Effectiveness of Oxygen-Saturated Seawater Injections and Air Sparging Technologies in Remediation of Coastal Marine Sediments from Sludge.

The occurrence of hypoxic muddy sediments on shallow beaches and other sheltered areas is a well-known environmental problem, which negatively affects coastal areas, tourism potential, the public use of beaches and sediment biodiversity. The usual solution is limited to dredging and removal of sludge to a landfill site. In this study, a laboratory-scale experiment was performed to determine the effectiveness of two technologies: a modification of air sparging and a new approach based on injecting oxygen-saturated seawater in hypoxic muddy sediments (oxygen-saturated seawater injections method), for remediating sludge in coastal sediments, minimizing environmental impact respect to dredging. Our results showed that both technologies significantly increased dissolved oxygen content in pore water, facilitating the oxidation of more than 90% of the organic matter, and other reduced inorganic compounds such as sulphide, with the consequent increase in sulphate concentration from 0.3 to 3.0 g·L-1. Moreover, a rise of redox potential from - 258 mV to above 200 mV, and a dramatic drop in chemical oxygen demand were also indicators that oxic conditions had been restored. After 65 days, soft, black, muddy and hypoxic sediment with high organic matter content and a characteristic foul odour was transformed into well-oxygenated sediment, which had a low organic matter content and had lost its initial shiny black colour and odour. The main difference between both technologies was the depth influenced by sediment remediation; oxygen-saturated seawater injections affected deeper areas than clean pressurized air injections.

Caveats on COVID-19 herd immunity threshold: the Spain case.

After a year of living with the COVID-19 pandemic and its associated consequences, hope looms on the horizon thanks to vaccines. The question is what percentage of the population needs to be immune to reach herd immunity, that is to avoid future outbreaks. The answer depends on the basic reproductive number, R0, a key epidemiological parameter measuring the transmission capacity of a disease. In addition to the virus itself, R0 also depends on the characteristics of the population and their environment. Additionally, the estimate of R0 depends on the methodology used, the accuracy of data and the generation time distribution. This study aims to reflect on the difficulties surrounding R0 estimation, and provides Spain with a threshold for herd immunity, for which we considered the different combinations of all the factors that affect the R0 of the Spanish population. Estimates of R0 range from 1.39 to 3.10 for the ancestral SARS-CoV-2 variant, with the largest differences produced by the method chosen to estimate R0. With these values, the herd immunity threshold (HIT) ranges from 28.1 to 67.7%, which would have made 70% a realistic upper bound for Spain. However, the imposition of the delta variant (B.1.617.2 lineage) in late summer 2021 may have expanded the range of R0 to 4.02-8.96 and pushed the upper bound of the HIT to 90%.

Identification of the first COVID-19 infections in the US using a retrospective analysis (REMEDID).

Accurate detection of early COVID-19 cases is crucial to reduce infections and deaths, however, it remains a challenge. Here, we used the results from a seroprevalence study in 50 US states to apply our Retrospective Methodology to Estimate Daily Infections from Deaths (REMEDID) with the aim of analyzing the initial spread of SARS-CoV-2 infections across the US. Our analysis revealed that the virus likely entered the country through California on December 28, 2019, which corresponds to 16 days prior to the officially recognized entry date established by the Centers of Disease Control and Prevention. Furthermore, the REMEDID algorithm provides evidence that SARS-CoV-2 entered, on average, a month earlier than previously reflected in official data for each US state. Collectively, our mathematical modeling provides more accurate estimates of the initial COVID-19 cases in the US, and has the ability to be extrapolated to other countries and used to retrospectively track the progress of the pandemic. The use of approaches such as REMEDID are highly recommended to better understand the early stages of an outbreak, which will enable health authorities to improve mitigation and preventive measures in the future.

Disruption of the actin cytoskeleton induces fluorescent glucose accumulation on the rat hepatocytes Clone 9.

