Genetic homogeneity of the invasive lionfish across the Northwestern Atlantic and the Gulf of Mexico based on Single Nucleotide Polymorphisms.

Despite the devastating impact of the lionfish (Pterois volitans) invasion on NW Atlantic ecosystems, little genetic information about the invasion process is available. We applied Genotyping by Sequencing techniques to identify 1,220 single nucleotide polymorphic sites (SNPs) from 162 lionfish samples collected between 2013 and 2015 from two areas chronologically identified as the first and last invaded areas in US waters: the east coast of Florida and the Gulf of Mexico. We used population genomic analyses, including phylogenetic reconstruction, Bayesian clustering, genetic distances, Discriminant Analyses of Principal Components, and coalescence simulations for detection of outlier SNPs, to understand genetic trends relevant to the lionfish's long-term persistence. We found no significant differences in genetic structure or diversity between the two areas (FST p-values > 0.01, and t-test p-values > 0.05). In fact, our genomic analyses showed genetic homogeneity, with enough gene flow between the east coast of Florida and Gulf of Mexico to erase previous signals of genetic divergence detected between these areas, secondary spreading, and bottlenecks in the Gulf of Mexico. These findings suggest rapid genetic changes over space and time during the invasion, resulting in one panmictic population with no signs of divergence between areas due to local adaptation.

Fine-scale genetic structure due to adaptive divergence among microhabitats.

It has been suggested that adaptive evolution on ecological timescales shapes communities. However, adaptation among environments relies on isolation or large selection coefficients that exceed migration effects. This reliance is tempered if adaptation is polygenic-does not depend on one allele completely replacing another but instead requires small allele frequency changes at many loci. Thus, whether individuals can evolve adaptation to fine-scale habitat variation (for example, microhabitats) is not resolved. Here we analyze the genetic divergence of the teleost fish, Fundulus heteroclitus, among microhabitats that are <200 m apart in three separate saltmarshes using 4741 single-nucleotide polymorphisms (SNPs). Among these SNPs, 1.3-2.3% have large and highly significant differences among microhabitats (mean FST=0.15; false discovery rate ⩽1%). The divergence among microhabitats for these outlier SNPs is larger than that among populations, exceeds neutral expectation and indicates surprising population structure among microhabitats. Thus, we suggest that polygenic selection is surprisingly effective in altering allele frequencies among many different SNPs that share similar biological functions in response to environmental and ecological differences over very small geographic distances. We acknowledge the evolutionary difficulty of large genetic divergence among well-connected habitats. Therefore, these studies are only the first step to discern whether natural selection is responsible and capable of effecting genetic divergence on such a fine scale.

Genomic variation among populations of threatened coral: Acropora cervicornis.

BACKGROUND: Acropora cervicornis, a threatened, keystone reef-building coral has undergone severe declines (>90 %) throughout the Caribbean. These declines could reduce genetic variation and thus hamper the species' ability to adapt. Active restoration strategies are a common conservation approach to mitigate species' declines and require genetic data on surviving populations to efficiently respond to declines while maintaining the genetic diversity needed to adapt to changing conditions. To evaluate active restoration strategies for the staghorn coral, the genetic diversity of A. cervicornis within and among populations was assessed in 77 individuals collected from 68 locations along the Florida Reef Tract (FRT) and in the Dominican Republic. RESULTS: Genotyping by Sequencing (GBS) identified 4,764 single nucleotide polymorphisms (SNPs). Pairwise nucleotide differences (π) within a population are large (~37 %) and similar to π across all individuals. This high level of genetic diversity along the FRT is similar to the diversity within a small, isolated reef. Much of the genetic diversity (>90 %) exists within a population, yet GBS analysis shows significant variation along the FRT, including 300 SNPs with significant FST values and significant divergence relative to distance. There are also significant differences in SNP allele frequencies over small spatial scales, exemplified by the large FST values among corals collected within Miami-Dade county. CONCLUSIONS: Large standing diversity was found within each population even after recent declines in abundance, including significant, potentially adaptive divergence over short distances. The data here inform conservation and management actions by uncovering population structure and high levels of diversity maintained within coral collections among sites previously shown to have little genetic divergence. More broadly, this approach demonstrates the power of GBS to resolve differences among individuals and identify subtle genetic structure, informing conservation goals with evolutionary implications.

