Assessing acute thermal assays as a rapid screening tool for coral restoration.

Escalating environmental threats to coral reefs coincides with global advancements in coral restoration programs. To improve long-term efficacy, practitioners must consider incorporating genotypes resilient to ocean warming and disease while maintaining genetic diversity. Identifying such genotypes typically occurs under long-term exposures that mimic natural stressors, but these experiments can be time-consuming, costly, and introduce tank effects, hindering scalability for hundreds of nursery genotypes used for outplanting. Here, we evaluated the efficacy of the acute Coral Bleaching Automated Stress System (CBASS) against long-term exposures on the bleaching response of Acropora cervicornis, the dominant restoration species in Florida's Coral Reef. Comparing bleaching metrics, Fv/Fm, chlorophyll, and host protein, we observed similar responses between the long-term heat and the CBASS treatment of 34.3 °C, which was also the calculated bleaching threshold. This suggests the potential of CBASS as a rapid screening tool, with 90% of restoration genotypes exhibiting similar bleaching tolerances. However, variations in acute bleaching phenotypes arose from measurement timing and experiment heat accumulation, cautioning against generalizations solely based on metrics like Fv/Fm. These findings identify the need to better refine the tools necessary to quickly and effectively screen coral restoration genotypes and determine their relative tolerance for restoration interventions.

Phytoplankton communities of the west coast of Florida - multiyear and seasonal responses to nutrient enrichment.

Blooms of the harmful algae species Karenia brevis are frequent off the southwest coast of Florida despite having relatively slow growth rates. The regional frequency of these harmful algal blooms led to the examination of the dominant estuarine outflows for effects on both K. brevis and the phytoplankton community in general. There is comparatively little information on the growth rates of non-Karenia taxonomic groups other than diatoms. A seasonally based series (Fall, Winter, and Spring) of bioassay experiments were conducted to determine the nutrient response of the coastal phytoplankton community. Treatments included estuarine waters (Tampa Bay, Charlotte Harbor, and the Caloosahatchee River) applied in a 1:25 dilution added to coastal water to mimic the influence of estuarine water in a coastal environment. Other treatments were 5-15 µM additions of nitrogen (N), phosphorus (P), and silica (Si) species, amino acids, and N (urea) + P added to coastal water. Incubations were conducted under ambient conditions with shading for 48 h. Analyses of dissolved and particulate nutrients were coupled with HPLC analysis of characteristic photopigments and taxonomic assignments of biomass via CHEMTAX. The coastal phytoplankton community, dominated by diatoms, cyanophytes and prasinophytes, was significantly different both by bioassay and by season, indicating little seasonal fidelity in composition. Specific growth rates of chlorophyll a indicated no significant difference between any controls, any estuarine treatment, P, or Si treatments. Conditions were uniformly N-limited with the highest growth rates in diatom biomass. Despite differing initial communities, however, there were seasonally reproducible changes in community due to the persistent growth or decline of the various taxa, including haptophytes, cyanophytes, and cryptophytes. For the one bioassay in which K. brevis was present, the slow growth of K. brevis relative to diatoms in a mixed community was evident, indicating that identifying the seasonally based behavior of other taxa in response to nutrients is critical for the simulation of phytoplankton competition and the successful prediction of the region's harmful algal blooms.

Gulf of Mexico blue hole harbors high levels of novel microbial lineages.

Exploration of oxygen-depleted marine environments has consistently revealed novel microbial taxa and metabolic capabilities that expand our understanding of microbial evolution and ecology. Marine blue holes are shallow karst formations characterized by low oxygen and high organic matter content. They are logistically challenging to sample, and thus our understanding of their biogeochemistry and microbial ecology is limited. We present a metagenomic and geochemical characterization of Amberjack Hole on the Florida continental shelf (Gulf of Mexico). Dissolved oxygen became depleted at the hole's rim (32 m water depth), remained low but detectable in an intermediate hypoxic zone (40-75 m), and then increased to a secondary peak before falling below detection in the bottom layer (80-110 m), concomitant with increases in nutrients, dissolved iron, and a series of sequentially more reduced sulfur species. Microbial communities in the bottom layer contained heretofore undocumented levels of the recently discovered phylum Woesearchaeota (up to 58% of the community), along with lineages in the bacterial Candidate Phyla Radiation (CPR). Thirty-one high-quality metagenome-assembled genomes (MAGs) showed extensive biochemical capabilities for sulfur and nitrogen cycling, as well as for resisting and respiring arsenic. One uncharacterized gene associated with a CPR lineage differentiated hypoxic from anoxic zone communities. Overall, microbial communities and geochemical profiles were stable across two sampling dates in the spring and fall of 2019. The blue hole habitat is a natural marine laboratory that provides opportunities for sampling taxa with under-characterized but potentially important roles in redox-stratified microbial processes.

