Recombinant Influenza Virus Carrying the Respiratory Syncytial Virus (RSV) F85-93 CTL Epitope Reduces RSV Replication in Mice

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants worldwide. Despite decades of research, there is still no registered vaccine available for this major pathogen. We investigated the protective efficacy of a recombinant influenza virus, PR8/NA-F_85–93, that carries the RSV CD8⁺ T cell epitope F_85–93 in its neuraminidase stalk. F_85–93-specific cytotoxic T lymphocytes (CTLs) were induced in mice after a single intranasal immunization with PR8/NA-F_85-93 virus, and these CTLs provided a significant reduction in the lung viral load upon a subsequent challenge with RSV. To avoid influenza-induced morbidity, we treated mice with matrix protein 2 (M2e)-specific monoclonal antibodies before PR8/NA-F_85-93 virus infection. Treatment with anti-M2e antibodies reduced the infiltration of immune cells in the lungs upon PR8/NA-F_85-93 infection, whereas the formation of inducible bronchus-associated lymphoid tissue was not affected. Moreover, this treatment prevented body weight loss yet still permitted the induction of RSV F-specific T cell responses and significantly reduced RSV replication upon challenge. These results demonstrate that it is possible to take advantage of the infection-permissive protection of M2e-specific antibodies against influenza A virus to induce heterologous CD8⁺ T cell-mediated immunity by an influenza A virus vector expressing the RSV F_85-93 epitope.     

The deSUMOylase SENP7 promotes chromatin relaxation for homologous recombination DNA repair

SUMO conjugation is known to occur in response to double‐stranded DNA breaks in mammalian cells, but whether SUMO deconjugation has a role remains unclear. Here, we show that the SUMO/Sentrin/Smt3‐specific peptidase, SENP7, interacts with the chromatin repressive KRAB‐associated protein 1 (KAP1) through heterochromatin protein 1 alpha (HP1α). SENP7 promotes the removal of SUMO2/3 from KAP1 and regulates the interaction of the chromatin remodeler CHD3 with chromatin. Consequently, in the presence of CHD3, SENP7 is required for chromatin relaxation in response to DNA damage, for homologous recombination repair and for cellular resistance to DNA‐damaging agents. Thus, deSUMOylation by SENP7 is required to promote a permissive chromatin environment for DNA repair.     

High Concordance of Bovine Single Nucleotide Polymorphism Genotypes Generated Using Two Independent Genotyping Strategies

Single nucleotide polymorphisms (SNPs) represent the most common form of DNA sequence variation in mammalian livestock genomes. While the past decade has witnessed major advances in SNP genotyping technologies, genotyping errors caused, in part, by the biochemistry underlying the genotyping platform used, can occur. These errors can distort project results and conclusions and can result in incorrect decisions in animal management and breeding programs; hence, SNP genotype calls must be accurate and reliable. In this study, 263 Bos spp. samples were genotyped commercially for a total of 16 SNPs. Of the total possible 4,208 SNP genotypes, 4,179 SNP genotypes were generated, yielding a genotype call rate of 99.31% (standard deviation ± 0.93%). Between 110 and 263 samples were subsequently re-genotyped by us for all 16 markers using a custom-designed SNP genotyping platform, and of the possible 3,819 genotypes a total of 3,768 genotypes were generated (98.70% genotype call rate, SD ± 1.89%). A total of 3,744 duplicate genotypes were generated for both genotyping platforms, and comparison of the genotype calls for both methods revealed 3,741 concordant SNP genotype call rates (99.92% SNP genotype concordance rate). These data indicate that both genotyping methods used can provide livestock geneticists with reliable, reproducible SNP genotypic data for in-depth statistical analysis.     

Obesity Is Associated with Lower Coronary Microvascular Density

Background

Obesity is associated with diastolic dysfunction, lower maximal myocardial blood flow, impaired myocardial metabolism and increased risk of heart failure. We examined the association between obesity, left ventricular filling pressure and myocardial structure.

Methods

We performed histological analysis of non-ischemic myocardium from 57 patients (46 men and 11 women) undergoing coronary artery bypass graft surgery who did not have previous cardiac surgery, myocardial infarction, heart failure, atrial fibrillation or loop diuretic therapy.