Population Genomics of the Euryhaline Teleost Poecilia latipinna.

Global climate change and increases in sea levels will affect coastal marine communities. The conservation of these ecologically important areas will be a challenge because of their wide geographic distribution, ecological diversity and species richness. To address this problem, we need to better understand how the genetic variation of the species in these communities is distributed within local populations, among populations and between distant regions. In this study we apply genotyping by sequencing (GBS) and examine 955 SNPs to determine Sailfin molly (Poecilia latipinna) genetic diversity among three geographically close mangrove salt marsh flats in the Florida Keys compared to populations in southern and northern Florida. The questions we are asking are whether there is sufficient genetic variation among isolated estuarine fish within populations and whether there are significant divergences among populations. Additionally, we want to know if GBS approaches agree with previous studies using more traditional molecular approaches. We are able to identify large genetic diversity within each saltmarsh community (π ≈ 36%). Additionally, among the Florida Key populations and the mainland or between southern and northern Florida regions, there are significant differences in allele frequencies seen in population structure and evolutionary relationships among individuals. Surprisingly, even though the cumulative FST value using all 955 SNPs within the three Florida Key populations is small, there are 29 loci with significant FST values, and 11 of these were outliers suggestive of adaptive divergence. These data suggest that among the salt marsh flats surveyed here, there is significant genetic diversity within each population and small but significant differences among populations. Much of the genetic variation within and among populations found here with GBS is very similar to previous studies using allozymes and microsatellites. However, the meaningful difference between GBS and these previous measures of genetic diversity is the number of loci examined, which allows more precise delineations of population structure as well as facilitates identifying loci with excessive FST values that could indicate adaptive divergence.

Robotic versus laparoscopic colectomy.

BACKGROUND: This study compared the experience and cost of the DaVinci Robotic system and laparoscopy for colon resections. METHODS: For this study, 30 consecutive robotic and 27 consecutive laparoscopic colectomies were divided into right and sigmoid colectomies for analysis. Comparisons included indications for surgery, gender, age, body mass index (BMI), estimated blood loss (EBL), length of operation, length of hospital stay (LOS), complications, operating room (OR) cost, OR personnel cost, OR supply cost, OR time cost, and total hospital cost. RESULTS: The comparison groups were similar in indications for surgery, gender, age, BMI, EBL, and LOS. The right colectomies included 17 robotic and 15 laparoscopic procedures. An intracorporeal anastomosis was performed in the robotic cases, and an extracorporeal anastomosis was performed in the laparoscopic cases. The total case time was 218.9 min for the robotic and 169.2 min for the laparoscopic procedures (p = 0.002). The total hospital cost was $9,255 for the robotic and $8,073 for the laparoscopic procedures (p = 0.430). The total OR cost was $5,823 for the robotic and $4,339 for the laparoscopic procedures (p < 0.000). The sigmoid colectomies included 13 robotic and 12 laparoscopic procedures. The robotic and laparoscopic cases were managed in similar sequence. The total case time was 225.2 min for the robotic and 199.4 min for the laparoscopic procedures (p = 0.128). The total hospital cost was $12,335 for the robotic and $10,697 for the laparoscopic procedures (p = 0.735). The total OR cost was $6,059 for the robotic and $4,974 for the laparoscopic procedures (p = 0.068). The complications in the robotic groups were more numerous, but were not attributable to equipment. CONCLUSIONS: The comparison groups were similar. The robotic cases were significantly longer for right colectomies because of the intracorporeal anastomosis instead of the extracorporeal anastomosis performed in the laparoscopy cases. Every cost category was higher for the robotic cases. The right colectomies showed significant increases in total OR cost, OR personnel cost, OR supply cost, and OR time cost. The sigmoid colectomies had significant increases in OR personnel cost and OR supply cost. The total hospital cost was higher for the robotic groups, but the difference was not statistically significant.