BACKGROUND: Glucose transport and metabolism are highly specialized in hepatocytes. Actin cytoskeleton is fundamental to the maintenance of their morphology as well as to ensure their functionality. Here we study the effect of the actin disrupting natural compounds cytochalasin B and latrunculin A on the glucose metabolism of the Clone 9 rat hepatocytes once the glucose molecule is inside them and the effects of two hormones which main function is regulating the glucose metabolism on the actin cytoskeleton of Clone 9 cells. METHODS: F-actin was labeled by using Oregon Green 514 ® phalloidin and glucose inside cells was monitored with the fluorescent D-glucose derivative; 2-NBDG. Observations and measurements were carried out by using a confocal microscope. RESULTS: Nor insulin neither glucagon was able to induce any significant effect in the quantity of F-actin present on Clone 9 cells. But insulin triggers a strong reorganization on the pattern of distribution of F-actin. However, the actin cytoskeleton disruption induced by CB and more efficiently by Lat A caused accumulation of 2-NBDG in cells. CONCLUSION: These results state that disruption of the actin cytoskeleton induces fluorescent glucose accumulation on the rat hepatocytes Clone 9 suggesting that actin disrupting agents cause a blockage in the glycolytic pathway of Clone 9 hepatocytes.

Retrospective methodology to estimate daily infections from deaths (REMEDID) in COVID-19: the Spain case study.

The number of new daily infections is one of the main parameters to understand the dynamics of an epidemic. During the COVID-19 pandemic in 2020, however, such information has been underestimated. Here, we propose a retrospective methodology to estimate daily infections from daily deaths, because those are usually more accurately documented. Given the incubation period, the time from illness onset to death, and the case fatality ratio, the date of death can be estimated from the date of infection. We apply this idea conversely to estimate infections from deaths. This methodology is applied to Spain and its 19 administrative regions. Our results showed that probable daily infections during the first wave were between 35 and 42 times more than those officially documented on 14 March, when the national government decreed a national lockdown and 9 times more than those documented by the updated version of the official data. The national lockdown had a strong effect on the growth rate of virus transmission, which began to decrease immediately. Finally, the first inferred infection in Spain is about 43 days before the official data were available during the first wave. The current official data show delays of 15-30 days in the first infection relative to the inferred infections in 63% of the regions. In summary, we propose a methodology that allows reinterpretation of official daily infections, improving data accuracy in infection magnitude and dates because it assimilates valuable information from the National Seroprevalence Studies.

Identification of the first COVID-19 infections in the US using a retrospective analysis.

Accurate detection of early COVID-19 cases is crucial to drastically reduce infection, hospitalization, and death rates. However, it remains a challenge and methods for identifying initial COVID-19 cases are urgently needed. Here, we used the results from a seroprevalence study in 50 US states to apply our Retrospective Methodology to Estimate Daily Infections from Deaths (REMEDID) with the aim of analyzing the initial stages and spread of SARS-CoV-2 infections across the United States (US). Our retrospective data analysis revealed that the virus likely entered the country through California on December 28, 2019, which corresponds to 16 days before the officially recognized entry date established by the CDC. Thus, REMEDID provides evidence that SARS-CoV-2 entered the U.S. earlier than previously reflected in official data. Collectively, our mathematical modeling more accurately estimates the initial COVID-19 cases in the US, may be extrapolated to other countries, and may be used to retrospectively track the progress of the pandemic. Approaches such as REMEDID may enable health authorities to accelerate preventative measures aimed at controlling pandemics within weeks of their onset.

Detection of 13,19-didesmethyl C spirolide by fluorescence polarization using Torpedo electrocyte membranes.

Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with alpha-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata. The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 +/- 1.9%. The quantification range for this toxin in mussel was 40-200 microg/kg of shellfish meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in shellfish as a screening assay.

Impact of the pectenotoxin C-43 oxidation degree on its cytotoxic effect on rat hepatocytes.

The metabolism of toxins that have accumulated in fish and shellfish is considered a detoxification process, as happens with pectenotoxins (PTXs) in the Japanese scallop Patinopecten yessoensis. PTXs are macrolactones that display hepatotoxicity in mice, principally by capping or sequestering actin, their molecular target. PTX-2, which is considered to be the parental compound, oxidizes progressively to PTX-1, PTX-3, and PTX-6 in the Japanese scallop. In this study, we observed that PTX-1, PTX-6, and PTX-9 induce dose-dependent damage in the actin cytoskeleton and in the viability of primary cultured rat hepatocytes. In Clone 9 rat hepatocytes, PTX-1 and PTX-9 also affect the morphology of cells, but surprisingly, PTX-6 induced no effect. In accordance with this lack of activity, the actin cytoskeleton of CaCo-2 cells, another epithelial cell line, is not affected by PTX-6. In conclusion, the order of cytotoxicity of the analogues is PTX-2 > PTX-1 > PTX-6 >PTX-9. From a structure-activity perspective, the increase in the level of oxidation of the PTX molecule on C-43 decreases its cytotoxicity. Furthermore, PTX-6 is not able to induce effects on immortal cells while retaining its toxicity against primary cultured cells, whereas PTX-9, a 7-S-isomer, is active in both cellular models. The different cytotoxicities exerted by PTX-6 on cell lines and primary cells could be determined by the presence of a carboxylic acid group on C43 of the PTX molecule.

