Ecotin protects Salmonella Typhimurium against the microbicidal activity of host proteases.

Salmonella enterica serovar Typhimurium causes acute diarrhea upon oral infection in humans. The harsh and proteolytic environment found in the gastrointestinal tract is the first obstacle that these bacteria face after infection. However, the mechanisms that allow Salmonella to survive the hostile conditions of the gut are poorly understood. The ecotin gene is found in an extensive range of known phyla of bacteria and it encodes a protein that has been shown to inhibit serine proteases. Thus, in the present work we studied the role of ecotin of Salmonella Typhimurium in host-pathogen interactions. We found that Salmonella Typhimurium Δ ecotin strain exhibited lower inflammation in a murine model of Salmonella induced colitis. The Δ ecotin mutant was more susceptible to the action of pancreatin and purified pancreatic elastase. In addition, the lack of ecotin led to impaired adhesion to Caco-2 and HT-29 cell lines, related to the proteolytic activity of brush border enzymes. Besides, Δ ecotin showed higher susceptibility to lysosomal proteolytic content and intracellular replication defects in macrophages. In addition, we found Ecotin to have a crucial role in Salmonella against the microbicide action of granules released and neutrophil extracellular traps from human polymorphonuclear leukocytes. Thus, the work presented here highlights the importance of ecotin in Salmonella as countermeasures against the host proteolytic defense system. IMPORTANCE: The gastrointestinal tract is a very complex and harsh environment. Salmonella is a successful food borne pathogen, but little is known about its capacity to survive against the proteolysis of the gut lumen and intracellular proteases. Here, we show that Ecotin, a serine protease inhibitor, plays an important role in protecting Salmonella against proteases present at different sites encountered during oral infection. Our results indicate that Ecotin is an important virulence factor in Salmonella , adding another tool to the wide range of features this pathogen uses during oral infection.

Prostatic Rhabdomyoma in a Toddler: A Case Report With Molecular Characterization.

We present a 29-month-old male patient in follow-up due to pyelocaliceal dilation with a prostatic nodule incidentally found during ultrasound evaluation. Cysto video endoscopy was performed and a prostate biopsy, obtained. Microscopic evaluation showed a haphazardly distributed population of muscular cells with cross striations without evidence of mitosis or necrosis. Immunohistochemistry was positive for myogenin and desmin and negative for smooth muscle actin. Next generation sequencing was performed without finding any pathogenic variant or fusion in the tumor RNA. The patient received no further treatment, remained asymptomatic and continues in follow up, 3 years after initial diagnosis. We report a case of prostate rhabdomyoma in a toddler, an exceptional location that raises concern about differential diagnosis with its malignant counterpart, rhabdomyosarcoma, especially at this age.

Whole transcriptome analysis of the pectoralis major muscle reveals molecular mechanisms involved with white striping in broiler chickens.

White striping (WS) is one of the most common myopathies identified in broiler chickens leading to substantial production losses, where the incidence reaches 12% in commercial chickens. It occurs primarily in heavier chickens being a modification of the breast muscle characterized by the presence of pale parallel streaks in the same orientation of the muscle fibers. Since the WS etiology remains unclear, we aimed to identify the biological and genetic mechanisms involved in its occurrence through the whole transcriptome analysis of WS in affected and unaffected chicken breast muscles. A total of 11,177 genes were expressed in the pectoralis major muscle. Out of those, 1,441 genes were differentially expressed (FDR ≤ 0.01) between the two analyzed groups, being, respectively, 772 genes upregulated and 669 downregulated in the WS affected group. A total of 36 significantly overrepresented GO terms related to WS myopathy were enriched, and the most relevant biological processes were activation of immune system, angiogenesis, hypoxia, cell death, and striated muscle contraction. The unbalance of those biological processes may trigger the occurrence of the WS phenotype in broilers. The possible lack of capillary blood supply homogeneously in the muscle triggers the hypoxia, following the activation of glycolysis, calcium signaling and apoptosis related genes facilitating the tissue damage and WS incidence.

The involvement of RUNX2 and SPARC genes in the bacterial chondronecrosis with osteomyelitis in broilers.

Economic losses due to an increase of leg disorders in broilers have become a major concern of the poultry industry. Despite the efforts to reduce skeletal abnormalities in chickens, insufficient progress has been made. Bacterial chondronecrosis with osteomyelitis (BCO) is one of the main disorders that affect bone integrity in broilers. However, the genetic pathways and genes involved in most bone problems, including BCO, remains unclear. In this study, femoral samples from male broilers with 45 days of age affected or not with BCO were used to compare the relative expression with a reverse transcription real time PCR approach of 13 candidate genes: SPP1 (osteopontin), TNFRSF11B (osteoprotegerin), SPARC (osteonectin), CALB1 (calbidin 1), CALM (Calmodulin 2), IBSP (sialoprotein), COL1A2 (collagen, type I, α 2), BMP2 (bone morphogenetic protein 2), BMP3 (bone morphogenetic protein 3), RANKL (κ-B nuclear factor ligand), SMAD1 (SMAD family member 1), LEPR (leptin receptor) and RUNX2 (related transcription factor Runt 2). Differential expression test between affected and non-affected groups was performed using the REST software. The RUNX2 and SPARC genes were downregulated (P<0.05) in the affected group, with reduced expression of fourfold when compared with the non-affected group. This result indicates that the downregulation of RUNX2 and SPARC can contribute to an increased incidence of BCO in broilers.

The cumulative impacts of repeated heavy rainfall, flooding and altered water quality on the high-latitude coral reefs of Hervey Bay, Queensland, Australia.

