Development and application of real-time PCR assays for quantification of genes encoding tetracycline resistance

We report here the development, validation, and use of three real-time PCR assays to quantify the abundance of the following three groups of tetracycline resistance genes: tet(A) and tet(C); tet(G); and tet genes encoding ribosomal protection proteins, including tet(M), tet(O), tetB(P), tet(Q), tet(S), tet(T), and tet(W). The assays were validated using known numbers of sample-derived tet gene templates added to microbiome DNA. These assays are both precise and accurate over at least 6 log tet gene copies. New tet gene variants were also identified from cloned tet amplicons as part of this study. The utility of these real-time PCR assays was demonstrated by quantifying the three tet gene groups present in bovine and swine manures, composts of swine manure, lagoons of hog house effluent, and samples from an Ekokan upflow biofilter system treating hog house effluent. The bovine manures were found to contain fewer copies of all three groups of tet genes than the swine manures. The composts of swine manures had substantially reduced tet gene abundance (up to 6 log), while lagoon storage or the upflow biofilter had little effect on tet gene abundance. These results suggest that the method of manure storage and treatment may have a substantial impact on the persistence and dissemination of tet genes in agricultural environments. These real-time PCR assays provide rapid, quantitative, cultivation-independent measurements of 10 major classes of tet genes, which should be useful for ecological studies of antibiotic resistance.     

SNP analysis of AMY2 and CTSL genes in Litopenaeus vannamei and Penaeus monodon shrimp

Genetic studies in shrimp have focused on disease, with production traits such as growth left unexamined. Two shrimp species, Litopenaeus vannamei and Penaeus monodon, which represent the majority of US shrimp imports, were selected for single nucleotide polymorphism (SNP) discovery in alpha-amylase (AMY2) and cathepsin-l (CTSL), both candidate genes for growth. In L. vannamei, four SNPs were found in AMY2 and one SNP was found in CTSL. In P. monodon, one SNP was identified in CTSL. The CTSL gene was mapped to linkage group 28 of P. monodon using the female map developed with the Australian P. monodon mapping population. Association analyses for the AMY2 and CTSL genes with body weight (BW) were performed in two L. vannamei populations. While neither gene was found to be significantly associated with BW in these populations, there was a trend in one population towards higher BW for allele G of CTSL SNP C681G.     

Mitochondrial DNA variation in Chinese and Indian rhesus macaques (Macaca mulatta)

DNA was extracted from the buffy coats or serum of 212 rhesus macaques (Macaca mulatta) sampled throughout the species' geographic range. An 835 base pair (bp) fragment of mitochondrial DNA (mtDNA) was amplified from each sample, sequenced, aligned, and used to estimate genetic distances from which phylogenetic trees were constructed. A tree that included sequences from rhesus macaques whose exact origins in China are known was used to determine the regional origin of clusters of haplotypes, or haplogroups, defined by the trees. Indian rhesus sequences formed one large homogeneous haplogroup with very low levels of nucleotide diversity and no geographic structure, and a second much smaller haplogroup apparently derived from Burma. The sequences from Burma and eastern and western China were quite divergent from those in the major haplogroup of India. Each of these sequences formed separate clusters of haplotypes that exhibited far greater nucleotide diversity and/or population structure. Correspondingly, sequences from Indian rhesus macaques that are considered to represent different subspecies (based on morphological differences) were intermingled in the tree, while those from China reflected some, but not all, aspects of subspecific taxonomy. Regional variation contributed 72% toward the paired differences between sequences in an analysis of molecular variance (AMOVA), and the average differences between the populations of eastern and western China were also statistically significant. These results suggest that Indian and Chinese rhesus macaques were reproductively isolated during most, if not all, of the Pleistocene, during which time Indian rhesus macaques experienced a severe genetic bottleneck, and that some gene flow westward into India was subsequently reestablished. Samples from breeding centers in three different provinces of China included sequences from rhesus macaques that originated in both eastern (or southern) and western China, confirming anecdotal reports that regional breeding centers in China exchange breeding stock. Genetic differences among rhesus macaques (even those acquired from the same regional breeding center) that originate in different geographic regions and are employed as subjects in biomedical experiments can contribute to phenotypic differences in the traits under study.     

