Significance of Relative Position of Cellulases in Designer Cellulosomes for Optimized Cellulolysis

Degradation of cellulose is of major interest in the quest for alternative sources of renewable energy, for its positive effects on environment and ecology, and for use in advanced biotechnological applications. Due to its microcrystalline organization, celluose is extremely difficult to degrade, although numerous microbes have evolved that produce the appropriate enzymes. The most efficient known natural cellulolytic system is produced by anaerobic bacteria, such as C. thermocellum, that possess a multi-enzymatic complex termed the cellulosome. Our laboratory has devised and developed the designer cellulosome concept, which consists of chimaeric scaffoldins for controlled incorporation of recombinant polysaccharide-degrading enzymes. Recently, we reported the creation of a combinatorial library of four cellulosomal modules comprising a basic chimaeric scaffoldin, i.e., a CBM and 3 divergent cohesin modules. Here, we employed selected members of this library to determine whether the position of defined cellulolytic enzymes is important for optimized degradation of a microcrystalline cellulosic substrate. For this purpose, 10 chimaeric scaffoldins were used for incorporation of three recombinant Thermobifida fusca enzymes: the processive endoglucanase Cel9A, endoglucanase Cel5A and exoglucanase Cel48A. In addition, we examined whether the characteristic properties of the T. fusca enzymes as designer cellulosome components are unique to this bacterium by replacing them with parallel enzymes from Clostridium thermocellum. The results support the contention that for a given set of cellulosomal enzymes, their relative position within a scaffoldin can be critical for optimal degradation of microcrystaline cellulosic substrates.     

CO2 and H2O: Understanding Different Stakeholder Perspectives on the Use of Carbon Credits to Finance Household Water Treatment Projects

Background

Carbon credits are an increasingly prevalent market-based mechanism used to subsidize household water treatment technologies (HWT). This involves generating credits through the reduction of carbon emissions from boiling water by providing a technology that reduces greenhouse gas emissions linked to climate change. Proponents claim this process delivers health and environmental benefits by providing clean drinking water and reducing greenhouse gases. Selling carbon credits associated with HWT projects requires rigorous monitoring to ensure households are using the HWT and achieving the desired benefits of the device. Critics have suggested that the technologies provide neither the benefits of clean water nor reduced emissions. This study explores the perspectives of carbon credit and water, sanitation and hygiene (WASH) experts on HWT carbon credit projects.

Methods

Thirteen semi-structured, in-depth interviews were conducted with key informants from the WASH and carbon credit development sectors. The interviews explored perceptions of the two groups with respect to the procedures applied in the Gold Standard methodology for trading Voluntary Emission Reduction (VER) credits.

Results

Agreement among the WASH and carbon credit experts existed for the concept of suppressed demand and parameters in the baseline water boiling test. Key differences, however, existed. WASH experts’ responses highlighted a focus on objectively verifiable data for monitoring carbon projects while carbon credit experts called for contextualizing observed data with the need for flexibility and balancing financial viability with quality assurance.

Conclusions

Carbon credit projects have the potential to become an important financing mechanism for clean energy in low- and middle-income countries. Based on this research we recommend that more effort be placed on building consensus on the underlying assumptions for obtaining carbon credits from HWT projects, as well as the approved methods for monitoring correct and consistent use of the HWT technologies in order to support public health impacts.     

CD24 and APC Genetic Polymorphisms in Pancreatic Cancers as Potential Biomarkers for Clinical Outcome

Background

There are no validated biomarkers that correlate with the prognosis of pancreatic ductal adenocarcinoma (PDA). The CD24 and adenomatous polyposis coli (APC) genes are important in the malignant transformation of gastrointestinal cells. This study examined APC and CD24 genetic polymorphisms and their possible impact on survival of patients with PDA.

Methods

Clinical and pathological data as well as blood samples for extracting DNA were obtained for 73 patients with PDA. Real-time PCR assessed genetic variants of APC (I1307K and E1317Q), and four different single nucleotide polymorphisms (SNPs) in the CD24 gene: C170T (rs52812045), TG1527del (rs3838646), A1626G (rs1058881) and A1056G (rs1058818).

Results

The median age at diagnosis was 64 (41–90) years. Thirty-one patients (42.5%) were operable, 16 (22%) had locally advanced disease and 26 (35.5%) had disseminated metastatic cancer. The malignancy-related mortality rate was 84%. Median survival was 14 months (11.25–16.74). Survival was similar for wild-type (WT), heterozygous and homozygous variants of the APC or CD24 genes. The three most frequent CD24 SNP combinations were: heterozygote for A1626G and WT for the rest of the alleles (14% of patients), heterozygote for C170T, A1626G, A1056G and WT for the rest (14% of patients), and heterozygote for C170T, A1056G and WT for the rest (10% of patients). All patients were APC WT. The first two groups were significantly younger at diagnosis than the third group.

