Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica: Expansion of a repertoire of virulence-associated factors

Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-Ser ↓ His motif for autocatalytic cleavage by cathepsin Ls were preserved. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc

Remission of hypertension after treatment of giant simple renal cyst: a case report

Renal cysts are common in old patients, and usually remain untreated. Giant renal cysts measuring more than 15 cm in greatest diameter are uncommon and the association with hypertension is very rare. We present a case of a 25-year-old woman with a giant right renal cyst associated with hypertension that was treated by laparoscopic excision, followed by resolution hypertension

Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported forhuman cathepsin K. The 1.4-Å three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and Kprovided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc

Female partners of patients after surgical prostate cancer treatment: interactions with physicians and support needs

<p>Abstract</p> <p>Background</p> <p>Few studies have explored the women's experiences as a result of a partners' diagnosis of prostate cancer. This study begins to explore women's interactions with physicians (primary care and urologist) and the support needs associated with the diagnosis and treatment of their partners' prostate cancer.</p> <p>Methods</p> <p>Two focus groups (n = 14) of women whose partners were diagnosed with prostate cancer (diagnoses' 1 - 18 months). A trained facilitator used open-ended questions to explore ideas. The framework approach was used to analyze the transcripts.</p> <p>Results</p> <p>Three main themes emerged: 1. <b>More support</b>. Validation and information is needed for women including emotional support and opportunities to share experiences. 2. <b>Role of the physician</b>. The transfer of care once specialized treatment is no longer needed remained poorly defined, which increased confusion and feelings of abandonment related to the role of the primary physician. 3. <b>Partners' relationship changes</b>. Men became more dependent on their partners for support and to act as the primary communicator and caregiver.</p> <p>Conclusions</p> <p>Additional research is needed in this field to confirm the importance of training primary care physicians to consider holistic treatment approaches that recognize the partner and family needs as important in the complete physical and emotional healing of their patients.</p

Hurricanes and climate in the Caribbean during the past 3700 years BP

International audienceA multiproxy analysis of lacustrine sediments cored in Grand-Case Pond at Saint-Martin, north of the Lesser Antilles archipelago, reveals three distinct climatic periods for the last 3700 years. From 3700 to ~2500 yr cal. BP and from 1150 yr cal. BP to the present, carbonate mud deposition occurred in connection with pond lowstands. These periods were also punctuated by severe drought events, marked by gypsum laminae, and hurricane landfalls, leading to marine sand inputs into the pond. The intermediate time interval, from 2500 to 1150 yr cal. BP, is typified by black organic mud deposition, suggesting that hypoxic to anoxic conditions prevailed at the pond bottom. These were probably linked with a perennial pond highstand and reflect more uniform and wetter climatic conditions than today. The carbon isotopic composition of the ostracod Perissocytheridea bisulcata shows that the lowest δ13C values are recorded during the hypoxic periods, as a consequence of bacterial recycling of isotopically depleted organic matter. Such a climatic history agrees closely with that documented from other records in the Caribbean area, such as the Cariaco Basin, central coast of Belize or Barbados. By constrast, discrepancies seem to emerge from the comparison between hurricane activity recorded at Saint-Martin on the one hand and Vieques (Puerto Rico) on the other hand. We explain this apparent contradiction by a balance between two distinct storm paths in response to latitudinal shifts of the Intertropical Convergence Zone (ITCZ). Stronger storm activity over the Gulf coast and the inner Caribbean Sea is favoured by a southern position of the ITCZ in connection with dry climatic conditions. Plausible links with the North Atlantic Oscillation (NAO) are also suggested

Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium.

BACKGROUND: Invasive fungal diseases (IFDs) remain important causes of morbidity and mortality. The consensus definitions of the Infectious Diseases Group of the European Organization for Research and Treatment of Cancer and the Mycoses Study Group have been of immense value to researchers who conduct clinical trials of antifungals, assess diagnostic tests, and undertake epidemiologic studies. However, their utility has not extended beyond patients with cancer or recipients of stem cell or solid organ transplants. With newer diagnostic techniques available, it was clear that an update of these definitions was essential. METHODS: To achieve this, 10 working groups looked closely at imaging, laboratory diagnosis, and special populations at risk of IFD. A final version of the manuscript was agreed upon after the groups' findings were presented at a scientific symposium and after a 3-month period for public comment. There were several rounds of discussion before a final version of the manuscript was approved. RESULTS: There is no change in the classifications of "proven," "probable," and "possible" IFD, although the definition of "probable" has been expanded and the scope of the category "possible" has been diminished. The category of proven IFD can apply to any patient, regardless of whether the patient is immunocompromised. The probable and possible categories are proposed for immunocompromised patients only, except for endemic mycoses. CONCLUSIONS: These updated definitions of IFDs should prove applicable in clinical, diagnostic, and epidemiologic research of a broader range of patients at high-risk