Mapping of quantitative trait loci controlling adaptive traits in coastal Douglas-fir. IV. Cold-hardiness QTL verification and candidate gene mapping

Quantitative trait locus (QTL) analyses are used by geneticists to characterize the genetic architecture of quantitative traits, provide a foundation for marker-aided-selection (MAS), and provide a framework for positional selection of candidate genes. The most useful QTL for breeding applications are those that have been verified in time, space, and/or genetic background. In this study, spring cold-hardiness of Douglas-fir foliar tissues was evaluated in two clonally replicated (n=170 and 383 clones) full-sib cohorts derived from the same parental cross in two different years (made 5 years apart). The cohorts were established in widely separated forest test sites and tissues were artificially freeze tested using different cold injury assessment methods. Four of six unique QTL detected for spring cold-hardiness in needles of Cohort 1 were tentatively verified in the second cohort. Four additional QTL were detected in Cohort 2, two on linkage groups (LGs) not previously represented in the smaller cohort. In total, 10 unique QTL were identified across both cohorts. Seventeen of twenty-nine putative cold-hardiness candidate genes (Douglas-fir ESTs) placed on the Douglas-fir linkage map locate within the 95% confidence intervals of spring needle cold-hardiness QTL from the two cohorts and thus represent priority targets for initiating association mapping in Douglas-fir.     

Lysophosphatidic acid affinity chromatography reveals pyruvate kinase as a specific LPA-binding protein

Lysophosphatidic acid is a pleiotropic lipid signaling molecule that evokes a broad array of cellular responses including proliferation, tumor cell invasion, neurite retraction, cytoskeletal rearrangements and smooth muscle contraction. Generally, lysophosphatidic acid triggers physiological responses through interaction with specific plasma membrane receptors called LPA 1-4. There is, however, increasing evidence in support of intracellular proteins that interact with LPA. We employed Affigel-immobilized LPA to isolate cytoplasmic proteins that interact with this lysophospholipid. Among the proteins retained by this affinity matrix, pyruvate kinase, clathrin heavy chain and heat shock protein 70 (Hsp70) were identified by mass spectrometry. Isothermal titration calorimetry showed that pyruvate kinase contains one binding site for LPA (Ka approx. 10(6) M(-1)). Furthermore, LPA dissociates enzymatically active pyruvate-kinase tetramers into less active dimers, and is maximally active at concentrations close to its critical micelle concentration. These effects were not mimicked by other lysophospholipids. Co-immunoprecipitation experiments showed that pyruvate kinase interacts with clathrin, and confocal imaging revealed co-localization between clathrin and pyruvate kinase in the perinuclear region of cells. Our data suggest that pyruvate kinase partly exists in complex with clathrin in subcellular membranous areas, and that locally increased LPA levels can trigger inactivation of the metabolic enzyme.     

Solving the heart mechanics equations with Newton and quasi Newton methods--a comparison

The non-linear elasticity equations of heart mechanics are solved while emulating the effects of a propagating activation wave. The dynamics of a 1 cm(3) slab of active cardiac tissue was simulated as the electrical wave traversed the muscular heart wall transmurally. The regular Newton (Newton-Raphson) method was compared to two modified Newton approaches, and also to a third approach that delayed update only of some selected Jacobian elements. In addition, the impact of changing the time step (0.01, 0.1 and 1 ms) and the relative non-linear convergence tolerance (10(-4), 10(-3) and 10(-2)) was investigated. Updating the Jacobian only when slow convergence occurred was by far the most efficient approach, giving time savings of 83-96%. For each of the four methods, CPU times were reduced by 48-90% when the time step was increased by a factor 10. Increasing the convergence tolerance by the same factor gave time savings of 3-71%. Different combinations of activation wave speed, stress rate and bulk modulus revealed that the fastest method became relatively even faster as stress rate and bulk modulus was decreased, while the activation speed had negligible influence in this respect.     

