Climate change, precipitation and impacts on an estuarine refuge from disease

Background

Oysters play important roles in estuarine ecosystems but have suffered recently due to overfishing, pollution, and habitat loss. A tradeoff between growth rate and disease prevalence as a function of salinity makes the estuarine salinity transition of special concern for oyster survival and restoration. Estuarine salinity varies with discharge, so increases or decreases in precipitation with climate change may shift regions of low salinity and disease refuge away from optimal oyster bottom habitat, negatively impacting reproduction and survival. Temperature is an additional factor for oyster survival, and recent temperature increases have increased vulnerability to disease in higher salinity regions.

Methodology/Principal Findings

We examined growth, reproduction, and survival of oysters in the New York Harbor-Hudson River region, focusing on a low-salinity refuge in the estuary. Observations were during two years when rainfall was above average and comparable to projected future increases in precipitation in the region and a past period of about 15 years with high precipitation. We found a clear tradeoff between oyster growth and vulnerability to disease. Oysters survived well when exposed to intermediate salinities during two summers (2008, 2010) with moderate discharge conditions. However, increased precipitation and discharge in 2009 reduced salinities in the region with suitable benthic habitat, greatly increasing oyster mortality. To evaluate the estuarine conditions over longer periods, we applied a numerical model of the Hudson to simulate salinities over the past century. Model results suggest that much of the region with suitable benthic habitat that historically had been a low salinity refuge region may be vulnerable to higher mortality under projected increases in precipitation and discharge.

Conclusions/Significance

Predicted increases in precipitation in the northeastern United States due to climate change may lower salinities past important thresholds for oyster survival in estuarine regions with appropriate substrate, potentially disrupting metapopulation dynamics and impeding oyster restoration efforts, especially in the Hudson estuary where a large basin constitutes an excellent refuge from disease.     

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.     

Stiffness gradients mimicking in vivo tissue variation regulate mesenchymal stem cell fate

Mesenchymal stem cell (MSC) differentiation is regulated in part by tissue stiffness, yet MSCs can often encounter stiffness gradients within tissues caused by pathological, e.g., myocardial infarction ～8.7±1.5 kPa/mm, or normal tissue variation, e.g., myocardium ～0.6±0.9 kPa/mm; since migration predominantly occurs through physiological rather than pathological gradients, it is not clear whether MSC differentiate or migrate first. MSCs cultured up to 21 days on a hydrogel containing a physiological gradient of 1.0±0.1 kPa/mm undergo directed migration, or durotaxis, up stiffness gradients rather than remain stationary. Temporal assessment of morphology and differentiation markers indicates that MSCs migrate to stiffer matrix and then differentiate into a more contractile myogenic phenotype. In those cells migrating from soft to stiff regions however, phenotype is not completely determined by the stiff hydrogel as some cells retain expression of a neural marker. These data may indicate that stiffness variation, not just stiffness alone, can be an important regulator of MSC behavior.     

Assessing historical fish community composition using surveys, historical collection data, and species distribution models

Accurate establishment of baseline conditions is critical to successful management and habitat restoration. We demonstrate the ability to robustly estimate historical fish community composition and assess the current status of the urbanized Barton Creek watershed in central Texas, U.S.A. Fish species were surveyed in 2008 and the resulting data compared to three sources of fish occurrence information: (i) historical records from a museum specimen database and literature searches; (ii) a nearly identical survey conducted 15 years earlier; and (iii) a modeled historical community constructed with species distribution models (SDMs). This holistic approach, and especially the application of SDMs, allowed us to discover that the fish community in Barton Creek was more diverse than the historical data and survey methods alone indicated. Sixteen native species with high modeled probability of occurrence within the watershed were not found in the 2008 survey, seven of these were not found in either survey or in any of the historical collection records. Our approach allowed us to more rigorously establish the true baseline for the pre-development fish fauna and then to more accurately assess trends and develop hypotheses regarding factors driving current fish community composition to better inform management decisions and future restoration efforts. Smaller, urbanized freshwater systems, like Barton Creek, typically have a relatively poor historical biodiversity inventory coupled with long histories of alteration, and thus there is a propensity for land managers and researchers to apply inaccurate baseline standards. Our methods provide a way around that limitation by using SDMs derived from larger and richer biodiversity databases of a broader geographic scope. Broadly applied, we propose that this technique has potential to overcome limitations of popular bioassessment metrics (e.g., IBI) to become a versatile and robust management tool for determining status of freshwater biotic communities.     

Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.     

Assessing the effectiveness of a community intervention for monkeypox prevention in the Congo basin

Background

In areas where health resources are limited, community participation in the recognition and reporting of disease hazards is critical for the identification of outbreaks. This is particularly true for zoonotic diseases such as monkeypox that principally affect people living in remote areas with few health services. Here we report the findings of an evaluation measuring the effectiveness of a film-based community outreach program designed to improve the understanding of monkeypox symptoms, transmission and prevention, by residents of the Republic of the Congo (ROC) who are at risk for disease acquisition.

Methodology/Principal Findings

During 90 days, monkeypox outreach was conducted for ∼23,860 people in northern ROC. Two hundred seventy-one attendees (selected via a structured sample) were interviewed before and after participating in a small-group outreach session. The proportion of interviewees demonstrating monkeypox-specific knowledge before and after was compared. Significant gains were measured in areas of disease recognition, transmission, and mitigation of risk. The ability to recognize at least one disease symptom and a willingness to take a family member with monkeypox to the hospital increased from 49 and 45% to 95 and 87%, respectively (p<0.001, both). Willingness to deter behaviors associated with zoonotic risk, such as eating the carcass of a primate found dead in the forest, remained fundamentally unchanged however, suggesting additional messaging may be needed.

Conclusions/Significance

These results suggest that our current program of film-based educational activities is effective in improving disease-specific knowledge and may encourage individuals to seek out the advice of health workers when monkeypox is suspected.     

Enzymatic shaving of the tegument surface of live schistosomes for proteomic analysis: a rational approach to select vaccine candidates

Background

The membrane-associated and membrane-spanning constituents of the Schistosoma mansoni tegument surface, the parasite''s principal interface with the host bloodstream, have recently been characterized using proteomic techniques. Biotinylation of live worms using membrane-impermeant probes revealed that only a small subset of the proteins was accessible to the reagents. Their position within the multilayered architecture of the surface has not been ascertained.

Methodology/Principal Findings

An enzymatic shaving approach on live worms has now been used to release the most accessible components, for analysis by MS/MS. Treatment with trypsin, or phosphatidylinositol-specific phospholipase C (PiPLC), only minimally impaired membrane integrity. PiPLC-enriched proteins were distinguished from those released in parasite vomitus or by handling damage, using isobaric tagging. Trypsin released five membrane proteins, Sm200, Sm25 and three annexins, plus host CD44 and the complement factors C3 and C4. Nutrient transporters and ion channels were absent from the trypsin fraction, suggesting a deeper location in the surface complex; surprisingly, two BAR-domain containing proteins were released. Seven parasite and two host proteins were enriched by PiPLC treatment, the vaccine candidate Sm29 being the most prominent along with two orthologues of human CD59, potentially inhibitors of complement fixation. The enzymes carbonic anhydrase and APD-ribosyl cyclase were also enriched, plus Sm200 and alkaline phosphatase. Host GPI-anchored proteins CD48 and CD90, suggest ‘surface painting’ during worm peregrination in the portal system.

Conclusions/Significance

Our findings suggest that the membranocalyx secreted over the tegument surface is not the inert barrier previously proposed, some tegument proteins being externally accessible to enzymes and thus potentially located within it. Furthermore, the detection of C3 and C4 indicates that the complement cascade is initiated, while two CD59 orthologues suggest a potential mechanism for its inhibition. The detection of several host proteins is a testimonial to the acquisitive properties of the tegument surface. The exposed parasite proteins could represent novel vaccine candidates for combating this neglected disease.