Molecular systematics of South American dolphins Sotalia: sister taxa determination and phylogenetic relationships, with insights into a multi-locus phylogeny of the Delphinidae

The evolutionary relationships among members of the cetacean family Delphinidae, the dolphins, pilot whales and killer whales, are still not well understood. The genus Sotalia (coastal and riverine South American dolphins) is currently considered a member of the Stenoninae subfamily, along with the genera Steno (rough toothed dolphin) and Sousa (humpbacked dolphin). In recent years, a revision of this classification was proposed based on phylogenetic analysis of the mitochondrial gene cytochrome b, wherein Sousa was included in the Delphininae subfamily, keeping only Steno and Sotalia as members of the Stenoninae subfamily. Here we investigate the phylogenetic placement of Sotalia using two mitochondrial genes, six autosomal introns and four Y chromosome introns, providing a total of 5,196 base pairs (bp) for each taxon in the combined dataset. Sequences from these genomic regions were obtained for 17 delphinid species, including at least one species from each of five or six currently recognized subfamilies plus five odontocete outgroup species. Maximum Parsimony, Maximum Likelihood and Bayesian phylogenetic analysis of independent (each fragment) and combined datasets (mtDNA, nuDNA or mtDNA+nuDNA) showed that Sotalia and Sousa fall within a clade containing other members of Delphininae, exclusive of Steno. Sousa was resolved as the sister taxon to Sotalia according to analysis of the nuDNA dataset but not analysis of the mtDNA or combined mtDNA+nuDNA datasets. Based on the results from our multi-locus analysis, we offer several novel changes to the classification of Delphinidae, some of which are supported by previous morphological and molecular studies.     

A quantitative analysis of microalgal lipids for optimization of biodiesel and omega‐3 production

The lipid characteristics of microalgae are known to differ between species and change with growth conditions. This work provides a methodology for lipid characterization that enables selection of the optimal strain, cultivation conditions, and processing pathway for commercial biodiesel production from microalgae. Two different microalgal species, Nannochloropsis sp. and Chlorella sp., were cultivated under both nitrogen replete and nitrogen depleted conditions. Lipids were extracted and fractionated into three major classes and quantified gravimetrically. The fatty acid profile of each fraction was analyzed using GC–MS. The resulting quantitative lipid data for each of the cultures is discussed in the context of biodiesel and omega‐3 production. This approach illustrates how the growth conditions greatly affect the distribution of fatty acid present in the major lipid classes and therefore the suitability of the lipid extracts for biodiesel and other secondary products. Biotechnol. Bioeng. 2013; 110: 2096–2104. © 2013 Wiley Periodicals, Inc.     

The herpes simplex virus type 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro

The replication of herpes simplex virus type 1 (HSV-1) DNA is associated with a high degree of homologous recombination. While cellular enzymes may take part in mediating this recombination, we present evidence for an HSV-1-encoded recombinase activity. HSV-1 alkaline nuclease, encoded by the UL12 gene, is a 5'-->3' exonuclease that shares homology with Redalpha, commonly known as lambda exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. The HSV-1 single-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Redbeta synaptase component of the phage lambda recombinase. Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda. Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products. The optimal conditions for strand exchange were 1 mM MgCl(2), 40 mM NaCl, and pH 7.5. Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end. Nuclease-defective UL12 could not support this reaction. These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism.     

Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world’s leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1–893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/c mice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocular mucosa was well tolerated without signs of inflammation. N-PmpC-specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFNγ immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage.     

Hypoxia-induced acute lung injury in murine models of sickle cell disease

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human alpha/sickle beta-globin (halphabetaS) transgene (SCD mice) or are heterozygous for the normal murine beta-globin gene and express the halphabetaS transgene (mbeta+/-, halphabetaS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing *NO generation and predisposing the lung to vaso-occlusion.     

