From water to edible fish. Transfer of metals and metalloids in the San Roque Reservoir (Córdoba,Argentina). Implications associated with fish consumption

The concentration of Mn, Fe, Zn, Cu, Cd, Cr, Ni, Ag, Mo, Nd, Al, Ce, As, Sr, Pb, Pt and Hg was analysed in water, sediments, and aquatic organisms from the San Roque Reservoir (Córdoba-Argentina), sampled during the wet and dry season, to evaluate their transfer through the food web. Stable nitrogen (δ¹⁵N) isotopes were used to investigate trophic interactions. According to this, samples were divided into three trophic groups: plankton, shrimp (Palaemonetes argentinus) and fish (Silverside, Odontesthes bonariensis). Liver and gills are the main heavy metal storage tissues in fish. Hg and As concentrations in the muscle of O. bonariensis exceed the Oral Reference doses for metals established by USEPA (2009). Trophic magnification factors (TMFs) for each element were determined from the slope of the regression between trace element concentrations and δ¹⁵N. Calculated TMFs showed fundamental differences in the trophodynamics of the studied elements during the wet and dry season in the San Roque Reservoir. Concentrations of Ni, Cd, Cr, Al, Mn, Fe, Mo, Ce, Nd, Pt and Pb during both seasons, and Sr during the dry season, showed statistically significant decreases (TMF < 1) with increasing trophic levels. Thus these elements were trophically diluted in the San Roque food chain. Conversely, Cu, Ag and As (dry season) showed no significant relationships with trophic levels. Among the elements studied, Hg in the wet season, and Zn in the dry season were the only ones showing a statistically significant increase (TMF > 1) in concentration with trophic level. Current results trigger the need for further studies to establish differential behaviour with different species within the aquatic web, particularly when evaluating the transfer of toxic elements to edible organisms, which could pose health risks to humans.     

δ15N and δ13C diet–tissue discrimination factors for large sharks under semi-controlled conditions

Stable isotopes (δ¹⁵N and δ¹³C) are being widely applied in ecological research but there has been a call for ecologists to determine species- and tissue-specific diet discrimination factors (?¹³C and ?¹⁵N) for their study animals. For large sharks stable isotopes may provide an important tool to elucidate aspects of their ecological roles in marine systems, but laboratory based controlled feeding experiments are impractical. By utilizing commercial aquaria, we estimated ?¹⁵N and ?¹³C of muscle, liver, vertebral cartilage and a number of organs of three large sand tiger (Carcharias taurus) and one large lemon shark (Negaprion brevirostris) under a controlled feeding regime. For all sharks mean ± SD for ?¹⁵N and ?¹³C in lipid extracted muscle using lipid extracted prey data were 2.29‰ ± 0.22 and 0.90‰ ± 0.33, respectively. The use of non-lipid extracted muscle and prey resulted in very similar ?¹⁵N and ?¹³C values but mixing of lipid and non-lipid extracted data produced variable estimates. Values of ?¹⁵N and ?¹³C in lipid extracted liver and prey were 1.50‰ ± 0.54 and 0.22‰ ± 1.18, respectively. Non-lipid extracted diet discrimination factors in liver were highly influenced by lipid content and studies that examine stable isotopes in shark liver, and likely any high lipid tissue, should strive to remove lipid effects through standardising C:N ratios, prior to isotope analysis. Mean vertebral cartilage ?¹⁵N and ?¹³C values were 1.45‰ ± 0.61 and 3.75‰ ± 0.44, respectively. Organ ?¹⁵N and ?¹³C values were more variable among individual sharks but heart tissue was consistently enriched by ~ 1–2.5‰. Minimal variability in muscle and liver δ¹⁵N and δ¹³C sampled at different intervals along the length of individual sharks and between liver lobes suggests that stable isotope values are consistent within tissues of individual animals. To our knowledge, these are the first reported diet–tissue discrimination factors for large sharks under semi-controlled conditions, and are lower than those reported for teleost fish.     

