Interview with Ella May Bard

Lisa M. Groesz recounts her interview with Ella May Bardhttps://digital.kenyon.edu/elfs_interviews/1048/thumbnail.jp

Interview with Ella May Bard

Ella May Bard talks the business side and the health aspects of producing apple cider.https://digital.kenyon.edu/elfs_interviews/1041/thumbnail.jp

Microbial community analysis of Acropora palamata mucus swabs, water and sediment samples from Hawksnest Bay, St. John, U.S. Virgin Islands

Colonies of the scleractinian coral Acropora palmata, listed as threatened under the US Endangered Species Act in 2006, have been monitored in Hawksnest Bay, within Virgin Islands National Park, St. John, from 2004 through 2010 by scientists with the US Geological Survey, National Park Service, and the University of the Virgin Islands. The focus has been on documenting the prevalence of disease, including white band, white pox (also called patchy necrosis and white patches), and unidentified diseases (Rogers et al., 2008; Muller et al., 2008). In an effort to learn more about the pathologies that might be involved with the diseases that were observed, samples were collected from apparently healthy and diseased colonies in July 2009 for analysis. Two different microbial assays were performed on Epicentre Biotechnologies DNA swabs containing A. palmata coral mucus, and on water and sediment samples collected in Hawksnest Bay. Both assays are based on polymerase chain reaction (PCR) amplification of portions of the small rRNA gene (16S). The objectives were to determine 1) if known coral bacterial pathogens Serratia marcescens (Acroporid Serratiosis), Vibrio coralliilyticus (temperature-dependent bleaching, White Syndrome), Vibrio shiloi (bleaching, necrosis), and Aurantimonas coralicida (White Plague Type II) were present in any samples, and 2) if there were any differences in microbial community profiles of each healthy, unaffected or diseased coral mucus swab. In addition to coral mucus, water and sediment samples were included to show ambient microbial populations. In the first test, PCR was used to separately amplify the unique and diagnostic region of the 16S rRNA gene for each of the coral pathogens being screened. Each pathogen test was designed so that an amplified DNA fragment could be seen only if the specific pathogen was present in a sample. A positive result was indicated by bands of DNA of the appropriate size on an agarose gel, which separates DNA fragments based on the size of the molecule. DNA from pure cultures of each of the pathogens was used as a positive control for each assay

Saline-saturated DMSO-EDTA as a storage medium for microbial DNA analysis from coral mucus swab samples

The mucus surface layer of corals plays a number of integral roles in their overall health and fitness. This mucopolysaccharide coating serves as vehicle to capture food, a protective barrier against physical invasions and trauma, and serves as a medium to host a community of microorganisms distinct from the surrounding seawater. In healthy corals the associated microbial communities are known to provide antibiotics that contribute to the coral’s innate immunity and function metabolic activities such as biogeochemical cycling. Culture-dependent (Ducklow and Mitchell, 1979; Ritchie, 2006) and culture-independent methods (Rohwer, et al., 2001; Rohwer et al., 2002; Sekar et al., 2006; Hansson et al., 2009; Kellogg et al., 2009) have shown that coral mucus-associated microbial communities can change with changes in the environment and health condition of the coral. These changes may suggest that changes in the microbial associates not only reflect health status but also may assist corals in acclimating to changing environmental conditions. With the increasing availability of molecular biology tools, culture-independent methods are being used more frequently for evaluating the health of the animal host. Although culture-independent methods are able to provide more in-depth insights into the constituents of the coral surface mucus layer’s microbial community, their reliability and reproducibility rely on the initial sample collection maintaining sample integrity. In general, a sample of mucus is collected from a coral colony, either by sterile syringe or swab method (Woodley, et al., 2008), and immediately placed in a cryovial. In the case of a syringe sample, the mucus is decanted into the cryovial and the sealed tube is immediately flash-frozen in a liquid nitrogen vapor shipper (a.k.a., dry shipper). Swabs with mucus are placed in a cryovial, and the end of the swab is broken off before sealing and placing the vial in the dry shipper. The samples are then sent to a laboratory for analysis. After the initial collection and preservation of the sample, the duration of the sample voyage to a recipient laboratory is often another critical part of the sampling process, as unanticipated delays may exceed the length of time a dry shipper can remain cold, or mishandling of the shipper can cause it to exhaust prematurely. In remote areas, service by international shipping companies may be non-existent, which requires the use of an alternative preservation medium. Other methods for preserving environmental samples for microbial DNA analysis include drying on various matrices (DNA cards, swabs), or placing samples in liquid preservatives (e.g., chloroform/phenol/isoamyl alcohol, TRIzol reagent, ethanol). These methodologies eliminate the need for cold storage, however, they add expense and permitting requirements for hazardous liquid components, and the retrieval of intact microbial DNA often can be inconsistent (Dawson, et al., 1998; Rissanen et al., 2010). A method to preserve coral mucus samples without cold storage or use of hazardous solvents, while maintaining microbial DNA integrity, would be an invaluable tool for coral biologists, especially those in remote areas. Saline-saturated dimethylsulfoxide-ethylenediaminetetraacetic acid (20% DMSO-0.25M EDTA, pH 8.0), or SSDE, is a solution that has been reported to be a means of storing tissue of marine invertebrates at ambient temperatures without significant loss of nucleic acid integrity (Dawson et al., 1998, Concepcion et al., 2007). While this methodology would be a facile and inexpensive way to transport coral tissue samples, it is unclear whether the coral microbiota DNA would be adversely affected by this storage medium either by degradation of the DNA, or a bias in the DNA recovered during the extraction process created by variations in extraction efficiencies among the various community members. Tests to determine the efficacy of SSDE as an ambient temperature storage medium for coral mucus samples are presented here