Telerobotic surgery for right and sigmoid colectomies: 30 consecutive cases.

BACKGROUND: This study aimed to evaluate the feasibility of using a robotic assistant for colon resections. This report describes the experience, advantages, and disadvantages of using the DaVinci system for a colectomy on the basis of 30 consecutive cases managed by a minimally invasive surgery fellowship-trained surgeon. METHODS: Data were prospectively collected on 30 consecutive colectomies performed using the DaVinci system from September 2002 to March 2005. RESULTS: A total of 13 sigmoid colectomies with splenic flexure mobilization and 17 right colectomies were performed for 14 men and 16 women. The preoperative diagnoses for the procedures were cancer (n = 5), diverticulitis (n = 8), polyps (n = 16), and carcinoid (n = 1). The right colectomies required 29.7 +/- 6.7 min (range, 22-44 min) for the port setup, 177.1 +/- 50.6 min (range, 103-306 min) for the robot, and 218.9 +/- 44.6 min (range, 167-340 min) for the total case. The length of stay was 5.2 +/- 5.8 days (range, 2-27 days). The robot portion was 80.9% of the total case time. The sigmoid colectomies required 30.1 +/- 9.6 min (range, 15-50 min) for the port setup, 103.2 +/- 29.4 min (range, 69-165 min) for the robot, and 225.2 +/- 37.1 min (range, 147-283 min) for the total case. The hospital length of stay was 6.0 +/- 7.3 days (range, 3-30 days). The robot portion was 45.8% of the total case time. Six complications occurred: left hip paresthesia, cecal injury, anastomotic leak, patient slipped from the operating table after the robotic portion of the case, transverse colon injury, and return of a patient to the office with urinary retention. Two sigmoid colectomies were converted to laparotomy. The specific advantages and disadvantages of using the DaVinci system for colectomies are discussed. CONCLUSIONS: The 30 consecutive cases demonstrated the technical feasibility of using the DaVinci system for a colectomy. The longevity of the DaVinci system's use for colectomy will be determined by comparison of its cost and outcomes with those for conventional laparoscopic colectomy.

Anaerobic biodegradation of pentachlorophenol in mixtures containing cadmium by two physiologically distinct microbial enrichment cultures.

Anaerobic biodegradation of pentachlorophenol (PCP), in mixtures containing cadmium (Cd), by sulfidogenic (SRB) and methanogenic (MET) enrichment cultures, was studied. Removal of 91-93% of PCP occurred in both SRB- and MET-enriched cultures, in the absence of Cd, within 82 days. The presence of soluble Cd initially decreased the rate of PCP removal by the enrichment cultures, but PCP removal rates improved as the Cd precipitated. GC-MS, 14C-PCP, and 13C-PCP studies confirmed mineralization of PCP by both enrichment cultures, as well as the incorporation of PCP carbon into specific phospholipid fatty acids (PLFAs) of the cell membranes of PCP-degrading anaerobes. This is the first report on anaerobic biodegradation of PCP by SRB- and MET-enriched cultures in the presence, with simultaneous precipitation, of the toxic heavy metal Cd, and of the incorporation of PCP carbons into specific PLFAs of the anaerobic bacterial cells.

Utility of natural populations for microarray analyses: isolation of genes necessary for functional genomic studies.

How much variation is there in gene expression? How is this variation partitioned within and among populations? How much variation is biologically important? That is, how much of this variation affects longevity, reproductive fitness, or probability of survival? Microarray analyses can be used to accurately quantify the expression of most, if not all, genes expressed in a tissue and thus address the first question. The latter questions can be investigated by examining the patterns of variation within and among natural populations of Fundulus. These populations are large and affected by historical, demographic, and selective constraints, providing a framework for the partition of variation in gene expression within and among populations. Additionally, the well established, phylogenetic relationship among Fundulus species can be used to discern adaptive change. A phylogenetic perspective reveals changes that are produced by natural selection and therefore indicates whether this variation affects longevity, reproductive fitness, or probability of survival, i.e., whether the variation is biologically important. However, a Fundulus microarray requires DNAs encoding specific Fundulus genes. This paper provides information on the production, isolation, and characterization of 4440 Fundulus cDNAs used in microarrays. Our approach was to pick random colonies from a normalized cDNA library and then PCR amplify and sequence these genes in a 96-well format. Periodically, the isolated and sequenced cDNAs were subtracted from the normalized library. Normalization reduced the number of redundant genes from 33% to 11%, increasing the effectiveness of this screening process. From 4440 sequenced cDNAs, 49% (2173) had a match in GenBank using BlastX searches. Of these, 53% were nonredundant, yielding 1149 identified genes. These data suggest that cDNAs necessary for microarray analyses can be produced effectively from most organisms.

