Timothy D. Sherman

Analysis of bacterial communities in seagrass bed sediments by double-gradient denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA genes.

Bacterial communities associated with seagrass bed sediments are not well studied. The work presented here investigated several factors and their impact on bacterial community diversity, including the presence or absence of vegetation, depth into sediment, and season. Double-gradient denaturing gradient gel electrophoresis (DG-DGGE) was used to generate banding patterns from the amplification products of 16S rRNA genes in 1-cm sediment depth fractions. Bioinformatics software and other statistical analyses were used to generate similarity scores between sections. Jackknife analyses of these similarity coefficients were used to group banding patterns by depth into sediment, presence or absence of vegetation, and by season. The effects of season and vegetation were strong and consistent, leading to correct grouping of banding patterns. The effects of depth were not consistent enough to correctly group banding patterns using this technique. While it is not argued that bacterial communities in sediment are not influenced by depth in sediment, this study suggests that the differences are too fine and inconsistent to be resolved using 1-cm depth fractions and DG-DGGE. The effects of vegetation and season on bacterial communities in sediment were more consistent than the effects of depth in sediment, suggesting they exert stronger controls on microbial community structure.

FIELD ASSAYS FOR MEASURING NITRATE REDUCTASE ACTIVITY IN ENTEROMORPHA SP. (CHLOROPHYCEAE), ULVA SP. (CHLOROPHYCEAE), AND GELIDIUM SP. (RHODOPHYCEAE) 1

In situ and in vitro nitrate reductase (NR) activity assays designed for use in the field on Enteromorpha sp., Ulva sp., and Gelidium sp. are described. In optimizing each assay, a variety of compounds and assay conditions were tested for their ability to extract NR and preserve its activity. Enteromorpha sp. had similar levels of in vitro NR activity after exposure to the in situ assay buffer, demonstrating that neither NR induction nor activation likely occurs during the in situ assay. Storing freshly collected Enteromorpha sp. led to a reduction in NR activity over time. However, the use of liquid nitrogen to freeze algal tissue on site and subsequent storage at −80° C preserved NR activity and allowed for later laboratory use of the optimized in vitro assay. Application of the in situ and in vitro assays to stands of Enteromorpha sp., Ulva sp., and Gelidium sp. in the field consistently found NR activity. In situ NR activity over 9 consecutive days in January demonstrated that Enteromorpha sp. responds to increases in nitrate availability. The influence of light on diel patterns of in vitro NR activity in the field was demonstrated for the first time as well. For the three species tested, these two assays provide a reliable tool for field investigation of the interaction between environmental signals (e.g. nutrient levels) and physiological signals (e.g. tissue metabolite levels) on nitrate reduction.

Nitrate reductase activity during desiccation and rehydration of the desiccation-tolerant moss Tortula ruralis

Abstract Water stress reduces the activity of many plant enzymes. When the stress is alleviated there is generally a delay in the recovery of metabolism to unstressed levels. The enzyme nitrate reductase (EC 1.6.6.1) declines in plants experiencing water deficits and upon the alleviation of water stress its activity recovers to control levels in 1–7 days. In desiccation-tolerant plants, metabolic recovery can occur within hours following rehydration. It has been proposed that metabolic characteristics of desiccation-tolerant plants are a source of information that can be used to improve the performance of crop plants under water deficits. In this study, the effect of desiccation and hydration on nitrate reductase activity in the desiccation-tolerant moss Tortula ruralis has been investigated. It was expected that the activity of nitrate reductase would decline during desiccation and recover rapidly following rehydration. Inclusion of nitrate in the hydration medium increased the activity in the tissue. Nitrate reductase activity increased with nitrate concentration in the bathing medium up to levels of 200 mM. The highest activity measured in rehydrated moss samples was 5.23 nkat g−1 dry weight (dw). The activity declined rapidly during dehydration and was not detectable in dried moss samples after 24 h of dehydration. The recovery of activity following rehydration was dependant on the rate of the preceding dehydration. In slowly dried moss, the activity recovered to control levels in less than 8 h while rapidly dried samples required 24 h for full recovery. Nitrite accumulated during slow dehydration but did not accumulate when desiccation was rapid. Following rehydration of slowly dried moss, the amount of nitrite declined and reached a control level within 1 h. It was proposed that nitrite accumulation might provide a source of nitrogen for metabolism during the time required for nitrate reduction to resume following rehydration. Attempts to measure immunologically reactive nitrate reductase protein levels or nitrate reductase mRNA levels using heterologous DNA probes were unsuccessful, suggesting that the Tortula nitrate reductase may be significantly different from that found in algae and higher plants.