Highly purified lipoteichoic acid induced pro-inflammatory signalling in primary culture of rat microglia through Toll-like receptor 2: selective potentiation of nitric oxide production by muramyl dipeptide

In contrast to the role of lipopolysaccharide from Gram-negative bacteria, the role of Gram-positive bacterial components in inducing inflammation in the CNS remains controversial. We studied the potency of highly purified lipoteichoic acid and muramyl dipeptide isolated from Staphylococcus aureus to activate primary cultures of rat microglia. Exposure of pure microglial cultures to lipoteichoic acid triggered a significant time- and dose-dependent production of pro-inflammatory cytokines (tumour-necrosis factor-alpha, interleukin-1beta, interleukin-6) and nitric oxide. Muramyl dipeptide strongly and selectively potentiated lipoteichoic acid-induced inducible nitric oxide synthase expression and nitric oxide production. However, it did not have any significant influence on the production of pro-inflammatory cytokines. As bacterial components are recognised by the innate immunity through Toll-like receptors (TLRs) we showed that lipoteichoic acid was recognised in microglia by the TLR2 and lipopolysaccharide by the TLR4, as cells isolated from mice lacking TLR2 or TLR4 did not produce pro-inflammatory cytokines and nitric oxide upon lipoteichoic acid or lipopolysaccharide stimulation, respectively. Lipoteichoic acid-induced glia activation was mediated by p38 and ERK1/2 MAP kinases, as pretreatment with inhibitor of p38 or ERK1/2 decreased lipoteichoic acid-induced cytokine release, iNOS mRNA expression and nitric oxide production. The observed pro-inflammatory response induced by lipoteichoic acid-activated microglia could play a major role in the inflammatory response of CNS induced by Gram-positive bacteria.     

Characterization of rumen ciliate community composition in domestic sheep, deer, and cattle, feeding on varying diets, by means of PCR-DGGE and clone libraries

The structure and variability of ciliate protozoal communities in the rumens of domestic New Zealand ruminants feeding on different diets was investigated. The relative abundance of ciliates compared with bacteria was similar across all samples. However, molecular fingerprinting of communities showed ruminant-specific differences in species composition. Community compositions of cattle were significantly influenced by diet. In contrast, diet effects in deer and sheep were weaker than the animal-to-animal variation. Cloning and sequencing of almost-full-length 18S rRNA genes from representative samples revealed that New Zealand ruminants were colonized by at least nine genera of ciliates and allowed the assignment of samples to two distinct community types. Cattle contained A-type communities, with most sequences closely related to those of the genera Polyplastron and Ostracodinium. Deer and sheep (with one exception) harboured B-type communities, with the majority of sequences belonging to the genera Epidinium and Eudiplodinium. It has been suggested that species composition of ciliate communities may impact methane formation in ruminants, with the B-type producing more methane. Therefore, manipulation of ciliate communities may be a means of mitigating methane emissions from grazing sheep and deer in New Zealand.     

Upregulation of IL-21 receptor on B cells and IL-21 secretion distinguishes novel 2009 H1N1 vaccine responders from nonresponders among HIV-infected persons on combination antiretroviral therapy

Mechanisms underlying failure of novel 2009 H1N1 influenza vaccine-induced Ab responses in HIV-infected persons are poorly understood. This study prospectively evaluated 16 HIV-infected patients on combination antiretroviral therapy and eight healthy controls (HC) who received a single 15 μg dose of nonadjuvanted novel 2009 H1N1 influenza vaccine during the 2009 H1N1 epidemic. Peripheral blood was collected at baseline (T0) and at 7 d (T1) and 28 d (T2) postvaccination for evaluation of immune responses. Prevaccination hemagglutination inhibition Ab titer was <1:20 in all except one study participant. At T2, all HC and 8 out of 16 patients (50%) developed a vaccine-induced Ab titer of ≥ 1:40. Vaccine responder (R) and vaccine nonresponder patients were comparable at T0 in age, CD4 counts, virus load, and B cell immunophenotypic characteristics. At T2, HC and R patients developed an expansion of phenotypic and functional memory B cells and ex vivo H1N1-stimulated IgG Ab-secreting cells in an ELISPOT assay. The memory B cell response was preceded by a significant expansion of plasmablasts and spontaneous H1N1-specific Ab-secreting cells at T1. At T2, HC and R patients also exhibited significant increases in serum IL-21 levels and in the frequency and mean fluorescence intensity of IL-21R-expressing B cells, which correlated with serum H1N1 Ab titers. Vaccine nonresponder patients failed to develop the above-described vaccine-induced immunologic responses. The novel association of novel 2009 H1N1 vaccine-induced Ab responses with IL-21/IL-21R upregulation and with development of memory B cells and plasmablasts has implications for future research in vaccine design.