Intraspecific variation in aerobic metabolism and glycolytic enzyme expression in heart ventricles.

A previous phylogenetic analysis among 15 taxa of the teleost fish Fundulus suggested that there should be thermal-adaptive differences in heart metabolism among populations. To test this hypothesis, the rate of oxygen consumption and the activities of all 11 glycolytic enzymes were measured in isolated heart ventricle from two populations of Fundulus heteroclitus. Heart ventricular metabolism is greater in a northern population versus a southern population of these fish. Analysis of the amount of glycolytic enzymes indicates that 87% of the variation in cardiac metabolism within and between populations is explained by the variation in three enzymes (pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase). These enzymes are the same three enzymes that were predicted to be important based on previously determined phylogenetic patterns of expression. Our data indicate that near-equilibrium enzymes, as well as classically defined rate-limiting enzymes, can also influence metabolism.

Inexpensive retraction technique for advanced laparoscopic procedures.

The described technique offers surgeons an inexpensive simple way to hold intraperitoneal structures in position for long periods with minimal trauma.

Thatch biodegradation and antifungal activities of two lignocellulolytic Streptomyces strains in laboratory cultures and in golf green turfgrass.

The use of lignocellulolytic Streptomyces spp. as biological agents, to enhance thatch degradation in turf and to slow its rate of accumulation while controlling fungal growth in the thatch layer, was studied. In flask scale studies, two lignocellulolytic Streptomyces violaceusniger (= hygroscopicus) strains (YCED9 and WYE53) decomposed thatch (> 30% dry weight) over a 12-week incubation period. Biodegradation was accompanied by production of extracellular cellulases, xylanases, and peroxidases. The accumulation of the polymeric, water-soluble lignin degradation intermediate acid, precipitable polymeric lignin (APPL), was also observed. Residual thatch from 12-week-old cultures had an increased lignin-to-carbohydrate ratio, an indication that although lignin was metabolized, carbohydrates were preferential carbon sources for these actinomycetes. A spore-containing soluble dry powder formulation was used as an inoculum in an in situ field experiment. This formulation was maintained in storage at 4 degrees C for over two years without viability loss. Results from the golf green experiment showed that although treated thatch layers in established greens were not appreciably reduced over the course of one summer, the Streptomyces were active and maintained their populations within the thatch, while fungal growth was suppressed as compared to controls. The results show that treatment of turfgrass with these Streptomyces may be useful for the long-term control of fungal populations within the thatch. Longer field studies are required to assess the long-term potential for also controlling thatch build-up and fungal pathogens.

Isolation and analysis of three peroxide sensor regulatory gene homologs ahpC, ahpX and oxyR in Streptomyces viridosporus T7A--a lignocellulose degrading actinomycete.

Increased lignolytic peroxidase activity has been demonstrated with the addition of sublethal doses of toxic H2O2 in Streptomyces viridosporus T7A. Until now, the effect of H2O2 at the molecular level has not been well characterized. Here, for the first time we report the isolation and analysis of three peroxide-induced gene homologs from S. viridosporus T7A; ahpC and ahpX (encoding alkyl hydroxyperoxidase subunits) and oxyR (encoding oxygen stress regulatory protein). The genome organization of these stress related genes were found to be divergently adjacent to each other. The protein sequence analysis of the oxyR homolog revealed a helix-turn-helix DNA-binding motif characteristic to the LysR of regulatory proteins induced by H2O2. The nucleotide sequence analysis of the intergenic region between ahpC and oxyR revealed that they shared a core T-n11-A, a signature protein-binding region of LysR family members. Based on similarities in sequence analysis, genetic organization, and the induction of lignin peroxidase activity upon exposure to hydrogen peroxide, we hypothesize a peroxide induction mechanism for the regulation of oxidative lignin biodegradation by S. viridosporus, possibly via use of OxyR which is also involved in regulating the peroxide stress response in this actinomycete.