Genotypic and phenotypic characterization of enterotoxigenic Escherichia coli strains isolated from Peruvian children.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines.

Risk factors for the spread of HIV and other sexually transmitted infections among men who have sex with men infected with HIV in Lima, Peru.

OBJECTIVES: To assess the prevalence of sexually transmitted infections (STIs), the frequency of sexual risk behaviours, and the relation between knowledge of HIV infection status and sexual risk behaviour among men who have sex with men (MSM) infected with HIV attending an STI clinic in Peru. METHODS: We recruited a convenience sample of 559 MSM from a municipal STI clinic in Lima, Peru. Participants completed a survey and provided blood for HIV, syphilis and HSV-2 antibody testing, and urine for gonorrhoea and chlamydia nucleic acid testing. RESULTS: Among 124 MSM with HIV, 72.6% were aware that they were infected with HIV. Active syphilis (RPR> or =1:8) was diagnosed in 21.0% of men infected with HIV, HSV-2 in 79.8%, urethral gonorrhoea in 1.6% and chlamydia in 1.6%. Among 41 participants reporting insertive anal intercourse with their last sex partner, 34.2% did not use a condom. Of the 86 participants reporting receptive anal intercourse, 25.6% did not use a condom. At least one episode of insertive unprotected anal intercourse (UAI) with a partner uninfected with HIV during the past 6 months was reported by 33.6% (35/104) of participants, and receptive UAI with a partner uninfected by HIV was reported by 44.6% (45/101). There was no difference in frequency of UAI with partners infected or uninfected with HIV observed between men who knew their serostatus compared with those who were previously undiagnosed (all p values >0.05). CONCLUSIONS: MSM with HIV in Peru engaged in high-risk behaviours for spreading HIV and STIs. Knowledge of whether someone was infected with HIV was not associated with a decreased frequency of UAI. Additional efforts to reduce risk behaviour after the diagnosis of HIV infection are necessary.

Serum prostacyclin binding defects in thrombotic thrombocytopenic purpura.

To understand the pathophysiologic significance of abnormal serum prostacyclin (PGI2) binding activities in thrombotic thrombocytopenic purpura (TTP), we evaluated the PGI2 binding characteristics in three chronic TTP sera and 19 normal sera. PGI2 binding by serum was rapid and reversible. The binding activity in TTP sera (22.1 +/- SD, 4.4%) was significantly lower than that of normal sera (42.2 +/- 6.2%). Moreover, the antiaggregating activity and 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) content in the gel filtrates representing the binding peak was proportionally lower in a TTP serum than normal serum. Although normal and TTP sera bound [14C]arachidonate with similar activity, and neither bound [3H]6KPGF1 alpha, there was a difference in prostaglandin E1 (PGE1) binding. Binding of [3H]PGE1 was subnormal in two TTP sera (W.J. and T.G.) and normal in the third (H.S.). Normal serum corrected the binding defects of TTP serum. Interestingly, the mixture of two TTP sera (W.J. and H.S.) mutually corrected their PGI2 binding defects. In addition, although in vivo plasma transfusions improved the PGI2 binding activity of W.J. and H.S., there existed a striking difference in the nature of their response. These observations indicate that there is at least two types of PGI2 binding defects in TTP. Our data indicate that TTP is associated with diminished serum binding of PGI2. This defect may reduce the availability of PGI2 to damaged vascular sites and decrease an important modulator of platelet thrombus formation at times of severe vascular insult.

Interaction between lymphocytes and platelets in the synthesis of prostacyclin.