A Field Study of Nitrogen Storage and Nitrate Reductase Activity in the Estuarine Macroalgae Enteromorpha lingulata (Chlorophyceae) and Gelidium pusillum (Rhodophyceae)

The purpose of this field study was to determine the relationship between environmental conditions, particularly high nitrate (NO3−), low salinity events, and both nitrogen (N) storage (NO3−, ammonium [NH4+], free amino acids [FAA], protein, and total N) and nitrate reductase (NR) activity in the macroalgaeEnteromorpha lingulata andGelidium pusillum in the lower Mobile Bay estuary (Alabama, USA). The environmental conditions at the collection site varied over the growing season with the most notable changes due to late winter and spring runoff entering the estuary (1–30 psu, 0.3–25.8 μM NO3−, 0.9–12.5 μM NH4+, 3–28°C, 61–2,375 μmol PAR m−2 s−1). Principal component analysis reduced the six environmental variables measured to three principal components. Stepwise, multiple regression analysis was then used to examine the relationship between the principal components and the internal NO3−, NH4+, and FAA pools and NR activity. The results indicate that changes in inorganic N availability and salinity rather than changes in irradiance determine patterns of N storage and NO3− reduction. BothE. lingulata andG. pusillum are capable of taking up and storing NO3− when it becomes available. Greater NO3− availability produced larger NH4+ and FAA pools along with higher rates of NR activity inE. lingulata, but notG. pusillum, suggesting thatE. lingulata is able to metabolize NO3− more rapidly during high NO3−, low salinity events. Differences in the susceptibility ofE. lingulata andG. pusillum to NH4+ inhibition and salinity stress combined with their different growth strategies help to explain the seasonal trends in total N. Total N inE. lingulata ranged from 2.57% to 6.39% dw, while the slower growingG. pusillum showed no significant variation in total N content (3.8–4.1% dw). These results led to the conclusion thatE. lingulata responds more quickly thanG. pusillum to high NO3−, low salinity events and that these events have a larger effect on the overall N content ofE. lingulata.

INVESTIGATIONS OF INTERNAL INTERACTIONS BETWEEN THE PARASITIC BARNACLE LOXOTHYLACUS TEXANUS (RHIZOCEPHALA: SACCULINIDAE) AND ITS HOST CALLINECTES SAPIDUS (BRACHYURA: PORTUNIDAE) USING PCR TECHNIQUES

We describe techniques that enable the preservation of tissues from the rhizocephalan barnacle, Loxothylacus texanus, inside the body cavities of blue crab hosts, Callinectes sapidus, in a manner that minimizes the degradative activities of hepatopancreatic enzymes. These procedures allow the extraction and amplification of both parasite and host 18S rDNA within the same sample and enable one to distinguish between parasitized and unparasitized crab tissue in as little as two weeks after infection, well before any external manifestations of the parasites. Two PCR-based approaches were taken to identify the presence of L. texanus. In the first approach, a set of primers specific for L. texanus was used to specifically amplify 18S sequence in a background of C. sapidus DNA or the DNA of other barnacle species. In the second approach, a set of general primers was used to amplify 18S sequence from C. sapidus and a variety of barnacle species. The products of this PCR were then digested with an enzyme that recognizes a restriction site present only in the L. texanus PCR product to yield a unique pattern of fragments. With these techniques, we could detect as few as five parasitic cypris larvae in water samples, as well as L. texanus in the tissue of a small crab collected from the field and in the four anterior periopods of a crab bearing the external stage of the parasite. In experiments with potential hosts of varying sizes and molt stages, we confirmed that the parasite was significantly more effective in infecting crabs less than 30 mm carapace width than larger individuals. These techniques will facilitate future investigations of ecological and physiological interactions between these important crustacean parasites and their hosts and will help to determine the economic impact of this parasite on blue crab fisheries.