Use of randomly amplified polymorphic DNA as a means of developing genus- and strain-specific Streptomyces DNA probes.

We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36 Streptomyces strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for Streptomyces, and a strain-specific probe for the biological control agent Streptomyces lydicus WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the S. lydicus WYEC108 in DNA isolated from soil.

C-terminal epitope tagging facilitates comparative ligand mapping from MHC class I positive cells.

Purification of specific class I molecules prior to peptide ligand characterization is complicated by the presence of multiple class I proteins in most cell lines. Immortalized B, T, and tumor cell lines typically express endogenous HLA-A, -B, and -C; and most individuals from which the cell lines are derived are heterozygous at these loci. Antibodies specific for a particular HLA molecule may be used for purification, but allele-specific antibodies can be biased by ligands occupying the peptide-binding groove. Through the use of C-terminal tagging, we have developed a method of soluble HLA production such that downstream purification does not skew the peptide analysis of the examined molecule. Comparison of peptides eluted from HLA class I molecules with and without C-terminal tags demonstrates that addition of a tag does not abrogate the peptide binding specificity of the original molecule. Both pooled Edman sequencing and mass spectrometric sequencing identified no substantial differences in peptides bound by untailed, 6-HIS-tailed, and FLAG-tailed class I molecules, demonstrating that the peptide specificity of a given molecule is not distorted by either tag. This production methodology bypasses problems with isolation of specific molecules and permits ligand mapping and epitope discovery in a variety of pathogen-infected and tumor cell lines.

Functional analyses of natural variation in Sp1 binding sites of a TATA-less promoter.

Within the lactate dehydrogenase-B (LdhB) proximal promoter is a region with multiple in vivo footprinted sites that resembles the binding site for the transcription factor SP1. Like many sequences that regulate transcription rate, these Sp1 binding sites are well conserved among species of the teleost fish Fundulus. The only exception is in the northern population of F. heteroclitus, where there are many changes in the Sp1 binding sites. These changes affect footprinting patterns, measures of promoter strength, and are associated with the adaptive increase in Ldh-B transcription rates. Reported here is data that demonstrates that Fundulus hepatocyctes have an SP1-like protein; in comparison to human SP1 protein, it has similar specificity and size and a greater affinity for the consensus Sp1 site. This Fundulus hepatocyte SP1-like protein as well as the human SP1 protein binds the Ldh-B Sp1 sites. Sequence variation in the northern Sp1 region eliminates the "preferred" Sp1 binding site, yet these northern Sp1 sites have significantly greater affinity for the SP1 protein than either the Sp1 sites from southern F. heteroclitus ( approximately 1.6-fold) or the consensus Sp1 site (GGGCGG; approximately 1.8-fold). Furthermore, the Ldh-B Sp1 sites also bind non-SP1 proteins, and the extent of binding is affected by the sequence variation in the proximal promoter. These data suggest that natural variation in Sp1 sites affect binding of transcription factors and may effect a modest change in transcription rates.

Magnesium deficiency-induced osteoporosis in the rat: uncoupling of bone formation and bone resorption.

