Characterization of Dinophysis spp. (Dinophyceae,Dinophysiales) from the mid-Atlantic region of the United States1

Due to the increasing prevalence of Dinophysis spp. and their toxins on every US coast in recent years, the need to identify and monitor for problematic Dinophysis populations has become apparent. Here, we present morphological analyses, using light and scanning electron microscopy, and rDNA sequence analysis, using a ~2-kb sequence of ribosomal ITS1, 5.8S, ITS2, and LSU DNA, of Dinophysis collected in mid-Atlantic estuarine and coastal waters from Virginia to New Jersey to better characterize local populations. In addition, we analyzed for diarrhetic shellfish poisoning (DSP) toxins in water and shellfish samples collected during blooms using liquid-chromatography tandem mass spectrometry and an in vitro protein phosphatase inhibition assay and compared this data to a toxin profile generated from a mid-Atlantic Dinophysis culture. Three distinct morphospecies were documented in mid-Atlantic surface waters: D. acuminata, D. norvegica, and a “small Dinophysis sp.” that was morphologically distinct based on multivariate analysis of morphometric data but was genetically consistent with D. acuminata. While mid-Atlantic D. acuminata could not be distinguished from the other species in the D. acuminata-complex (D. ovum from the Gulf of Mexico and D. sacculus from the western Mediterranean Sea) using the molecular markers chosen, it could be distinguished based on morphometrics. Okadaic acid, dinophysistoxin 1, and pectenotoxin 2 were found in filtered water and shellfish samples during Dinophysis blooms in the mid-Atlantic region, as well as in a locally isolated D. acuminata culture. However, DSP toxins exceeded regulatory guidance concentrations only a few times during the study period and only in noncommercial shellfish samples.     

Comparative chemical attributes of native North American hop,Humulus lupulus var. lupuloides E. Small

The genetic diversity of 159 representative genotypes of native hop (Humulus lupulus var. lupuloides E. Small, Cannabaceae) from 34 selected populations was assessed by relative magnitudes and ranges of alpha acids (AA), beta acids (BA), and the cohumulone (CoH) component of alpha acids, with reference to temporal changes between 1989-1990 and 2001, and to the same attributes in American and European hop cultivars, principally H. lupulus var. lupulus L. Chemical profiles of these genotypes were generated by high pressure liquid chromatography (HPLC) of methanol extracts from their processed samples (cones). The alpha ratio (AR, alpha acids / alpha+beta acids) measured the degree to which alpha acids predominated in cone extracts. Synchronous ranges of AR and CoH were also selected for graphic portrayals of native hop genotypic diversity. Cones sampled and analyzed from eight populations that were accessible in both 1989 and 2001 were distinct in chemical attributes, indicating a succession of genotypes, and suggesting temporal cycling of H. lupulus var.lupuloides germplasm. The principal distinctions between the two sub-species were a markedly higher proportion of CoH (38-88% vs. 19-41%) in alpha acids of H. l. var. lupuloides, and generally higher concentrations of AA in cultivars of both American and European commercial hop cultivars, predominantly H. lupulus var. lupulus. All of the 159 native hop genotypes also contained detectable levels of xanthohumol and xanthogalenol, prenylflavonoids recently reported to have mammalian anti-cancer activity. Some native genotypes had previously exhibited natural repellence of insect and mite pests; thus H. lupulus var. lupuloides germplasm offers a diverse resource of underutilized and yet undefined biochemicals.     

Forecasted range shifts of arid-land fishes in response to climate change

Climate change is poised to alter the distributional limits, center, and size of many species. Traits may influence different aspects of range shifts, with trophic generality facilitating shifts at the leading edge, and greater thermal tolerance limiting contractions at the trailing edge. The generality of relationships between traits and range shifts remains ambiguous however, especially for imperiled fishes residing in xeric riverscapes. Our objectives were to quantify contemporary fish distributions in the Lower Colorado River Basin, forecast climate change by 2085 using two general circulation models, and quantify shifts in the limits, center, and size of fish elevational ranges according to fish traits. We examined relationships among traits and range shift metrics either singly using univariate linear modeling or combined with multivariate redundancy analysis. We found that trophic and dispersal traits were associated with shifts at the leading and trailing edges, respectively, although projected range shifts were largely unexplained by traits. As expected, piscivores and omnivores with broader diets shifted upslope most at the leading edge while more specialized invertivores exhibited minimal changes. Fishes that were more mobile shifted upslope most at the trailing edge, defying predictions. No traits explained changes in range center or size. Finally, current preference explained multivariate range shifts, as fishes with faster current preferences exhibited smaller multivariate changes. Although range shifts were largely unexplained by traits, more specialized invertivorous fishes with lower dispersal propensity or greater current preference may require the greatest conservation efforts because of their limited capacity to shift ranges under climate change.     