Use of stable isotopes to understand food webs in Macao wetlands

In this study, components of the food-web in Macao wetlands were quantified using stable isotope ratio techniques based on carbon and nitrogen values. The δ¹³C and δ¹⁵N values of particulate organic matter (δ¹³C_POM and δ¹⁵N_POM, respectively) ranged from ?30.64 ± 1.0 to ?28.1 ± 0.7 ‰, and from ?1.11 ± 0.8 to 3.98 ± 0.7 ‰, respectively. The δ¹³C values of consumer species ranged from ?33.94 to ?16.92 ‰, showing a wide range from lower values in a freshwater lake and inner bay to higher values in a mangrove forest. The distinct dietary habits of consumer species and the location-specific food source composition were the main factors affecting the δ¹³C values. The consumer ¹⁵N-isotope enrichment values suggested that there were three trophic levels; primary, secondary, and tertiary. The primary consumer trophic level was represented by freshwater herbivorous gastropods, filter-feeding bivalves, and plankton-feeding fish, with a mean δ¹⁵N value of 5.052 ‰. The secondary consumer level included four deposit-feeding fish species distributed in Fai Chi Kei Bay and deposit-feeding gastropods in the Lotus Flower Bridge flat, with a mean δ¹⁵N value of 6.794 ‰. The tertiary consumers group consisted of four crab species, one shrimp species, and four fish species in the Lotus Flower Bridge Flat, with a mean δ¹⁵N value of 13.473 ‰. Their diet mainly comprised organic debris, bottom fauna, and rotten animal tissues. This study confirms the applicability of the isotopic approach in food web studies.     

Influence of daylight surface aggregation behavior on nutrient cycling during a Karenia brevis (Davis) G. Hansen & Ø. Moestrup bloom: Migration to the surface as a nutrient acquisition strategy

The toxic HAB dinoflagellate Karenia brevis (Davis) G. Hansen & Ø. Moestrup (formerly Gymnodinium breve) exhibits a migratory pattern atypical of dinoflagellates: cells concentrate in a narrow (∼0–5 cm) band at the water surface during daylight hours due to phototactic and negative geotactic responses, then disperse downward at night via non-tactic, random swimming. The hypothesis that this daylight surface aggregation behavior significantly influences bacterial and algal productivity and nutrient cycling within blooms was tested during a large, high biomass (chlorophyll a >19 μg L⁻¹) K. brevis bloom in October of 2001 by examining the effects of this surface layer aggregation on inorganic and organic nutrient concentrations, cellular nitrogen uptake, primary and bacterial productivity and the stable isotopic signature (δ¹⁵N, δ¹³C) of particulate material. During daylight hours, concentrations of K. brevis and chlorophyll a in the 0–5 cm surface layer were enhanced by 131% (±241%) and 32.1% (±86.1%) respectively compared with an integrated water sample collection over a 0–1 m depth. Inorganic (NH₄, NO₃₊₂, PO₄, SiO₄) and organic (DOP, DON) nutrient concentrations were also elevated within the surface layer as was both bacterial and primary productivity. Uptake of nitrogen (NH₄⁺, NO₃⁻, urea, dissolved primary amines, glutamine and alanine) compounds by K. brevis was greatest in the surface layer for all compounds tested, with the greatest enhancement evident in urea uptake rates, from 0.08 × 10⁻⁵ ng N K. brevis cell⁻¹ h⁻¹ to 3.1 × 10⁻⁵ ng N K. brevis cell⁻¹ h⁻¹. These data suggests that this surface aggregation layer is not only an area of concentrated cells within K. brevis blooms, but also an area of increased biological activity and nutrient cycling, especially of nitrogen. Additionally, the classic dinoflagellate migration paradigm of a downward migration for access to elevated NO₃⁻ concentrations during the dark period may not apply to certain dinoflagellates such as K. brevis in oligotrophic nearshore areas with no significant nitricline. For these dinoflagellates, concentration within a narrow surface layer in blooms during daylight hours may enhance nutrient supply through biological cycling and photochemical nutrient regeneration.     