People of the British Isles: preliminary analysis of genotypes and surnames in a UK control population

There is a great deal of interest in fine scale population structure in the UK, both as a signature of historical immigration events and because of the effect population structure may have on disease association studies. Although population structure appears to have a minor impact on the current generation of genome-wide association studies, it is likely to play a significant part in the next generation of studies designed to search for rare variants. A powerful way of detecting such structure is to control and document carefully the provenance of the samples involved. Here we describe the collection of a cohort of rural UK samples (The People of the British Isles), aimed at providing a well-characterised UK control population that can be used as a resource by the research community as well as providing fine scale genetic information on the British population. So far, some 4,000 samples have been collected, the majority of which fit the criteria of coming from a rural area and having all four grandparents from approximately the same area. Analysis of the first 3,865 samples that have been geocoded indicates that 75% have a mean distance between grandparental places of birth of 37.3km, and that about 70% of grandparental places of birth can be classed as rural. Preliminary genotyping of 1,057 samples demonstrates the value of these samples for investigating fine scale population structure within the UK, and shows how this can be enhanced by the use of surnames

Oxidative Stress in Zebrafish (Danio rerio) Sperm

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line

The clinical significance of serum and bronchoalveolar lavage inflammatory cytokines in patients at risk for Acute Respiratory Distress Syndrome

BACKGROUND: The predictive role of many cytokines has not been well defined in Acute Respiratory Distress Syndrome (ARDS). METHODS: We measured prospectively IL-4, IL-6, IL-6 receptor, IL-8, and IL-10, in the serum and bronchoalveolar lavage fluid (BALF) in 59 patients who were admitted to ICU in order to identify predictive factors for the course and outcome of ARDS. The patients were divided into three groups: those fulfilling the criteria for ARDS (n = 20, group A), those at risk for ARDS and developed ARDS within 48 hours (n = 12, group B), and those at risk for ARDS but never developed ARDS (n = 27, group C). RESULTS: An excellent negative predictive value for ARDS development was found for IL-6 in BALF and serum (100% and 95%, respectively). IL-8 in BALF and IL-8 and IL-10 serum levels were higher in non-survivors in all studied groups, and were associated with a high negative predictive value. A significant correlation was found between IL-8 and APACHE score (r = 0.60, p < 0.0001). Similarly, IL-6 and IL-6r were highly correlated with PaO2/FiO2 (r = -0.27, p < 0.05 and r = -0.55, p < 0.0001, respectively). CONCLUSIONS: BALF and serum levels of the studied cytokines on admission may provide valuable information for ARDS development in patients at risk, and outcome in patients either in ARDS or in at risk for ARDS

Early Epidemiological Assessment of the Virulence of Emerging Infectious Diseases: A Case Study of an Influenza Pandemic

Background: The case fatality ratio (CFR), the ratio of deaths from an infectious disease to the number of cases, provides an assessment of virulence. Calculation of the ratio of the cumulative number of deaths to cases during the course of an epidemic tends to result in a biased CFR. The present study develops a simple method to obtain an unbiased estimate of confirmed CFR (cCFR), using only the confirmed cases as the denominator, at an early stage of epidemic, even when there have been only a few deaths. Methodology/Principal Findings: Our method adjusts the biased cCFR by a factor of underestimation which is informed by the time from symptom onset to death. We first examine the approach by analyzing an outbreak of severe acute respiratory syndrome in Hong Kong (2003) with known unbiased cCFR estimate, and then investigate published epidemiological datasets of novel swine-origin influenza A (H1N1) virus infection in the USA and Canada (2009). Because observation of a few deaths alone does not permit estimating the distribution of the time from onset to death, the uncertainty is addressed by means of sensitivity analysis. The maximum likelihood estimate of the unbiased cCFR for influenza may lie in the range of 0.16-4.48% within the assumed parameter space for a factor of underestimation. The estimates for influenza suggest that the virulence is comparable to the early estimate in Mexico. Even when there have been no deaths, our model permits estimating a conservative upper bound of the cCFR. Conclusions: Although one has to keep in mind that the cCFR for an entire population is vulnerable to its variations among sub-populations and underdiagnosis, our method is useful for assessing virulence at the early stage of an epidemic and for informing policy makers and the public. © 2009 Nishiura et al.published_or_final_versio