Effects of environmental variables on the distribution of juvenile cubomedusae Carybdea marsupialis in the coastal Western Mediterranean.

Relationships between environmental factors and oscillations in jellyfish abundance, especially in the early life stages, could help to interpret past increases and also predict scenarios in a changing future. For the first time, we present cubozoan spatial and temporal distributions in the earliest stages and their relationships with different factors. Abundances of Carybdea marsupialis medusae showed high interannual variability from 2008 to 2014 along the Dénia coast (SE Spain, W Mediterranean). During 2015, samples were collected from 11 beaches along 17 km of coastline, 8 times from January to November in order to determine the effects of environmental factors on the distribution of juvenile C. marsupialis. Juveniles (≤ 15 mm diagonal bell width) were present from May to July, with more sampled near shore (0-15 m). Most of them occurred in June when their numbers were unequal among beaches (average 0.05 ind m-3, maximum 6.71 ind m-3). We tested distributions of juveniles over time and space versus temperature, salinity, nutrients (N, P and Si), chlorophyll-a (Chl-a), and zooplankton abundance. Temperature and cladocerans (zooplankton group) were significantly positively correlated with juvenile distribution, whereas Chl-a concentration was weakly negative. By contrast, in 2014, high productivity areas (Chl-a and zooplankton) overlapped the maximum adult abundance (5.2 ind m-3). The distribution of juveniles during 2015 did not spatially coincide with the areas where ripe adults were located the previous year, suggesting that juveniles drift with the currents upon release from the cubopolyps. Our results yield important insights into the complexity of cubozoan distributions.

Detection of gymnodimine-A and 13-desmethyl C spirolide phycotoxins by fluorescence polarization.

The gymnodimines and spirolides are phycotoxins classified into a heterogeneous group of marine biocompounds called cyclic imines. Although there is no clear evidence of their toxicity to humans, gymnodimines and spirolides are highly toxic to rodents and constitute a source of false positives in lipophilic toxin detection by the mouse bioassay. Using nicotinic acetylcholine receptor-enriched membranes of Torpedo, and fluorescent alpha-bungarotoxin, we developed a fluorescence polarization assay to detect and quantify gymnodimine-A and 13-desmethyl C spirolide. The presence of these cyclic imines in solution inhibited the interaction of fluorescent-labeled alpha-bungarotoxin with nicotinic acetylcholine receptors in a concentration-dependent manner. The sensitivity of the assay is in the order of nanomolar concentrations of gymnodimine and 13-desmethyl C spirolide. Okadaic acid, yessotoxin, and brevetoxin-2, three lipophilic marine toxins, did not interfere with this assay. A suitable extraction method in shellfish was also developed. The gymnodimine-A and 13-desmethyl C spirolide recovery rates of mussel matrix extraction with acetone/chloroform were 63.6% +/- 3.5% and 87.4% +/- 5.3%, respectively. In summary, this inhibition assay is capable of gymnodimine-A and 13-desmethyl C spirolide detection in mussel extracts with enough sensitivity and specificity to quantify these toxins in the range of 50-2000 microg/kg and 70-700 microg/kg of shellfish meat, respectively.

Use of biosensors as alternatives to current regulatory methods for marine biotoxins.

Marine toxins are currently monitored by means of a bioassay that requires the use of many mice, which poses a technical and ethical problem in many countries. With the exception of domoic acid, there is a legal requirement for the presence of other toxins (yessotoxin, saxitoxin and analogs, okadaic acid and analogs, pectenotoxins and azaspiracids) in seafood to be controlled by bioassay, but other toxins, such as palytoxin, cyclic imines, ciguatera and tetrodotoxin are potentially present in European food and there are no legal requirements or technical approaches available to identify their presence. The need for alternative methods to the bioassay is clearly important, and biosensors have become in recent years a feasible alternative to animal sacrifice. This review will discuss the advantages and disadvantages of using biosensors as alternatives to animal assays for marine toxins, with particular focus on surface plasmon resonance (SPR) technology.