To test the hypothesis that prostacyclin (PGI2) is formed via a biochemical interaction between platelets and lymphocytes, we measured eicosanoids by high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). A distinct 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) peak was noted when [14C]arachidonic acid ([14C]AA) was added to the mixed cell preparations which was increased by pretreating platelets with 1-benzylimidazole (1-BI). Lymphocytes prelabeled with [14C]AA failed to form 6KPGF1 alpha when stimulated with phytohemagglutinin (PHA) or ionophore A23187. When the prelabeled platelets were suspended together with aspirin-treated lymphocytes and stimulated with ionophore, thrombin, or collagen, a 6KPGF1 alpha peak was detected and enhanced by 1-BI. These results were supported by quantifying the 6KPGF1 alpha content in the HPLC-purified fraction by RIA. Adding prostaglandin H2 (PGH2) directly to lymphocytes led to 6KPGF1 alpha production. Platelet aggregation and release were inhibited by lymphocytes in a dose-related manner. We conclude that lymphocytes possess PGI2 synthase activity which is capable of converting platelet-derived PGH2 into PGI2. PGI2 formed is sufficient to inhibit platelet function.

Parameters governing permeate flux in an anaerobic membrane bioreactor treating low-strength municipal wastewaters: a literature review.

The objective of this review was to conduct a comprehensive literature survey to identify the parameters that govern the permeate flux in an anaerobic membrane bioreactor (AnMBR) treating municipal wastewater. Based on the survey, research to date indicates that the optimal membrane system for an AnMBR consists of an organic, hydrophilic, and negatively charged membrane with a pore size of approximately 0.1 microm. The use of both external and submerged membrane configurations shows promise. The operating parameters that affect permeate flux in an external membrane system are transmembrane pressure (TMP) and cross-flow velocity. The operating parameters that affect permeate flux in a submerged membrane system are TMP, sparging intensity, and duration of the relaxation period. Both cross-flow velocity and sparging intensity impart a significant amount of shear force on the biomass in an AnMBR. High shear forces can reduce the microbial activity in an AnMBR. In addition, high shear forces can reduce the size of the biosolids in the mixed liquor and increase the release of soluble microbial products. In this respect, external and submerged membrane systems are expected to perform differently because the magnitude of the shear forces to which the biomass is exposed in an external membrane system is significantly greater than that in a submerged system. The size of the biosolid particles and concentration of soluble microbial products in the mixed liquor affect permeate flux. Higher concentrations of soluble microbial products may be present in the mixed liquor when an AnMBR is operated at relatively low operating temperatures. Aerobic polishing following anaerobic treatment can potentially significantly reduce the concentration of some components of the soluble microbial products in the mixed liquor. It is not possible to remove the foulant layer on an organic membrane with caustic cleaning alone. Acidic cleaning or acidic cleaning followed by caustic cleaning is required to remove the foulant layer. This suggests that both biological/organic and inorganic material contribute to membrane fouling.

Characterization of fouled membranes from a membrane enhanced biological phosphorus removal system.

Characterization of fouled membranes is the first step towards a good understanding of membrane fouling nature and thus formulating effective engineering measures for fouling prevention and control. In this study, fouled membrane fibres collected from a pilot scale membrane enhanced biological phosphorus removal (MEBPR) process were systematically examined. Several analytical tools, including scanning electron microscopy (SEM), conventional optical microscopy (COM), energy dispersive X-ray (EDX) microanalysis, matrix assisted laser desorption/ionization--mass spectrometry (MALDI-MS) analysis, and conventional chemical analysis techniques were used. The results indicated that membrane fouling in the MEBPR process was mainly of an organic nature, and most extractable foulants were carbohydrates and humic or humic-like substances. Unlike in other wastewater treatment membrane bioreactors, microbial growth on fouled membranes was not substantial, probably due to the vigorous aeration applied and the strong hydrodynamic conditions within the membrane pore structure. After a period of sludge filtration, membrane surfaces became more hydrophobic and the resultant hydrophobic interactions between the fouled membranes and mixed liquor constituents might have accelerated the fouling process.

Prostaglandin production by murine tumors as a predictor for therapeutic response to indomethacin.

We investigated whether there is a relationship between the production of eicosanoids by murine solid tumors and their response to the prostaglandin H (PGH) synthase inhibitor indomethacin. Three sarcomas, designated FSA, NFSA, and SA-NH, and two carcinomas, designated MCA-K and HCA-I, syngeneic to C3Hf/Kam mice were used. In general, FSA and NFSA produced more PGH synthase products than lipoxygenase products, whereas HCA-I produced both types of metabolites in large quantities. All three tumors responded well to indomethacin treatment by slowing their growth. In contrast, MCA-K and SA-NH tumors produced insignificant quantities of PGH synthase products, but substantial amounts of lipoxygenase products. Their growth was not affected by treatment with indomethacin. Indomethacin did not influence tumor cell survival either in vitro or in vivo, but it reduced the proportion of S-phase cells in the tumors. The antitumor effect of indomethacin was not reduced by immunosuppression of the tumor host and was independent of tumor immunogenicity, implying that indomethacin acted through nonimmunological mechanisms. Thus, the effectiveness of indomethacin was directly related to the ability of tumors to produce PGs. Consequently, the eicosanoid profile of tumors could serve as a valuable way to select patients likely to respond to indomethacin and other PGH synthase inhibiting agents.