More than skin and bones: Comparing extraction methods and alternative sources of DNA from avian museum specimens

Next‐generation sequencing has greatly expanded the utility and value of museum collections by revealing specimens as genomic resources. As the field of museum genomics grows, so does the need for extraction methods that maximize DNA yields. For avian museum specimens, the established method of extracting DNA from toe pads works well for most specimens. However, for some specimens, especially those of birds that are very small or very large, toe pads can be a poor source of DNA. In this study, we apply two DNA extraction methods (phenol–chloroform and silica column) to three different sources of DNA (toe pad, skin punch and bone) from 10 historical avian museum specimens. We show that a modified phenol–chloroform protocol yielded significantly more DNA than a silica column protocol (e.g., Qiagen DNeasy Blood & Tissue Kit) across all tissue types. However, extractions using the silica column protocol contained longer fragments on average than those using the phenol–chloroform protocol, probably as a result of loss of small fragments through the silica column. While toe pads yielded more DNA than skin punches and bone fragments, skin punches proved to be a reliable alternative source of DNA and might be especially appealing when toe pad extractions are impractical. Overall, we found that historical bird museum specimens contain substantial amounts of DNA for genomic studies under most extraction scenarios, but that a phenol–chloroform protocol consistently provides the high quantities of DNA required for most current genomic protocols.     

Directed Mutation of the Rubisco Large Subunit of Tobacco Influences Photorespiration and Growth

The gene for the large subunit of Rubisco was specifically mutated by transforming the chloroplast genome of tobacco (Nicotiana tabacum). Codon 335 was altered to encode valine instead of leucine. The resulting mutant plants could not grow without atmospheric CO2 enrichment. In 0.3% (v/v) CO2, the mutant and wild-type plants produced similar amounts of Rubisco but the extent of carbamylation was nearly twice as great in the mutants. The mutant enzyme's substrate-saturated CO2-fixing rate and its ability to distinguish between CO2 and O2 as substrates were both reduced to 25% of the wild type's values. Estimates of these parameters obtained from kinetic assays with the purified mutant enzyme were the same as those inferred from measurements of photosynthetic gas exchange with leaves of mutant plants. The Michaelis constants for CO2, O2, and ribulose-1,5-bisphosphate were reduced and the mutation enhanced oxygenase activity at limiting O2 concentrations. Consistent with the reduced CO2 fixation rate at saturating CO2, the mutant plants grew slower than the wild type but they eventually flowered and reproduced apparently normally. The mutation and its associated phenotype were inherited maternally. The chloroplast-transformation strategy surmounts previous obstacles to mutagenesis of higher-plant Rubisco and allows the consequences for leaf photosynthesis to be assessed.     

Paternal behavior influences development of aggression and vasopressin expression in male California mouse offspring

Parental care has been demonstrated to have important effects on offspring behavioral development. California mice (Peromyscus californicus) are biparental, and correlational evidence suggests that pup retrieving by fathers has important effects on the development of aggressive behavior and extra-hypothalamic vasopressin systems. We tested whether retrievals affected these systems by manipulating paternal retrieval behavior between day 15 and 21 postpartum. Licking and grooming behavior affect behavioral development in rats, so we also experimentally reduced huddling and grooming behavior by castrating a subset of fathers. Experimentally increasing the frequency of paternal pup retrieving behavior decreased attack latency in resident-intruder in both male and female adult offspring, whereas experimental reduction of huddling and grooming had no effect. In a separate group of male offspring, we examined vasopressin immunoreactivity (AVP-ir) in two regions of the posterior bed nucleus of the stria terminalis (BNST): the dorsal fiber tracts (dBNST) and the ventral cell body-containing region (vBNST). Experimentally increasing retrievals led to an apparent shift in AVP-ir distribution. Specifically, offspring from the high retrieval group had more AVP-ir than offspring from the sham retrieval group in the dBNST, whereas the opposite was observed in the vBNST. Experimental reduction of paternal grooming was associated with increased AVP-ir in the paraventricular nucleus and also increased corticosterone and progesterone, similar to observed effects of maternal grooming on HPA function. This study provides further evidence that paternal behavior influences the development of aggression and associated neural substrates.     