Conclusions

Specific polymorphisms in the APC and CD24 genes may play a role in pancreatic cancer development. Correlation with survival requires a larger cohort.     

Verbal Autopsy: Evaluation of Methods to Certify Causes of Death in Uganda

To assess different methods for determining cause of death from verbal autopsy (VA) questionnaire data, the intra-rater reliability of Physician-Certified Verbal Autopsy (PCVA) and the accuracy of PCVA, expert-derived (non-hierarchical) and data-driven (hierarchal) algorithms were assessed for determining common causes of death in Ugandan children. A verbal autopsy validation study was conducted from 2008-2009 in three different sites in Uganda. The dataset included 104 neonatal deaths (0-27 days) and 615 childhood deaths (1-59 months) with the cause(s) of death classified by PCVA and physician review of hospital medical records (the ‘reference standard’). Of the original 719 questionnaires, 141 (20%) were selected for a second review by the same physicians; the repeat cause(s) of death were compared to the original,and agreement assessed using the Kappa statistic.Physician reviewers’ refined non-hierarchical algorithms for common causes of death from existing expert algorithms, from which, hierarchal algorithms were developed. The accuracy of PCVA, non-hierarchical, and hierarchical algorithms for determining cause(s) of death from all 719 VA questionnaires was determined using the reference standard. Overall, intra-rater repeatability was high (83% agreement, Kappa 0.79 [95% CI 0.76-0.82]). PCVA performed well, with high specificity for determining cause of neonatal (>67%), and childhood (>83%) deaths, resulting in fairly accurate cause-specific mortality fraction (CSMF) estimates. For most causes of death in children, non-hierarchical algorithms had higher sensitivity, but correspondingly lower specificity, than PCVA and hierarchical algorithms, resulting in inaccurate CSMF estimates. Hierarchical algorithms were specific for most causes of death, and CSMF estimates were comparable to the reference standard and PCVA. Inter-rater reliability of PCVA was high, and overall PCVA performed well. Hierarchical algorithms performed better than non-hierarchical algorithms due to higher specificity and more accurate CSMF estimates. Use of PCVA to determine cause of death from VA questionnaire data is reasonable while automated data-driven algorithms are improved.     

Paleoceanographic Insights on Recent Oxygen Minimum Zone Expansion: Lessons for Modern Oceanography

Climate-driven Oxygen Minimum Zone (OMZ) expansions in the geologic record provide an opportunity to characterize the spatial and temporal scales of OMZ change. Here we investigate OMZ expansion through the global-scale warming event of the most recent deglaciation (18-11 ka), an event with clear relevance to understanding modern anthropogenic climate change. Deglacial marine sediment records were compiled to quantify the vertical extent, intensity, surface area and volume impingements of hypoxic waters upon continental margins. By integrating sediment records (183-2,309 meters below sea level; mbsl) containing one or more geochemical, sedimentary or microfossil oxygenation proxies integrated with analyses of eustatic sea level rise, we reconstruct the timing, depth and intensity of seafloor hypoxia. The maximum vertical OMZ extent during the deglaciation was variable by region: Subarctic Pacific (~600-2,900 mbsl), California Current (~330-1,500 mbsl), Mexico Margin (~330-830 mbsl), and the Humboldt Current and Equatorial Pacific (~110-3,100 mbsl). The timing of OMZ expansion is regionally coherent but not globally synchronous. Subarctic Pacific and California Current continental margins exhibit tight correlation to the oscillations of Northern Hemisphere deglacial events (Termination IA, Bølling-Allerød, Younger Dryas and Termination IB). Southern regions (Mexico Margin and the Equatorial Pacific and Humboldt Current) exhibit hypoxia expansion prior to Termination IA (~14.7 ka), and no regional oxygenation oscillations. Our analyses provide new evidence for the geographically and vertically extensive expansion of OMZs, and the extreme compression of upper-ocean oxygenated ecosystems during the geologically recent deglaciation.     

The Prognostic Significance of Metabolic Response Heterogeneity in Metastatic Colorectal Cancer

Background

Tumoral heterogeneity is a major determinant of resistance in solid tumors. FDG-PET/CT can identify early during chemotherapy non-responsive lesions within the whole body tumor load. This prospective multicentric proof-of-concept study explores intra-individual metabolic response (mR) heterogeneity as a treatment efficacy biomarker in chemorefractory metastatic colorectal cancer (mCRC).

Methods

Standardized FDG-PET/CT was performed at baseline and after the first cycle of combined sorafenib (600mg/day for 21 days, then 800mg/day) and capecitabine (1700 mg/m²/day administered D1-14 every 21 days). MR assessment was categorized according to the proportion of metabolically non-responding (non-mR) lesions (stable FDG uptake with SUVmax decrease <15%) among all measurable lesions.