Results

Non-obese (body mass index, BMI, ≤30 kg/m², n=33) and obese patients (BMI >30 kg/m², n=24) did not differ with respect to myocardial total, interstitial or perivascular fibrosis, arteriolar dimensions, or cardiomyocyte width. Obese patients had lower capillary length density (1145±239, mean±SD, vs. 1371±333 mm/mm³, P=0.007) and higher diffusion radius (16.9±1.5 vs. 15.6±2.0 μm, P=0.012), in comparison with non-obese patients. However, the diffusion radius/cardiomyocyte width ratio of obese patients (0.73±0.11 μm/μm) was not significantly different from that of non-obese patients (0.71±0.11 μm/μm), suggesting that differences in cardiomyocyte width explained in part the differences in capillary length density and diffusion radius between non-obese and obese patients. Increased BMI was associated with increased pulmonary capillary wedge pressure (PCWP, P<0.0001), and lower capillary length density was associated with both increased BMI (P=0.043) and increased PCWP (P=0.016).

Conclusions

Obesity and its accompanying increase in left ventricular filling pressure were associated with lower coronary microvascular density, which may contribute to the lower maximal myocardial blood flow, impaired myocardial metabolism, diastolic dysfunction and higher risk of heart failure in obese individuals.     

Loss of Protein Kinase C-δ Protects against LPS-Induced Osteolysis Owing to an Intrinsic Defect in Osteoclastic Bone Resorption

Bone remodeling is intrinsically regulated by cell signaling molecules. The Protein Kinase C (PKC) family of serine/threonine kinases is involved in multiple signaling pathways including cell proliferation, differentiation, apoptosis and osteoclast biology. However, the precise involvement of individual PKC isoforms in the regulation of osteoclast formation and bone homeostasis remains unclear. Here, we identify PKC-δ as the major PKC isoform expressed among all PKCs in osteoclasts; including classical PKCs (−α, −β and −γ), novel PKCs (−δ, −ε, −η and −θ) and atypical PKCs (−ι/λ and −ζ). Interestingly, pharmacological inhibition and genetic ablation of PKC-δ impairs osteoclastic bone resorption in vitro. Moreover, disruption of PKC-δ activity protects against LPS-induced osteolysis in mice, with osteoclasts accumulating on the bone surface failing to resorb bone. Treatment with the PKC-δ inhibitor Rottlerin, blocks LPS-induced bone resorption in mice. Consistently, PKC-δ deficient mice exhibit increased trabeculae bone containing residual cartilage matrix, indicative of an osteoclast-rich osteopetrosis phenotype. Cultured ex vivo osteoclasts derived from PKC-δ null mice exhibit decreased CTX-1 levels and MARKS phosphorylation, with enhanced formation rates. This is accompanied by elevated gene expression levels of cathepsin K and PKC −α, −γ and −ε, as well as altered signaling of pERK and pcSrc416/527 upon RANKL-induction, possibly to compensate for the defects in bone resorption. Collectively, our data indicate that PKC-δ is an intrinsic regulator of osteoclast formation and bone resorption and thus is a potential therapeutic target for pathological osteolysis.     

S-Nitrosylation of platelet alphaIIbbeta3 as revealed by Raman spectroscopy

The exact mechanisms regulating conformational changes in the platelet-specific integrin alphaIIbbeta3 are not fully understood. However, a role exists for thiol/disulfide exchange in integrin conformational changes leading to altered disulfide bonding patterns, via its endogenous thiol isomerase activity. Nitric oxide (NO) accelerates this intrinsic enzymatic activity and, in doing so, reverses the activational state of the integrin on the platelet surface toward a more unactivated one. We propose that it is an S-nitrosylation-induced "shuffling" of thiol/disulfide exchange that regulates this reversal of the activated state of the integrin. In this study, we use Raman spectroscopy to explore S-nitrosylation of purified alphaIIbbeta3. Using S-nitrosoglutathione (GSNO) as a model system, we identify Raman markers which show a direct interaction between NO and the thiol groups of the integrin and reveal many of the structural changes that occur in alphaIIbbeta3 in the course of not only its activation but also its deactivation. Key conformational changes are detected within the integrin when treated with manganese (Mn2+), occurring mainly in the cysteine and disulfide regions of the protein, confirming the importance of thiol/disulfide exchange in integrin activation. These changes are subsequently shown to be reversed in the presence of NO.     