Porphyrin detection in denatured cnidarian tissue extracts

Porphyrin metabolic disruption from exposure to xenobiotic contaminants such as heavy metals, dioxins, and aromatic hydrocarbons can elicit overproduction of porphyrins. Measurement of porphyrin levels, when used in conjunction with other diagnostic assays, can help elucidate an organism’s physiological condition and provide evidence for exposure to certain toxicants. A sensitive microplate fluorometric assay has been optimized for detecting total porphyrin levels in detergent solubilized protein extracts from symbiotic, dinoflagellate containing cnidarian tissues. The denaturing buffer used in this modified assay contains a number of potentially interfering components (e.g., sodium dodecyl sulfate (SDS), dithiothreitol (DTT), protease inhibitors, and chlorophyll from the symbiotic zooxanthellae), which required examination and validation. Examination of buffer components were validated for use in this porphyrin assay; while the use of a specific spectrofluorometric filter (excitation 400 ± 15 nm; emission 600 ± 20 nm) minimized chlorophyll interference. The detection limit for this assay is 10 fmol of total porphyrin per μg of total soluble protein and linearity is maintained up to 5000 fmol. The ability to measure total porphyrins in a SDS protein extract now allows a single extract to be used in multiple assays. This is an advantage over classical methods, particularly when tissue samples are limiting, as is often the case with coral due to availability and collection permit restrictions

Proteins associated with pancreatic cancer survival in patients with resectable pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a dismal prognosis. However, while most patients die within the first year of diagnosis, very rarely, a few patients can survive for >10 years. Better understanding the molecular characteristics of the pancreatic adenocarcinomas from these very-long-term survivors (VLTS) may provide clues for personalized medicine and improve current pancreatic cancer treatment. To extend our previous investigation, we examined the proteomes of individual pancreas tumor tissues from a group of VLTS patients (survival ≥10 years) and short-term survival patients (STS, survival <14 months). With a given analytical sensitivity, the protein profile of each pancreatic tumor tissue was compared to reveal the proteome alterations that may be associated with pancreatic cancer survival. Pathway analysis of the differential proteins identified suggested that MYC, IGF1R and p53 were the top three upstream regulators for the STS-associated proteins, and VEGFA, APOE and TGFβ-1 were the top three upstream regulators for the VLTS-associated proteins. Immunohistochemistry analysis using an independent cohort of 145 PDAC confirmed that the higher abundance of ribosomal protein S8 (RPS8) and prolargin (PRELP) were correlated with STS and VLTS, respectively. Multivariate Cox analysis indicated that 'High-RPS8 and Low-PRELP' was significantly associated with shorter survival time (HR=2.69, 95% CI 1.46-4.92, P=0.001). In addition, galectin-1, a previously identified protein with its abundance aversely associated with pancreatic cancer survival, was further evaluated for its significance in cancer-associated fibroblasts. Knockdown of galectin-1 in pancreatic cancer-associated fibroblasts dramatically reduced cell migration and invasion. The results from our study suggested that PRELP, LGALS1 and RPS8 might be significant prognostic factors, and RPS8 and LGALS1 could be potential therapeutic targets to improve pancreatic cancer survival if further validated

Intelligence quotient–adjusted memory impairment is associated with abnormal single photon emission computed tomography perfusion

Cognitive reserve among highly intelligent older individuals makes detection of early Alzheimer's disease (AD) difficult. We tested the hypothesis that mild memory impairment determined by IQ-adjusted norms is associated with single photon emission computed tomography (SPECT) perfusion abnormality at baseline and predictive of future decline. Twenty-three subjects with a Clinical Dementia Rating (CDR) score of 0, were reclassified after scores were adjusted for IQ into two groups, 10 as having mild memory impairments for ability (IQ-MI) and 13 as memory-normal (IQ-MN). Subjects underwent cognitive and functional assessments at baseline and annual follow-up for 3 years. Perfusion SPECT was acquired at baseline. At follow-up, the IQ-MI subjects demonstrated decline in memory, visuospatial processing, and phonemic fluency, and 6 of 10 had progressed to a CDR of 0.5, while the IQ-MN subjects did not show decline. The IQ-MI group had significantly lower perfusion than the IQ-MN group in parietal/precuneus, temporal, and opercular frontal regions. In contrast, higher perfusion was observed in IQ-MI compared with IQ-MN in the left medial frontal and rostral anterior cingulate regions. IQ-adjusted memory impairment in individuals with high cognitive reserve is associated with baseline SPECT abnormality in a pattern consistent with prodromal AD and predicts subsequent cognitive and functional decline