Magnesium (Mg) intake has been linked to bone mass and/or rate of bone loss in humans. Experimental Mg deficiency in animal models has resulted in impaired bone growth, osteopenia, and increased skeletal fragility. In order to assess changes in bone and mineral homeostasis that may be responsible, we induced dietary Mg deficiency in adult Simonsen albino rats for 16 weeks. Rats were fed either a low Mg diet (0.002 percent) or a normal control Mg diet (0.063 percent). Blood was obtained at baseline, 4 weeks, 8 weeks, 12 weeks and 16 weeks in both groups for serum Mg, calcium, PTH, and 1.25(OH)2-vitamin D determinations. Femora were harvested at 4 weeks and 16 weeks for mineral analysis and histomorphometry. Serum Mg fell in the Mg depleted group to 0.6 mg/dl (mean) by 16 weeks (controls = 2.0 mg/dl). The serum calcium (Ca) concentration was higher in the Mg depleted animals at 16 weeks, 10.8 mg/dl (controls = 8.9 mg/dl). Serum PTH concentration fell progressively in the Mg deficient rats to 30 pg/ml by week 16 (control = 96 pg/ml). Serum concentration of 1.25(OH)2-vitamin D also fell progressively in the Mg deficient animals by 16 weeks to 14 pg/ml (control = 30 pg/ml). While the percent ash weights of Ca and phosphorus in the femur were not different at any time point, the percent ash weight of Mg progressively fell to 0.54 percent vs control (0.74 percent) by 16 weeks. The percent ash weight of potassium also fell progressively in the Mg deficient group to approximately 30 percent of control by 16 weeks. Histomorphometric analyses showed a significant drop in trabecular bone volume in Mg deficient animals by 16 weeks (percent BV/TV = 13.2 percent vs 17.3 percent in controls). Evaluation of the endosteal bone surface features showed significantly greater bone resorption in the Mg depleted group as reflected in increased number of tartrate-resistant positive osteoclasts/mm bone surface (7.8 vs 4.0 in controls) and an elevated percent of bone surface occupied by osteoclasts (percent OcS/BS = 12.2 percent vs 6.7 percent in controls. This increased resorption occurred in the presence of an inappropriate lowered bone forming surface relative to controls; a decreased number of osteoblasts per mm bone surface (0.23 vs 0.94 in control) and a decrease in percent trabecular surface lined by osteoid (percent OS/BS = 0.41 vs 2.27 percent in controls) were also noted. Our findings demonstrate a Mg-deficiency induced uncoupling of bone formation and bone resorption resulting in a loss of bone mass. While the fall in PTH and/or 1.25(OH)2-D may explain a decrease in osteoblast activity, the mechanism for increased osteoclast activity is unclear. These data suggest that Mg deficiency may be a risk factor for osteoporosis.

In vitro and in vivo antagonism of pathogenic turfgrass fungi by Streptomyces hygroscopicus strains YCED9 and WYE53.

Disease prevention is a current practice used to minimize fungal diseases of turfgrasses in lawns and golf greens. Prevention is accomplished through fungicide applications, and by periodic thatch removal. During the development of a microbial biodethatch product utilizing the lignocellulose-degrading Streptomyces hygroscopicus strains YCED9 and WYE53, we demonstrated using in vitro plate antagonism bioassays that both strains are antagonists of various turfgrass fungal pathogens. These activities were present when the cultures were growing on thatch, as demonstrated by antifungal antagonism bioassays with culture filtrates. Experiments conducted using a growth chamber demonstrated that a bio-dethatch formulation containing spores of strains YCED9 and WYE53 in a zeolite carrier, provided protection for Kentucky bluegrass seedlings against turfgrass pathogens, including Pythium ultimum, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia homeocarpa, Gaeumannomyces graminis and Microdochium nivale. Results showed that by integrating the use of the S. hygroscopicus YCED9/WYE53 bio-dethatch formulation into routine turf management practices, it should be possible to both minimize thatch build-up while also controlling fungal turfgrass diseases by way of the antifungal biocontrol activity of these strains. This in turn would help control fungal pathogens in turfgrass while minimizing the need for routine chemical fungicide applications.

Laparoscopic common bile duct exploration.

Laparoscopic cholecystectomy has become the procedure of choice for laparoscopically skilled surgeons when dealing with both chronic and acute cholecystitis. When choledocholithiasis is encountered in the treatment of these patients the skilled laparoscopist has several treatment options available to treat the patient in one stage and avoid the morbidity of endoscopic sphincterotomy. Although still controversial, laparoscopic common bile duct exploration has been shown to be safe, applicable, and cost-effective in the treatment of choledocholithiasis. This report details several laparoscopic treatment alternatives for choledocholithiasis.