenchymal stem cell-dependent suppression of urethral stricture

The effective treatment of urethral stricture remains a medical problem. The use of proinflammatory cytokines as stimuli to improve the reparative efficacy of mesenchymal stem cells (MSCs) towards damaged tissues represents an evolving field of investigation. However, the therapeutic benefits of this strategy in the treatment of urethral stricture remain unknown. Here, we enriched exosomes derived from human umbilical cord-derived MSCs pretreated with or without tumor necrosis factor alpha (TNF-α) to evaluate their therapeutic effects in an in vivo model of TGFβ1-induced urethral stricture. Male Sprague-Dawley rats received sham (saline) or TGFβ1 injections to urethral tissues followed by incisions in the urethra. Animals in the TGFβ1 injection (urethral fibrosis) cohort were subsequently injected with vehicle control, or with exosomes derived from MSCs cultured with or without TNF-α. After 4 weeks, rats underwent ultrasound evaluation and, following euthanasia, urethral tissues were harvested for histological and molecular analysis. In vitro, the effects of MSC-derived exosomes on fibroblast secretion of collagen and cytokines were studied by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. Exosomes derived from MSCs pretreated with TNF-α were more effective in suppressing urethral fibrosis and stricture than exosomes from untreated MSCs. We found that miR-146a, an anti-inflammatory miRNA, was strongly upregulated in TNF-α-stimulated MSCs and was selectively packaged into exosomes. Moreover, miR-146a-containing exosomes were taken up by fibroblasts and inhibited fibroblast activation and associated inflammatory responses, a finding that may underlie the therapeutic mechanism for suppression of urethral stricture. Inhibition of miR-146a in TNF-α-treated MSCs partially reduced antifibrotic effects and increased the release of proinflammatory factors of exosomes derived from these cells. Together these findings demonstrate that exosomes derived from TNF-α-treated MSCs are of therapeutic benefit in urethral fibrosis, suggesting that this strategy may have utility as an adjuvant therapy in the treatment of urethral stricture diseases.     

Morphological studies of polycystic mouse ovaries induced by dehydroepiandrosterone

Summary Morphological alterations induced by dehydroepiandrosterone (DHA) were studied in polycystic mouse ovaries (PCO). Treated mice showed ovulatory failure and cystic changes; cysts and follicles in various stages of growth and atresia were present although corpora lutea were absent. The levels of testosterone, dihydrotestosterone, 3- and 3-androstanediol, estrone and androstenedione increased, whereas estradiol was not detectable.The ultrastructure of granulosa cells in healthy and atretic follicles was similar to that of control animals, although the membrana granulosa in cysts was reduced to a monolayer of flattened cells. The theca interna of healthy and atretic follicles and ovarian cysts showed ultrastructural signs of abnormal steroidogenic stimulation.No significant differences (0.7<P<0.8) were found between the extensive surface area of gap junctions of healthy follicles of control and DHA-treated animals. On the P-face of granulosa cells of large healthy follicles, meandering strands of tight junctional particles were observed; their average length was significantly longer than those in healthy follicles of control animals (P<0.001). This increase was probably related to the large amounts of androgens present in the treated animals.Theca interna cells possessed small gap junctions; no significant differences (P>0.9) in gap-junction surface area were observed between DHA-treated and control animals. These results suggest that the size of gap junctions is probably unrelated to the steroidogenic activities of theca cells.The following trivial names have been used: Dihydrotestosterone: 5-androstan 17 ol-13 one; 3-androstanediol: 5-androstan 3,17 diol; 3-androstanediol: 5-androstan 3,17 diol     

Contrasting Mode of Evolution at a Coat Color Locus in Wild and Domestic Pigs

Despite having only begun ~10,000 years ago, the process of domestication has resulted in a degree of phenotypic variation within individual species normally associated with much deeper evolutionary time scales. Though many variable traits found in domestic animals are the result of relatively recent human-mediated selection, uncertainty remains as to whether the modern ubiquity of long-standing variable traits such as coat color results from selection or drift, and whether the underlying alleles were present in the wild ancestor or appeared after domestication began. Here, through an investigation of sequence diversity at the porcine melanocortin receptor 1 (MC1R) locus, we provide evidence that wild and domestic pig (Sus scrofa) haplotypes from China and Europe are the result of strikingly different selection pressures, and that coat color variation is the result of intentional selection for alleles that appeared after the advent of domestication. Asian and European wild boar (evolutionarily distinct subspecies) differed only by synonymous substitutions, demonstrating that camouflage coat color is maintained by purifying selection. In domestic pigs, however, each of nine unique mutations altered the amino acid sequence thus generating coat color diversity. Most domestic MC1R alleles differed by more than one mutation from the wild-type, implying a long history of strong positive selection for coat color variants, during which time humans have cherry-picked rare mutations that would be quickly eliminated in wild contexts. This pattern demonstrates that coat color phenotypes result from direct human selection and not via a simple relaxation of natural selective pressures.     