Influence of negative energy balance on cyclicity and fertility in the high producing dairy cow

The peripartum period is of critical importance to subsequent health and fertility. Most cows enter a state of negative energy balance (NEB) associated with many metabolic changes which have carry over effects on the resumption and normality of estrous cyclicity and the success of subsequent inseminations. A dataset on 500 lactations explored the relationships between metabolic traits measured before and after calving with fertility. Stepwise multiple regression analysis showed that longer calving to conception intervals were associated with altered profiles of IGF-I, urea and body condition score. These relationships between metabolic profiles and fertility differed between first lactation cows (which are still growing but produce less milk) and mature animals. Early postpartum the liver undergoes extensive biochemical and morphological modifications to adapt to NEB, the uterus is extensively remodeled and must clear bacterial infections, and the ovary must resume ovulatory cycles. RNA isolated from liver and uterine tissues harvested 2 weeks postpartum from cows in mild (MNEB) and severe (SNEB) energy balance was used to screen the Affymetrix 23K bovine microarray. In liver, SNEB resulted in differential expression of key genes involved in lipid catabolism, gluconeogenesis, and the synthesis and stability of IGF-I. This was accompanied by reduced systemic concentrations of IGF-I which is likely to impact on ovarian function and early embryo development. Within endometrium, cows in SNEB showed histological evidence for higher levels of inflammation and the microarray analysis identified groups of differentially expressed genes involved in tissue remodeling and immune response. This may delay uterine repair after calving, likely contributing to the observed reduction in fertility.     

Partial purification and chemical characterization of a glycoprotein (putative hydrocolloid) emulsifier produced by a marine bacterium Antarctobacter

During screening for novel emulsifiers and surfactants, a marine alphaproteobacterium, Antarctobacter sp. TG22, was isolated and selected for its production of an extracellular emulsifying agent, AE22. This emulsifier was produced optimally in a low-nutrient seawater medium supplemented with glucose and was extractable by cold ethanol precipitation of the high-molecular-weight fraction (>100 kDa). Production of AE22 commenced towards the late exponential phase of growth, with maximum emulsifying activity detected after approximately 4 days of the cells entering the death phase. Chemical, chromatographic and nuclear magnetic resonance spectroscopic analysis confirmed AE22 to be a high-molecular-weight (>2,000 kDa) glycoprotein with high uronic acids content, thus denoting an apparent polyanionic structure. Functional characterization showed this polymer to compare well to xanthan gum and gum arabic as an emulsion-stabilizing agent for a range of different food oils. However, AE22 exhibited better stabilizing than emulsifying properties, which could be conferred by its viscosifying effect in solution or from certain chemical groups found on the polysaccharide or protein moieties of the polymer. This new high-molecular-weight glycoprotein exhibits interesting functional qualities that are comparable to other biopolymers of this type and shows particular promise as an emulsion-stabilizing agent in biotechnological applications.     

A trait-based approach across the native and invaded range to understand plant invasiveness and community impact

Alien plant species invasiveness and impact on diversity (i.e. species richness and composition) can be driven by the altered competitive interactions experienced by the invader in its invaded range compared to its native range. Trait-based competition effects on invasiveness can be mediated through size-asymmetric competition, i.e. a trait suit of the invader that drives competitive dominance, and through ‘niche differences', i.e. trait differentiation and thus minimized competition between invader and the invaded community. In terms of invasion impact, size-asymmetric competition is expected to result in competitive exclusion of co-occurring subordinate species, whereas ‘niche differences' might result in competitive exclusion of the most functionally similar co-occurring species. Although observational work does not allow the full disentanglement of both trait-based effects, it does allow to verify the occurrence of expected theoretical trait patters. In this study, we explored the trait-based competition effects on invasiveness and diversity impact for Rosa rugosa in both its invaded range in Belgium and its native range in Japan, based on seven functional traits across 100 vegetation plots. Following the predictions for enhanced invasiveness, we found much lower functional overlap between R. rugosa and the co-occurring species in the invaded range compared to the native range. This likely also explains the absence of diversity impact in its native range. Despite the absence of changes in species richness in the invaded range, the invader did strongly impact species composition of invaded communities. This impact occurred through strong shade tolerance responses, suggesting size-asymmetric competition effects and cover loss of co-occurring dominant species, next to exclusion of co-occurring species most functionally similar to the invader; suggesting niche difference effects. In conclusion, this case-study illustrates how exploring functional trait patterns across a species native and invaded range can help in understanding how trait-based competition processes can affect invasiveness and community impact.