Sequencing,Annotation, and Characterization of the Influenza Ferret Infectome

Ferrets have become an indispensable tool in the understanding of influenza virus virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza infectome during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals was subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pandemic H1N1 (pdmH1N1) influenza virus infection were analyzed by digital gene expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days postinfection, the innate immune gene expression was replaced by the adaptive immune response, which correlates with viral clearance. Reinfection with homologous pandemic influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas.     

Genetic relatedness does not retain spatial pattern across multiple spatial scales: dispersal and colonization in the coral,Pocillopora damicornis

Patterns of isolation by distance are uncommon in coral populations. Here, we depart from historical trends of large‐scale, geographical genetic analyses by scaling down to a single patch reef in Kāne‘ohe Bay, Hawai‘i, USA, and map and genotype all colonies of the coral, Pocillopora damicornis. Six polymorphic microsatellite loci were used to assess population genetic and clonal structure and to calculate individual colony pairwise relatedness values. Our results point to an inbred, highly clonal reef (between 53 and 116 clonal lineages of 2352 genotyped colonies) with a much skewed genet frequency distribution (over 70% of the reef was composed of just seven genotypes). Spatial autocorrelation analyses revealed that corals found close together on the reef were more genetically related than corals further apart. Spatial genetic structure disappears, however, as spatial scale increases and then becomes negative at the largest distances. Stratified, random sampling of three neighbouring reefs confirms that reefs are demographically open and inter‐reef genetic structuring was not detected. Attributing process to pattern in corals is complicated by their mixed reproductive strategies. Separate autocorrelation analyses, however, show that the spatial distribution of both clones and nonclones contributes to spatial genetic structure. Overall, we demonstrate genetic structure on an intrareef scale and genetic panmixia on an inter‐reef scale indicating that, for P. damicornis, the effect of small‐ and large‐scale dispersal processes on genetic diversity are not the same. By starting from an interindividual, intrareef level before scaling up to an inter‐reef level, this study demonstrates that isolation‐by‐distance patterns for the coral P. damicornis are limited to small scales and highlights the importance of investigating genetic patterns and ecological processes at multiple scales.     

Differential responses by two closely related native fishes to restoration actions

Globally, river degradation has decimated freshwater fish populations. To help reverse this trend in a southeastern Australia river, we used multiple restoration actions, including reintroduction of instream woody habitat, riparian revegetation, removal of a weir hindering fish movement, fencing out livestock, and controlling riparian weeds. We monitored the responses of native fish at the segment scale (20 km) and reach scale (0.3 km) over 7 years to assess the effectiveness of the different restoration strategies. Two closely related species, Murray cod Maccullochella peeli and trout cod Maccullochella macquariensis, increased at the restored segment compared with the control segment. However, inherent differences between river segments and low sample size hampered assessment of the mechanisms responsible for segment‐scale changes in fish abundance. In contrast, at the reach scale, only M. peeli abundance significantly increased in reaches supplemented with wood. These differential responses by 2 closely related fish species likely reflect species‐specific responses to increased habitat availability and enhanced longitudinal connectivity when the weir improved passage around a fishway. Changes in M. peeli abundance in segments supplemented with and without wood suggest an increase in carrying capacity and not simply a redistribution of individuals within the segment, facilitated the observed expansion. Our findings confirm the need to consider individual fish species' habitat preferences carefully when designing restoration interventions. Further, species‐specific responses to restoration actions provide waterway managers with precise strategies to target fish species for recovery and the potential to predict fish outcomes based on ecological preferences.     

Modulating biochemical perturbations during 72-hour machine perfusion of kidneys: role of preservation solution

This study documents renal biochemistry during hypothermic machine perfusion of kidneys. It is intended to demonstrate that a comprehensive evaluation of organ viability during ex-vivo preservation is needed to increase the number of organs available for transplantation and to reduce the current renal discard rate. Porcine kidneys were hypothermically machine perfused for 72 h with either Unisol-UHK or Belzer-Machine Perfusion Solution, (Belzer-MPS). Renal perfusate samples were periodically collected and biochemically analyzed. Significant differences were measured in the renal metabolic activity between the two experimental groups while similar values for traditional parameters such as renal flow rate and vascular resistance values were recorded. The effluent of UHK perfused kidneys showed strong metabolites and NH(4)(+) dynamics (P<0.05 vs. baseline), while the Belzer-MPS kidneys metabolic activity led to little or no change of the effluent biochemistry relative to baseline.     