δ15N tracks changes in the assimilation of sewage-derived nutrients into a riverine food web before and after major process alterations at two municipal wastewater treatment plants

Stable isotopes (δ¹⁵N and δ¹³C) were used to assess the changes in exposure and assimilation of sewage-derived nutrients in an aquatic food web following changes in effluent quality over an 8 year period at two municipal wastewater treatment plants (WWTPs) that discharge to the Grand River, in southern Ontario. Upgrades at the Kitchener WWTP started in late 2012 to enhance nitrification, while the Waterloo WWTP had a series of construction issues at the plant that resulted in a deterioration of its effluent quality over the study period (2007–2014). Fish (rainbow darter, Etheostoma caeruleum) and primary consumers (benthic invertebrates) were sampled in the receiving waters associated with each outfall. Upgrades at the Kitchener WWTP resulted in improved effluent quality with total annual ammonia output dropping by nearly sixfold (583–100 t), while the Waterloo WWTP increased its total annual ammonia output by nearly fourfold (135–500 t) over the duration of the study. Downstream of the Kitchener WWTP, the reduction in total ammonia output negatively correlated with changes in δ¹⁵N of rainbow darter from being depleted (prior to the upgrade) to reflecting signatures similar to those at the upstream reference site. The biota downstream of the Waterloo WWTP showed the opposite trend, going from slightly enriched, to being depleted relative to the upstream reference sites. δ¹³C was consistently higher downstream of both WWTPs regardless of changing effluent quality, and annual variability in δ¹³C was associated with annual river discharge. In a laboratory based dietary switch study conducted with rainbow darter, the isotope half-life in muscle (29 days for δ¹⁵N and 33 days for δ¹³C) were determined and these rapid changes were consistent with responses in muscle of wild fish. This is a unique study that was able to contrast two WWTPs in the same watershed as they underwent major changes in treatment processes. Stable isotopes were very effective as a tool to trace the changes in aquatic biota due to changes in wastewater effluent quality, both improvements and deterioration over time.     

Effect of tertiary sewage effluent additions on Prymnesium parvum cell toxicity and stable isotope ratios

We investigated the ability of the ichthyotoxic haptophyte Prymnesium parvum to use sewage-originated nutrients applying stable carbon (C) and nitrogen (N) isotope techniques. P. parvum was cultured under N and phosphorus (P) sufficient and deficient conditions in either sewage effluent-based medium or in a nitrate- and phosphate-based control. Cell densities and toxicities were monitored and stable carbon N isotopes signatures (δ¹³C and δ¹⁵N) of P. parvum and the sewage effluent analysed. Nitrogen and P sufficient cultures achieved the highest biomass followed by P and N deficient cultures, regardless of sewage effluent additions. The P deficient cultures with sewage effluent had higher toxicity, estimated as haemolytic activity (9.4 ± 0 × 10^?5 mg Saponin equiv. cell^?1) compared to the P deficient control and to all N deficient and NP sufficient cultures. Nutrient deficient conditions had no effect on the cell δ¹⁵N, but a decreasing effect on δ¹³C in the inorganic N deficient treatment. Growth in sewage-based media was followed by a substantial increase in the cell δ¹⁵N (10.4–16.1‰) compared to the control treatments (2.4–4.9‰), showing that P. parvum is capable of direct use of sewage-originated N, inorganic as well as organic. Uptake of terrestrial derived C in the sewage treatments was confirmed by a decrease in cell δ¹³C, implying that P. parvum is able to utilize organic nutrients in sewage effluent.     

Evaluation of the efficiency of metabolism of dinoflagellate phosphorus and carbon by a planktonic ciliate