Thermal adaptations to extreme freeze–thaw cycles in the high tropical Andes

Temperature plays a key role in the biology of ectotherms, including anurans, which are found at higher elevations in the tropics than anywhere in the temperate zone. High elevation tropical environments are characterized by extreme daily thermal fluctuation including high daily maxima and nightly freezing. Our study investigated the contrasting operative temperatures of the anurans Telmatobius marmoratus and Pleurodema marmoratum in different environmental contexts at the same elevation and biome above 5,200 m. Telmatobius marmoratus avoids extremes of daily temperature fluctuation by utilizing thermally buffered aquatic habitat at all life stages, with minimal operative temperature variation (range: 4.6–8.0°C). Pleurodema marmoratum, in contrast, experienced operative temperatures from ?3.5 to 44°C and has one of the widest thermal breadths reported for any tropical frog, from >32°C (critical thermal maximum) to surviving freezing periods of 1 and 6 hr down to ?3.0°C. Our findings expand experimental evidence of frost tolerance in amphibians to the widespread Neotropical family Leptodactylidae, the first such evidence of frost tolerance in a tropical amphibian. Our study identifies three strategies (wide thermal tolerance breadth, use of buffered microhabitats, and behavioral thermoregulation), which allow these tropical frogs to withstand the current wide daily thermal fluctuation above 5,000 m.a.s.l. and which may help them adapt to future climatic changes. Abstract in Spanish is available with online material     

GABA concentrations in the striatum following repetitive cerebral ischemia

GABAergic neurons in the striatum are very sensitive to the effects of ischemia. The progressive decline in striatal GABA following transient forebrain ischemia in gerbils may be secondary to either a decreased production or an increase in reuptake mechanisms or both. The current experiment was designed to evaluate release of GABA by stimulation with K+ or inhibition of its uptake with nipecotic acid or their combination (K+ nipecotic) after repetitive forebrain ischemia in gerbils by in-vivo microdialysis on Days 1, 3, 5, and 14 following the insult. Infusion of nipecotic acid or potassium chloride, resulted in a significant increase in extracellular GABA. This response was significantly decreased in the post-ischemic animals. The synergistic effect of increased GABA concentrations by the infusion of nipecotic acid+potassium chloride seen in the controls was not evident in the post-ischemic animals. In conclusion, though there is a reduction in the extracellular GABA concentrations in the first week following an ischemic insult, restorative mechanisms are operative in the second week as seen by the increasing GABA concentrations.     

Identification of a broad-spectrum arenavirus entry inhibitor

Several arenaviruses, including Lassa virus (LASV), are causative agents of hemorrhagic fever, for which effective therapeutic options are lacking. The LASV envelope glycoprotein (GP) gene was used to generate lentiviral pseudotypes to identify small-molecule inhibitors of viral entry. A benzimidazole derivative with potent antiviral activity was identified from a high-throughput screen utilizing this strategy. Subsequent lead optimization for antiviral activity identified a modified structure, ST-193, with a 50% inhibitory concentration (IC(50)) of 1.6 nM against LASV pseudotypes. ST-193 inhibited pseudotypes generated with other arenavirus envelopes as well, including the remaining four commonly associated with hemorrhagic fever (IC(50)s for Junín, Machupo, Guanarito, and Sabiá were in the 0.2 to 12 nM range) but exhibited no antiviral activity against pseudotypes incorporating either the GP from the LASV-related arenavirus lymphocytic choriomeningitis virus (LCMV) or the unrelated G protein from vesicular stomatitis virus, at concentrations of up to 10 microM. Determinants of ST-193 sensitivity were mapped through a combination of LASV-LCMV domain-swapping experiments, genetic selection of viral variants, and site-directed mutagenesis. Taken together, these studies demonstrate that sensitivity to ST-193 is dictated by a segment of about 30 amino acids within the GP2 subunit. This region includes the carboxy-terminal region of the ectodomain and the predicted transmembrane domain of the envelope protein, revealing a novel antiviral target within the arenavirus envelope GP.