Spinner dolphins in a remote Hawaiian atoll: social grouping and population structure

Spinner dolphins (Stenella longirostris) commonly use inshoreisland and atoll habitats for daytime rest and social interactionsand forage over deep waters at night. In Hawaii, they occurthroughout the archipelago. We applied photoidentification mark-recapturetechniques to study the population structure of spinner dolphinsassociated with remote Midway Atoll, far-western Hawaii. AtMidway, spinner dolphins live in stable bisexually bonded societiesof long-term associates, with strong geographic fidelity, noobvious fission-fusion, and limited contacts with other populations.Their large cohesive groups change little over time and arebehaviorally/socially discrete from other spinner dolphin groups.This social pattern differs considerably from the fluid fission-fusionmodel proposed previously for spinner dolphins associated witha large island habitat in the main Hawaiian Archipelago. Thesedifferences correspond to geographic separation and habitatvariation. While in the main islands there are several daytimeresting places available at each island habitat; in far-westernHawaii, areas of suitable habitat are limited and separatedby large stretches of open pelagic waters with potentially highrisk of shark predation. We hypothesize that with deepwaterfood resources in close proximity and other atolls relativelyfar away for easy (day-to-day) access, it is energetically morebeneficial in the remote Hawaiian atolls to remain "at home"than to travel to other atolls, so there is stability insteadof variability; there is no fission-fusion effect. Thus, thegeographic isolation and small size of remote atolls triggera process in which the fluidity of the fission-fusion spinnerdolphin society is replaced with long-term group fidelity andsocial stability.     

Involvement of mtDNA damage in free fatty acid-induced apoptosis

A growing body of evidence indicates that free fatty acids (FFA) can have deleterious effects on beta-cells. It has been suggested that the beta-cell dysfunction and death observed in diabetes may involve exaggerated activation of the inducible form of nitric oxide synthase (iNOS) by FFA, with the resultant generation of excess nitric oxide (NO). However, the cellular targets with which NO interact have not been fully identified. We hypothesized that one of these targets might be mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by exposure of INS-1 cells to FFA (2/1 oleate/palmetate). The results showed that FFA caused a dose-dependent increase in mtDNA damage. Additionally, using ligation-mediated PCR, we were able to show that the DNA damage pattern at the nucleotide level was identical to the one induced by pure NO and different from damage caused by peroxynitrite or superoxide. Following exposure to FFA, apoptosis was detected by DAPI staining and cytochrome c release. Treatment of INS-1 cells with the iNOS inhibitor aminoguanidine protected these cells from mtDNA damage and diminished the appearance of apoptosis. These studies suggest that mtDNA may be a sensitive target for NO-induced toxicity which may provoke apoptosis in beta-cells following exposure to FFA.     

Survival, growth, settlement and metamorphosis of refrigerated larvae of the mussel Mytilus galloprovincialis Lamarck and their use in settlement and antifouling bioassays

Straight-hinge veliger and pediveliger larvae of the mussel Mytilus galloprovincialis were refrigerated for varying periods for use in bioassays. Straight-hinge veliger larvae grew to the umbo-veliger stage after 2 months in the refrigerator, but no pediveligers were observed during the 3-month refrigeration period. The average survival rate of larvae in the refrigerator was 79% after 1 month, but gradually decreased with the refrigeration period, and was as low as 22% after 3 months. All refrigerated larvae grew to the pediveliger stage in the incubator at 17 degrees C at the same rate as that of the control larvae that were not refrigerated. Settlement and metamorphosis of pediveligers from both refrigerated and control groups were facilitated by microbial film and epinephrine and inhibited by phentolamine. Thus, refrigeration can be used as an effective method of storing larvae of M. galloprovincialis for use in assays to assess candidate settlement inducers and antifouling substances.