The trophic transfer of nutrients through the microbial food web is a key top-down control in aquatic ecosystems which is notoriously difficult to evaluate, particularly for planktonic protists. In this study, a sensitive dual-radioactive tracer technique was developed to simultaneously assess the ingestion rate, and carbon- and phosphorus-specific assimilation efficiencies, of the marine planktonic ciliate Strobilidium neptuni feeding on the autotrophic dinoflagellate Heterocapsa triquetra. Dinoflagellate prey were simultaneously 16 h pulse labelled with NaH¹⁴CO₃ and H₃³³PO₄ before being fed to the ciliate, and radioactive labels were traced into ciliate biomass and the experimental medium, as well as being monitored in the prey cells. Rates measured in short-term (10 min) incubations, as commonly used to estimate protist uptake of fluorescently labelled prey, were approximately 6 times higher and 3–6 times more variable than rates measured in longer 3–5 h incubations. The efficiency of accumulation of prey carbon (54±9%) by ciliates was lower than that of prey phosphorus (68±3%) suggesting that the phosphorus to carbon ratio in the ciliates was 1.3 times higher than in the labelled dinoflagellate biomass. Rates of phosphorus accumulation and release were combined to reveal that ciliates consumed 3.2±0.6 dinoflagellates cell^?1 h^?1. The assessment of carbon tracer release by ciliates was less reliable due to ¹⁴CO₂ exchange between the experimental media and air. The study concludes that the dual phosphorus–carbon radioactive tracer labelling of algal prey allowed the quantification of protist herbivory and nutrient remineralisation in laboratory experiments, thereby providing a potential technique for studying planktonic microbial trophic interactions in situ.     

Improving estimates of trophic shift (Δδtrophic) for diet reconstruction studies using enzyme activities

For diet reconstruction studies using stable isotopes, accurate estimates of trophic shift (Δδ_trophic) are necessary to get reliable results. Several factors have been identified which affect the trophic shift. The goal of the present experiment was to test whether measurements of the activities of enzymes could improve the accuracy of estimation of trophic shift in fish. Forty-eight Nile tilapia (Oreochromis niloticus) were fed under controlled conditions with two diets differing in their protein content (21 and 41%) each at four different levels (4, 8, 12 and 16 g kg^? 0.8 d^? 1). At the end of the feeding experiment, proximate composition, whole body δ¹³C and δ¹⁵N as well as the activities of enzymes involved in anabolism and catabolism were measured. Step-wise regression specified contributing variables for Δδ¹⁵N (malic enzyme, aspartate aminotransferase and protein content) and Δδ¹³C_{lipid-free material} (aspartate aminotransferase and protein content). Explained variation by using the significant main effects was about 70% for Δδ¹⁵N and Δδ¹³C_{lipid-free material}, respectively. The results of the present study indicate that enzyme activities are suitable indicators to improve estimates of trophic shift.     

Use of stable isotope ratios of fish larvae as indicators to assess diets and patterns of anthropogenic nitrogen pollution in estuarine ecosystems

A stable isotope study was carried out to investigate the feeding ecology of the common goby Pomatoschistus microps larvae (Krøyer, 1838), and to assess differences in the response of planktonic food web to nutrient enrichment, in two ecosystems from the Southern European coast with different levels of historical pollution (estuaries of the Minho and Lima Rivers). At each estuary and time (July 2012, November 2012, February 2013, and June 2013), the fish larvae of two size classes (class 0: 0–10 mm; class 1: 10–15 mm), particulate organic matter (POM), and pelagic zooplankton were collected. The stable isotope mixing model SIAR revealed that, despite temporal differences in the relative proportion of prey items ingested, in both estuaries P. microps larvae feed on both planktonic-hyperbenthic food sources, predating mainly on copepods (from 34% to 60%), Mysidacea (from 16% to 28%), and brachyuran zoea (from 14% to 29%). Fish larvae size classes did not differ significantly for δ¹⁵N, and exhibited a very narrow range of the δ¹³C signature. Enriched δ¹⁵N values of all biota in the Lima estuary throughout the study period, with a marked nitrogen enrichment in colder months, are indicative of higher anthropogenic inputs of nitrogen (e.g. sewage and industrial discharges, agriculture) into this system. The δ¹⁵N values of fish larvae and other planktonic groups can be a sensitive bioindicator, because they are highly correlated with the nitrogen content of water (ammonium), indicating that this element has transferred through the planktonic food web. Enriched carbon isotope ratios were observed in warmer months, in both estuaries, and the heavier δ¹³C values in Lima are best explained by differences in the degree of marine influence. This research emphasises the utility of stable isotopes in trophic interactions studies, highlighting the relevance of the stable nitrogen isotope of zooplanktonic communities as a reliable bioindicator to detect patterns of anthropogenic nitrogen contamination in estuarine ecosystems.     