Localization of estrogen-induced DNA adducts and cytochrome P-450 activity at the site of renal carcinogenesis in the hamster kidney.

Renal carcinoma in male Syrian hamsters, induced by chronic administration of estradiol for 5-7 months, is known to arise in the cortex at the cortico-medullary junction. In this in vivo model for hormonal carcinogenesis, estrogen-induced covalent DNA adducts have previously been observed in whole kidney and have been postulated to be involved in tumor induction. In the present study, the intrarenal distribution of estrogen-induced DNA modification and estrogen metabolizing enzymes were investigated in male Syrian hamsters to ascertain a role of metabolism and adduct formation in estrogen-induced carcinogenesis. The highest estrogen-induced DNA adduct concentrations as measured by 32P-postlabeling analysis were found in the renal cortex of hamsters treated with estradiol for 7 months. Total adduct levels in medullary DNA were approximately one-half of those found in cortex. Cytochrome P-450 enzymes were detected only in microsomes of kidney cortex (approximately 0.8 +/- 0.6 nmol P-450/mg protein) but not medulla of untreated male Syrian hamsters. Prostaglandin endoperoxide synthase activity in kidney cortical microsomes was 1/5 of the activity found in medullary microsomes. Thus, microsomal cytochrome P-450 levels and estrogen-induced DNA adduct formation were highest in hamster kidney cortex, the origin of renal tumorigenesis. It is postulated that estrogen metabolism by cytochrome P-450 enzymes leading to covalent DNA modification plays a role in hormonal carcinogenesis in the hamster kidney.

Purification of thromboxane synthetase and evidence of two distinct mechanisms for the formation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid by porcine lung microsomes.

Synthesis of thromboxane B2 and 12-L-hydroxy-5,8,10-heptadecatrienoic acid by crude microsomes and by partially purified thromboxane synthetase from porcine lung has been studied. Formation of both compounds catalyzed by the purified enzyme is inhibited similarly by imidazole or increased temperature (52 degrees C). However, during the initial stages of purification only the production of thromboxane B2 was diminished by imidazole or increased temperature, indicating the presence of an additional mechanism for the formation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid. This novel mechanism is attributed to the effect of nondialyzable heat-stable factor(s) present in the crude preparation which stimulates the formation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid from prostaglandin H2 in a time-dependent manner.

The stimulation of arachidonic acid metabolism in human platelets by hydrodynamic stresses.

Even though shear-induced platelet activation and aggregation have been studied for about 20 years, there remains some controversy concerning the arachidonic acid metabolites formed during stress activation and the role of thromboxane A2 in shear-induced platelet aggregation. In this study, platelets were labelled with [1-14C]arachidonic acid to follow the metabolism of arachidonic acid in stimulated platelets using HPLC and scintillation counting. Platelets activated by thrombin formed principally thromboxane A2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). In contrast, for platelets activated by shear--though arachidonic acid metabolism was stimulated--only 12-HETE was formed and essentially no cyclooxygenase metabolites were detected. This indicates that physical forces may initiate a different pathway for eicosanoid metabolism than most commonly used chemical stimuli and perhaps also implies that regulation of the cyclooxygenase activity may be a secondary level of regulation in eicosanoid metabolism.

The effect of shear stress on the uptake and metabolism of arachidonic acid by human endothelial cells.

The uptake and metabolism of arachidonic acid (AA) by human umbilical vein endothelial cells was studied for cells in stationary culture and for cells exposed to physiological levels of shear stress. For cells grown in stationary culture, the initial incorporation of arachidonic acid was primarily into diacylglycerol and phospholipids. Cells exposed to flow incorporated labeled arachidonic acid at a similar rate as cells maintained in stationary culture; however, the distribution of the label was altered by flow. The incorporation of arachidonic acid into diacylglycerol and phosphatidylinositol was increased in cells exposed to flow. The largest increase occurred for cells exposed to arterial levels of shear stress for the shortest time period studied, 0.5 h. Prostacyclin (PGI2) and PGF2 alpha were the principal arachidonic acid metabolites formed. Shear stress-stimulated cells preferentially produced PGI2 relative to other eicosanoid products. The initiation of flow caused a burst of AA metabolism which was highly specific for PGI2. This might represent an increase in the turnover of phosphatidylinositol-bound arachidonic acid which is specifically converted to PGI2 as a result of flow-induced membrane stresses.