Terrestrial runoff and flooding have resulted in major impacts on coral communities worldwide, but we lack detailed understanding of flood plume conditions and their ecological effects. Over the course of repeated flooding between 2010 and 2013, we measured coral cover and water quality on the high-latitude coral reefs of Hervey Bay, Queensland, Australia. In 2013, salinity, total suspended solids, total nitrogen and total phosphorus were altered for up to six months post-flooding. Submarine groundwater caused hypo-saline conditions for a further four months. Despite the greater magnitude of flooding in 2013, declines in coral abundance (∼28%) from these floods were lower than the 2011 flood (∼40%), which occurred immediately after a decade of severe drought. There was an overall cumulative decrease of coral by ∼56% from 2010 to 2013. Our study highlights the need for local scale monitoring and research to facilitate informed management and conservation of catchments and marine environments.

Purinergic signaling modulates human visceral adipose inflammatory responses: implications in metabolically unhealthy obesity.

Obesity is accompanied by chronic inflammation of VAT, which promotes metabolic changes, and purinergic signaling has a key role in a wide range of inflammatory diseases. Therefore, we addressed whether fat inflammation could be differentially modulated by this signaling pathway in the MUO and in individuals who remain MHO. Our results show that the necrotized VAT of both groups released greater levels of ATP compared with lean donors. Interestingly, MUO tissue SVCs showed up-regulation and engagement of the purinergic P2X7R. The extracellular ATP concentration is regulated by an enzymatic process, in which CD39 converts ATP and ADP into AMP, and CD73 converts AMP into adenosine. In VAT, the CD73 ectoenzyme was widely distributed in immune and nonimmune cells, whereas CD39 expression was restricted to immune CD45PAN+ SVCs. Although the MUO group expressed the highest levels of both ectoenzymes, no difference in ATP hydrolysis capacity was found between the groups. As expected, MUO exhibited the highest NLRP3 inflammasome expression and IL-1ß production. MUO SVCs also displayed up-regulation of the A2AR, allowing extracellular adenosine to increase IL-1ß local secretion. Additionally, we demonstrate that metabolic parameters and BMI are positively correlated with purinergic components in VAT. These findings indicate that purinergic signaling is a novel mechanism involved in the chronic inflammation of VAT underlying the metabolic changes in obesity. Finally, our study reveals a proinflammatory role for adenosine in sustaining IL-1ß production in this tissue.

Hopping hotspots: global shifts in marine biodiversity.

Hotspots of high species diversity are a prominent feature of modern global biodiversity patterns. Fossil and molecular evidence is starting to reveal the history of these hotspots. There have been at least three marine biodiversity hotspots during the past 50 million years. They have moved across almost half the globe, with their timing and locations coinciding with major tectonic events. The birth and death of successive hotspots highlights the link between environmental change and biodiversity patterns. The antiquity of the taxa in the modern Indo-Australian Archipelago hotspot emphasizes the role of pre-Pleistocene events in shaping modern diversity patterns.

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae.

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking.

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking.

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil.

OBJECTIVES: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined. METHODS: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Löwenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 bp upstream from the start codon was determined by DNA sequencing of purified PCR products. RESULTS: Of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region. Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity. CONCLUSIONS: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.

Climate change, human impacts, and the resilience of coral reefs.

The diversity, frequency, and scale of human impacts on coral reefs are increasing to the extent that reefs are threatened globally. Projected increases in carbon dioxide and temperature over the next 50 years exceed the conditions under which coral reefs have flourished over the past half-million years. However, reefs will change rather than disappear entirely, with some species already showing far greater tolerance to climate change and coral bleaching than others. International integration of management strategies that support reef resilience need to be vigorously implemented, and complemented by strong policy decisions to reduce the rate of global warming.

Historical overfishing and the recent collapse of coastal ecosystems.

Ecological extinction caused by overfishing precedes all other pervasive human disturbance to coastal ecosystems, including pollution, degradation of water quality, and anthropogenic climate change. Historical abundances of large consumer species were fantastically large in comparison with recent observations. Paleoecological, archaeological, and historical data show that time lags of decades to centuries occurred between the onset of overfishing and consequent changes in ecological communities, because unfished species of similar trophic level assumed the ecological roles of overfished species until they too were overfished or died of epidemic diseases related to overcrowding. Retrospective data not only help to clarify underlying causes and rates of ecological change, but they also demonstrate achievable goals for restoration and management of coastal ecosystems that could not even be contemplated based on the limited perspective of recent observations alone.

Insertion time of the pulmonary artery catheter in critically ill patients.

OBJECTIVES: Measurement of the time elapsed from the decision to use a pulmonary artery catheter to the onset of the adapted treatment. DESIGN: Prospective study. SETTING: Critical care unit of a university hospital. PATIENTS: A total of 104 critically ill patients. INTERVENTIONS: The time elapsed from the decision to use a pulmonary artery catheter to the onset of the adapted treatment. Five time intervals (availability, preparation, catheterization, data collection, and therapeutic intervals) were individualized according to the times of decision of pulmonary artery catheter insertion, operator's hand washing, venipuncture, postoperative dressing, data collection, and the effective onset of subsequent therapy. MEASUREMENTS AND MAIN RESULTS: Among 120 used pulmonary artery catheters, seven could not be inserted. The time to use the pulmonary artery catheter was never shorter than 45 mins (median value = 120 mins). For availability, preparation, catheterization, data collection, and therapeutic intervals, the median values were 30, 20, 20, 20, and 10 mins, respectively. The availability and data collection intervals were shortened during the night period and the fourth quarter of the study, respectively. CONCLUSIONS: The pulmonary artery catheter use is time consuming. However, the availability and data collection intervals could be shortened.