Feasibility of gymnodimine and 13-desmethyl C spirolide detection by fluorescence polarization using a receptor-based assay in shellfish matrixes.

The detection of toxins in shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by intraperitoneal injection causing false positives in lipophilic toxin detection by the mouse bioassay. Based on the detection of molecular interactions by fluorescence polarization an inhibition assay was developed using fluorescent alpha-bungarotoxin and nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata to detect gymnodimine and 13-desmethyl C spirolide. Both toxins, classified into the cyclic imine group, inhibit the interaction of alpha-bungarotoxin with Torpedo nicotinic acetylcholine receptors in the nM range. In this study we analyze the matrix effect of four shellfish species on the fluorescence polarization assay. Mussels, clams, cockles and scallops were extracted with acetone and sequentially partitioned with n-hexane and chloroform. The interference of these shellfish extracts with the alpha-bungarotoxin fluorescence or its binding to the nicotinic acetylcholine receptor was lower than 11%. The average recovery rates of gymnodimine and 13-desmethyl C spirolide using these solvents were 90.6+/-7.8% and 89.6+/-3.2%, respectively with variations among species. The quantification range of this fluorescence polarization assay for gymnodimine and 13-desmethyl C spirolide in all tested species was 80-2000 microg kg(-1) and 85-700 microg kg(-1) of shellfish meat, respectively. This assay format can be used to detect gymnodimine and 13-desmethyl C spirolide in shellfish as a screening assay.

Feasibility of using a surface plasmon resonance-based biosensor to detect and quantify yessotoxin.

Yessotoxin (YTX) is a disulfated polyether toxin produced by marine dinoflagellates. Although there is no clear evidence that YTX is toxic to humans, it is a major cause of false positives in DSP toxin detection by mouse bioassay. We developed a new detection and quantification method for yessotoxin using a BiaCore X Surface plasmon resonance (SPR)-based biosensor. The assay is based in the interaction of YTX with phosphodiesterase enzymes (PDE), one of its cellular targets. The injection of several YTX concentrations (3-12 microM) over immobilized PDE I, showed a dose dependent binding signal, which K(obs) (observed rate constant) allowed us to obtain a calibration curve with a linear fit. The detection of yessotoxin using SPR-based biosensor allows the quantification of the toxin with an automated and repetitive method at concentrations in the range of the 1 mg kg(-1) European regulatory limit.

Paralytic shellfish poisoning detection by surface plasmon resonance-based biosensors in shellfish matrixes.

The detection of paralytic shellfish poisoning (PSP) toxins in contaminated shellfish is essential for human health preservation. Ethical and technical reasons have prompted the search for new detection procedures as an alternative to the mouse bioassay. On the basis of the detection of molecular interactions by surface plasmon resonance (SPR) biosensors, an inhibition assay was developed using an anti-GTX2/3 antibody (GT13-A) and a saxitoxin-CM5 chip. This assay allowed for quantification of saxitoxin (STX), decarbamoyl saxitoxin (dcSTX), gonyautoxin 2,3 (GTX2/3), decarbamoyl gonyautoxin 2,3 (dcGTX2/3), gonyautoxin 5 (GTX5), and C 1,2 (C1/2) at concentrations from 2 to 50 ng/mL. The interference of five shellfish matrixes with the inhibition assay was analyzed. Mussels, clams, cockles, scallops, and oysters were extracted with five published methods. Ethanol extracts and acetic acid/heat extracts (AOAC Lawrence method) performed adequately in terms of surface regeneration and baseline interference, did not inhibit antibody binding to the chip surface significantly, and presented STX calibration curves similar to buffer controls in all matrixes tested. Hydrochloric acid/heat extracts (AOAC mouse bioassay method) presented surface regeneration problems, and although ethanol-acetic acid/dichloromethane extracts performed well, they were considered too laborious for routine sample testing. Overall the best results were obtained with the ethanol extraction method with calibration curves prepared in blank matrix extracts. STX recovery rate with the ethanol extraction method was 60.52+/-3.72%, with variations among species. The performance of this biosensor assay in natural samples, compared to two AOAC methods for PSP toxin quantification (mouse bioassay and HPLC), suggests that this technology can be useful as a PSP screening assay. In summary, the GT13-A-STX chip inhibition assay is capable of PSP toxin detection in ethanol shellfish extracts, with sufficient sensitivity to quantify the toxin in the range of the European regulatory limit of 80 microg/100 g of shellfish meat.