Influence of experimental,environmental, and geographic factors on nutrient‐diffusing substrate experiments in running waters

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Predation on snails by three species of triclad and its bearing on the distribution of Planaria torva in Britain

Dugesia polychroa, D. lugubris and Planaria torva preyed upon snails of differing morphologies and while the Dugesia spp. fed approximately in proportion to snail abundance, PL torva was more selective on Potamopyrgus jenkinsi. The incidence of feeding on four snail taxa by the Dugesia spp. was significantly correlated and differed strikingly from that of PI. torva where feeding was related to its annual life cycle. Large peaks of feeding coincided in the Dugesia spp. for the four snail types; this was attributed to snail vulnerability being increased by environmental disturbance. Comparison of feeding by Dugesia spp. in the presence and absence of PI. torva indicated that severe competition was occurring for food with PI. torva having a food refuge in P. jenkinsi. The distribution of PI. torva in Britain is largely confined to productive habitats in the Glasgow-Edinburgh and East Anglia regions. It is concluded that historical rather than ecological events have played the major part in its distribution to date. The strong association of PL torva with port areas and the canal system suggests that it is a recent introduction via the timber trade with Fennoscania.     

Kaposi's sarcoma-associated herpesvirus latent gene vFLIP inhibits viral lytic replication through NF-kappaB-mediated suppression of the AP-1 pathway: a novel mechanism of virus control of latency

Kaposi's sarcoma-associated herpesvirus (KSHV) latency is central to the evasion of host immune surveillances and induction of KSHV-related malignancies. The mechanism of KSHV latency remains unclear. Here, we show that the KSHV latent gene vFLIP promotes viral latency by inhibiting viral lytic replication. vFLIP suppresses the AP-1 pathway, which is essential for KSHV lytic replication, by activating the NF-kappaB pathway. Thus, by manipulating two convergent cellular pathways, vFLIP regulates both cell survival and KSHV lytic replication to promote viral latency. These results also indicate that the effect of the NF-kappaB pathway on KSHV replication is determined by the status of the AP-1 pathway and hence provide a mechanistic explanation for the contradictory role of the NF-kappaB pathway in KSHV replication. Since the NF-kappaB pathway is commonly activated during infection of gammaherpesviruses, these findings might have general implications for the control of gammaherpesviral latency.     

Compound profiling using a panel of steroid hormone receptor cell-based assays

A major focus in the current discovery of drugs targeting nuclear receptors (NRs) is identifying drugs with reduced side effects by improving selectivity, not only from other receptors but also by selective modulation of the NR of interest. Cellular assays not only provide valuable information on functional activity, potency, and selectivity but also are ideally suited for differentiating partial agonists and antagonists. The ability to partially activate a receptor is believed to be closely tied to the ability to selectively modulate the NR, resulting in expression of a subset of the normally regulated genes. To this end, the authors have built a complete panel of cell-based steroid hormone receptor assays for the androgen receptor, estrogen receptor alpha, estrogen receptor beta, glucocorticoid receptor, mineralocorticoid receptor, and progesterone receptor by stably engineering a Gal4 DNA-binding domain/nuclear receptor ligand-binding domain fusion protein into an upstream activation sequence beta-lactamase reporter cell line. Each assay was validated with known agonists and antagonists for correct pharmacology and high-throughput compatibility. To demonstrate the utility of these assays, the authors profiled 35 pharmacologically relevant compounds in a dose-response format against the panel in both agonist and antagonist modes. The results demonstrated that selective estrogen receptor modulators can be identified and differentiated, as well as mixed and partial agonists and antagonists easily detected in the appropriate assays. Importantly, a comparison of the chimeric assays with full-length reporter gene assay data from the literature shows a good degree of correlation in terms of selectivity and pharmacology of important ligands. Taken together, these steroid hormone receptor assays provide good selectivity, sensitivity, and appropriate pharmacology for high-throughput screening and selectivity profiling of modulators of steroid hormone receptors.