Results

Ninety-two patients were included. The median overall survival (OS) and progression-free survival (PFS) were 8.2 months (95% CI: 6.8–10.5) and 4.2 months (95% CI: 3.4–4.8) respectively. In the 79 assessable patients, early PET-CT showed no metabolically refractory lesion in 47%, a heterogeneous mR with at least one non-mR lesion in 32%, and a consistent non-mR or early disease progression in 21%. On exploratory analysis, patients without any non-mR lesion showed a significantly longer PFS (HR 0.34; 95% CI: 0.21–0.56, P-value <0.001) and OS (HR 0.58; 95% CI: 0.36–0.92, P-value 0.02) compared to the other patients. The proportion of non-mR lesions within the tumor load did not impact PFS/OS.

Conclusion

The presence of at least one metabolically refractory lesion is associated with a poorer outcome in advanced mCRC patients treated with combined sorafenib-capecitabine. Early detection of treatment-induced mR heterogeneity may represent an important predictive efficacy biomarker in mCRC.

Trial Registration

ClinicalTrials.gov NCT01290926     

Endogenous Murine BST-2/Tetherin Is Not a Major Restriction Factor of Influenza A Virus Infection

BST-2 (tetherin, CD317, HM1.24) restricts virus growth by tethering enveloped viruses to the cell surface. The role of BST-2 during influenza A virus infection (IAV) is controversial. Here, we assessed the capacity of endogenous BST-2 to restrict IAV in primary murine cells. IAV infection increased BST-2 surface expression by primary macrophages, but not alveolar epithelial cells (AEC). BST-2-deficient AEC and macrophages displayed no difference in susceptibility to IAV infection relative to wild type cells. Furthermore, BST-2 played little role in infectious IAV release from either AEC or macrophages. To examine BST-2 during IAV infection in vivo, we infected BST-2-deficient mice. No difference in weight loss or in viral loads in the lungs and/or nasal tissues were detected between BST-2-deficient and wild type animals. This study rules out a major role for endogenous BST-2 in modulating IAV in the mouse model of infection.     

Resilience in the General Population: Standardization of the Resilience Scale (RS-11)

Background

The objectives of the study were to generate normative data for the RS-11 for different age groups for men and women and to further investigate the construct validity and factor structure in the general population.

Methods

Nationally representative face-to face household surveys were conducted in Germany in 2006 (n = 5,036).

Results

Normative data for the RS-11 were generated for men and women (53.7% female) and different age levels (mean age (SD) of 48.4 (18.0) years). Men had significantly higher mean scores compared with women (60.0 [SD = 10.2] vs. 59.3 [SD = 11.0]). Results of CFA supported a one-factor model of resilience. Self-esteem (standardized β = .50) and life satisfaction (standardized β =.20) were associated with resilience.

Conclusions

The normative data provide a framework for the interpretation and comparisons of resilience with other populations. Results demonstrate a special importance of self-esteem in the understanding of resilience.     

Understanding Low-Acuity Visits to the Pediatric Emergency Department

Background

Canadian pediatric emergency department visits are increasing, with a disproportionate increase in low-acuity visits locally (33% of volume in 2008-09, 41% in 2011-12). We sought to understand: 1) presentation patterns and resource implications; 2) parents’ perceptions and motivations; and 3) alternate health care options considered prior to presenting with low-acuity problems.

Methods

We conducted a prospective cohort study at our tertiary pediatric emergency department serving two provinces to explore differences between patients with and without a primary care provider. During four, 2-week study periods over 1 year, parents of low-acuity visits received an anonymous survey. Presentation times, interventions, diagnoses and dispositions were captured on a data collection form linked to the survey by study number.

Results

Parents completed 2,443 surveys (74.1% response rate), with survey-data collection form pairs available for 2,146 visits. Overall, 89.7% of respondents had a primary care provider; 68% were family physicians. Surprisingly, 40% of visits occurred during weekday office hours and 27.3% occurred within 4 hours of symptom onset; 67.5% of those early presenters were for injuries. Few parents sought care from their primary care provider (25%), health information line (20.7%), or urgent care clinic (18.5%); 36% reported that they believed their child’s problem required the emergency department. Forty-five percent required only a history, physical exam and reassurance; only 11% required an intervention not available in an office setting. Patients without a primary care provider were significantly more likely to present during weekday office hours (p = 0.003), have longer symptom duration (p<0.001), and not know of other options (p = 0.001).

Conclusions

Many parents seek pediatric emergency department care for low-acuity problems despite their child having a primary care provider. Ensuring timely access to these providers may help reduce pediatric emergency department overuse. Educational initiatives should inform parents about low-acuity problems and where appropriate care can/should be accessed.