Human snoRNA-93 is processed into a microRNA-like RNA that promotes breast cancer cell invasion

Genetic searches for tumor suppressors have recently linked small nucleolar RNA misregulations with tumorigenesis. In addition to their classically defined functions, several small nucleolar RNAs are now known to be processed into short microRNA-like fragments called small nucleolar RNA-derived RNAs. To determine if any small nucleolar RNA-derived RNAs contribute to breast malignancy, we recently performed a RNA-seq-based comparison of the small nucleolar RNA-derived RNAs of two breast cancer cell lines (MCF-7 and MDA-MB-231) and identified small nucleolar RNA-derived RNAs derived from 13 small nucleolar RNAs overexpressed in MDA-MB-231s. Importantly, we find that inhibiting the most differentially expressed of these small nucleolar RNA-derived RNAs (sdRNA-93) in MDA-MB-231 cells results primarily in a loss of invasiveness, whereas increased sdRNA-93 expression in either cell line conversely results in strikingly enhanced invasion. Excitingly, we recently determined sdRNA-93 expressions in small RNA-seq data corresponding to 116 patient tumors and normal breast controls, and while we find little sdRNA-93 expression in any of the controls and only sporadic expression in most subtypes, we find robust expression of sdRNA-93 in 92.8% of Luminal B Her2+tumors. Of note, our analyses also indicate that at least one of sdRNA-93’s endogenous roles is to regulate the expression of Pipox, a sarcosine metabolism-related protein whose expression significantly correlates with distinct molecular subtypes of breast cancer. We find sdRNA-93 can regulate the Pipox 3′UTR via standard reporter assays and that manipulating endogenous sdRNA-93 levels inversely correlates with altered Pipox expression. In summary, our results strongly indicate that sdRNA-93 expression actively contributes to the malignant phenotype of breast cancer through participating in microRNA-like regulation.RNA: Small nucleolar-derived RNA contributes to tumor invasivenessA short microRNA-like fragment excised from a small nucleolar RNA (called a snoRNA-derived RNA or sdRNA) contributes to the invasiveness of breast cancer cells. Glen Borchert from the University of South Alabama, USA, and colleagues compared the expression of sdRNAs between a primary breast cancer cell line and a metastatic one. They identified 13 sdRNAs with markedly different abundances. Blocking the sdRNA with the biggest expression differential sdRNA-93-decreased cellular invasion, whereas increasing its abundance enhanced tumor cell invasiveness. Looking at human tissues, sdRNA-93 was routinely expressed in biopsies taken from patients with the luminal HER2-positive form of breast cancer, less often in other tumor types and almost never in healthy breast samples. Hinting at its function, the researchers showed that sdRNA-93 targets and regulates a gene contributing to specific molecular subtypes of breast cancer.

ADAR Mediated RNA Editing Modulates MicroRNA Targeting in Human Breast Cancer.

RNA editing by RNA specific adenosine deaminase acting on RNA (ADAR) is increasingly being found to alter microRNA (miRNA) regulation. Editing of miRNA transcripts can affect their processing, as well as which messenger RNAs (mRNAs) they target. Further, editing of target mRNAs can also affect their complementarity to miRNAs. Notably, ADAR editing is often increased in malignancy with the effect of these RNA changes being largely unclear. In addition, numerous reports have now identified an array of miRNAs that directly contribute to various malignancies although the majority of their targets remain largely undefined. Here we propose that modulating the targets of miRNAs via mRNA editing is a frequent occurrence in cancer and an underappreciated participant in pathology. In order to more accurately characterize the relationship between these two regulatory processes, this study examined RNA editing events within mRNA sequences of two breast cancer cell lines (MCF-7 and MDA-MB-231) and determined whether or not these edits could modulate miRNA associations. Computational analyses of RNA-Seq data from these two cell lines identified over 50,000 recurrent editing sites within human mRNAs, and many of these were located in 3’ untranslated regions (UTRs). When these locations were screened against the list of currently-annotated miRNAs we discovered that editing caused a subset (~9%) to have significant alterations to mRNA complementarity. One miRNA in particular, miR-140–3p, is known to be misexpressed in many breast cancers, and we found that mRNA editing allowed this miRNA to directly target the apoptosis inducing gene DFFA in MCF-7, but not in MDA-MB-231 cells. As these two cell lines are known to have distinct characteristics in terms of morphology, invasiveness and physiological responses, we hypothesized that the differential RNA editing of DFFA in these two cell lines could contribute to their phenotypic differences. Indeed, we confirmed through western blotting that inhibiting miR-140–3p increases expression of the DFFA protein product in MCF-7, but not MDA-MB-231, and further that inhibition of miR-140–3p also increases cellular growth in MCF-7, but not MDA-MB-231. Broadly, these results suggest that the creation of miRNA targets may be an underappreciated function of ADAR and may help further elucidate the role of RNA editing in tumor pathogenicity.