O-fucosylation is required for ADAMTS13 secretion

ADAMTS13 is a plasma metalloproteinase that cleaves von Willebrand factor to smaller, less thrombogenic forms. Deficiency of ADAMTS13 activity in plasma leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains eight thrombospondin type 1 repeats (TSR), seven of which contain a consensus sequence for the direct addition of fucose to the hydroxyl group of serine or threonine. Mass spectral analysis of tryptic peptides derived from human ADAMTS13 indicate that at least six of the TSRs are modified with an O-fucose disaccharide. Analysis of [(3)H]fucose metabolically incorporated into ADAMTS13 demonstrated that the disaccharide has the structure glucose-beta1,3-fucose. Mutation of the modified serine to alanine in TSR2, TSR5, TSR7, and TSR8 reduced the secretion of ADAMTS13. Mutation of more than one site dramatically reduced secretion regardless of the sites mutated. When the expression of protein O-fucosyltransferase 2 (POFUT2), the enzyme that transfers fucose to serines in TSRs, was reduced using siRNA, the secretion of ADAMTS13 decreased. A similar outcome was observed when ADAMTS13 was expressed in a cell line unable to synthesize the donor for fucose addition, GDP-fucose. Although overexpression of POFUT2 did not affect the secretion of wild-type ADAMTS13, it did increase the secretion of the ADAMTS13 TSR1,2 double mutant but not that of ADAMTS13 TSR1-8 mutant. Together these findings indicate that O-fucosylation is functionally significant for secretion of ADAMTS13.     

RCAN1-1L is overexpressed in neurons of Alzheimer's disease patients

At least two different isoforms of RCAN1 mRNA are expressed in neuronal cells in normal human brain. Although RCAN1 mRNA is elevated in brain regions affected by Alzheimer's disease, it is not known whether the disease affects neuronal RCAN1, or if other cell types (e.g. astrocytes or microglia) are affected. It is also unknown how many protein isoforms are expressed in human brain and whether RCAN1 protein is overexpressed in Alzheimer's disease. We explored the expression of both RCAN1-1 and RCAN1-4 mRNA isoforms in various cell types in normal and Alzheimer's disease postmortem samples, using the combined technique of immunohistochemistry and in situ hybridization. We found that both exon 1 and exon 4 are predominantly expressed in neuronal cells, and no significant expression of either of the exons was observed in astocytes or microglial cells. This was true in both normal and Alzheimer's disease brain sections. We also demonstrate that RCAN1-1 mRNA levels are approximately two-fold higher in neurons from Alzheimer's disease patients versus non-Alzheimer's disease controls. Using western blotting, we now show that there are three RCAN1 protein isoforms expressed in human brain: RCAN1-1L, RCAN1-1S, and RCAN1-4. We have determined that RCAN1-1L is expressed at twice the level of RCAN1-4, and that there is very minor expression of RCAN1-1S. We also found that the RCAN1-1L protein is overexpressed in Alzheimer's disease patients, whereas RCAN1-4 is not. From these results, we conclude that RCAN1-1 may play a role in Alzheimer's disease, whereas RCAN1-4 may serve another purpose.     

Strong genetic isolation despite wide distribution in a commercially exploited coastal shark

The common smoothhound, Mustelus mustelus, is an epibenthic species targeted by fisheries around the world driven by the increasing demand for shark products. Given the wide-spread occurrence of this species and corresponding lack of molecular data in many areas of said distribution, baseline molecular assessments of this commercially important shark may contribute to finer-scale analyses in areas in which this species is targeted. Therefore, population genetic analyses were conducted along the East Atlantic, from the Mediterranean Sea to the south-east coast of Africa, using microsatellite markers and the mitochondrial control region (mtCR). Overall, M. mustelus displayed low to moderate genetic diversity, with the Mediterranean populations appearing to exhibit the lowest mitochondrial diversity, and the west African populations displaying the lowest nuclear diversity. Microsatellite analysis indicated strong genetic differentiation between the three regions, with finer-scale population structure in each region, without correlation between genetic and geographical distance. For the mtCR sequences, a total of 18 haplotypes were identified, with a high degree of divergence discernable between the regions, largely in accordance with the microsatellite data. The study documents a remarkable level of population isolation across a vast area, suggesting little or no present-day connectivity among extant populations. The findings may serve as an essential baseline for global population management and commercial traceability of this threatened shark.