Regulation of intracellular arachidonate in normal and stressed endothelial cells.

The uptake of arachidonate and stearate from serum-free media by endothelial cells was investigated over a 48 h period. Arachidonate was rapidly incorporated into both the phospholipids and triacylglycerols. Triacylglycerol incorporation reached a maximum at 2 h and then rapidly declined with a concomitant increase in phospholipid incorporation. High initial arachidonate incorporation into phosphatidylcholine was followed by a partial transfer of that arachidonate to phosphatidylethanolamine. In contrast, stearate was slowly incorporated into all of the phospholipids and was not incorporated into the triacylglycerols. Cells stimulated with A23187 for 24 h cleaved stearate from all the phospholipids equally, whereas more arachidonate was cleaved from phosphatidylethanolamine than from the other phospholipids. Released arachidonate was both metabolized and reacylated into the triacylglycerols. Our results suggest that triacylglycerols serve as a modulator of intracellular arachidonate concentrations in endothelial cells.

Biofilm reactors in anaerobic wastewater treatment.

Attached biofilm reactors provide the means for implementing energy-efficient anaerobic wastewater treatment at full scale. Progress has been made in the development of fixed, expanded and fluidized bed anaerobic processes by addressing fundamental reactor design issues. Several new biofilm reactor concepts have evolved from recent studies.

Biotechnology developments for the treatment of toxic and inhibitory wastewaters.

The cost-effectiveness of biological processes has encouraged many researchers to consider biotreatment for the stabilization of toxic or recalcitrant wastewaters. However, to ensure adequate removal of trace contaminants and satisfactory performance with high strength inhibitory industrial wastewaters, conventional biotechnology is being re-evaluated. This review summarizes selected recent contributions to the development of appropriate biotechnology for toxic wastewater treatment. Microbiological constraints and potential solutions are examined. Assessments of conventional biological processes for contaminant control are reviewed, and several new developments in bioreactor design for inhibitory wastes are presented.

Activated microglia are the principal glial source of thromboxane in the central nervous system.

Thromboxane A2(TxA2) is a potent vasoconstrictor associated with cerebrovascular disease and is thought to be synthesized within tissues of the brain. In order to determine the cellular sources of TxA2 in the central nervous system (CNS), we measured the release of the stable metabolite TxB2 in cultures of mixed or highly enriched populations of brain glia. Using techniques which isolated large numbers of highly enriched microglia and astroglia, we found that only microglia release TxB2. Moreover, microglia, not astroglia, contain the requisite synthetic enzyme thromboxane synthase. Phagocytic signals and lipopolysaccharide are potent stimulants of microglial release of thromboxane, with lesser effects shown by platelet activating factor and substance P. We conclude that microglia, when activated, are the principal source of brain-derived thromboxane and may help to control vascular flow at sites of acute CNS injury.

Providing a Microenvironment for the Development of Human CD34+ Hematopoietic Cells in SCID Mice.

In order to develop a convenient small-animal model that can support the differentiation of human bone-marrow-derived CD34+ cells, we transplanted SCID mice with an immortalized human stromal cell line, Lof(11-10). The Lof(11-10) cell line has been characterized to produce human cytokines capable of supporting primitive human hematopoietic cell proliferation in vitro. Intraperitoneal injection of Lof(11-10) cells into irradiated SCID mice by itself resulted in a dose-dependent survival of the mice from lethal irradiation. The radioprotective survival was reflected by an increase in the growth and number of mouse bone-marrow-derived committed hematopoietic progenitors. The Lof(11-10) cells localized to the spleen, but not to the bone marrow of these animals and resulted in detectable levels of circulating human IL-6 in their plasma. Secondary intravenous injections of either human or simian CD34+ cells into the Lof(11-10)-transplanted SCID mice resulted in engraftment of injected cells within the bone marrow of these mice. The utility of this small-animal model that allows the growth and differentiation of human CD34+ cells and its potential use in clinical gene therapy protocols are discussed. Copyright 1997 S. Karger AG, Basel