Identifying carbon sources and trophic position of coral reef fishes using diet and stable isotope (δ15N and δ13C) analyses in two contrasted bays in Moorea, French Polynesia

Stable isotope ratios (δ¹⁵N and δ¹³C) and diet of three fish species, Stegastes nigricans, Chaetodon citrinellus and Epinephelus merra, were analyzed on the fringing coral reefs of two bays that are differentially exposed to river runoff on Moorea Island, French Polynesia. S. nigricans and C. citrinellus relied mostly on turf algae and presented similar trophic levels and δ¹⁵N values, whereas E. merra fed on large invertebrates (crabs and shrimps) and had higher trophic levels and δ¹⁵N values. Discrepancies existed between stomach content and stable isotope analyses for the relative importance of food items. Bayesian mixing models indicated that sedimented organic matter was also an important additional food for S. nigricans and C. citrinellus, and fishes for E. merra. The main sources of organic matter involved in the food webs ending with these species were algal turfs and surface sediments, while water particulate organic matter was barely used. Significant spatial differences in C and N isotopic ratios for sources and fishes were found within and between bays. Lower ¹³C and higher ¹⁵N values were observed for various compartments of the studied trophic network at the end of each bay than at the entrance. Differences were observed between bays, with organic sources and consumers being, on average, slightly more ¹³C-depleted and ¹⁵N-enriched in Cook’s Bay than in Opunohu Bay, linked with a higher mean annual flow of the river at Cook’s Bay. Our results suggest that rivers bring continental material into these two bays, which is partly incorporated into the food webs of fringing coral reefs at least close to river mouths. Thus, continental inputs can influence the transfer of organic matter within coral reef food webs depending on the diet of organisms.     

Paenibacillus ihbetae sp. nov., a cold-adapted antimicrobial producing bacterium isolated from high altitude Suraj Tal Lake in the Indian trans-Himalayas

The assessment of bacterial diversity and bioprospection of the high-altitude lake Suraj Tal microorganisms for potent antimicrobial activities revealed the presence of two Gram-stain-variable, endospore-forming, rod-shaped, aerobic bacteria, namely IHBB 9852^T and IHBB 9951. Phylogenetic analysis based on 16S rRNA gene sequence showed the affiliation of strains IHBB 9852^T and IHBB 9951 within the genus Paenibacillus, exhibiting the highest sequence similarity to Paenibacillus lactis DSM 15596^T (97.8% and 97.7%) and less than 95.9% similarity to other species of the genus Paenibacillus. DNA-DNA relatedness among strains IHBB 9852^T and IHBB 9951 was 90.2%, and with P. lactis DSM 15596^T, was 52.7% and 52.4%, respectively. The novel strains contain anteiso-C_15:0, iso-C_15:0, C_16:0 and iso-C_16:0 as major fatty acids, and phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol were predominant polar lipids. The DNA G+C content for IHBB 9852T and IHBB 9951 was 52.1 and 52.2 mol%. Based on the results of phenotypic and genomic characterisations, we concluded that strains IHBB 9852^T and IHBB 9951 belong to a novel Paenibacillus species, for which the name Paenibacillus ihbetae sp. nov. is proposed. The type strain is IHBB 9852^T (=MTCC 12459^T = MCC 2795^T = JCM 31131^T = KACC 19072^T; DPD TaxonNumber TA00046) and IHBB 9951 (=MTCC 12458 = MCC 2794 = JCM 31132 = KACC 19073) is a reference strain.     

Feeding ecology of pelagic fish larvae and juveniles in slope waters of the Gulf of Mexico

Stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) were used to investigate feeding patterns of larval and early juvenile pelagic fishes in slope waters of the Gulf of Mexico. Contribution of organic matter supplied to fishes and trophic position within this pelagic food web was estimated in 2007 and 2008 by comparing dietary signatures of the two main producers in this ecosystem: phytoplankton [based on particulate organic matter (POM)] and Sargassum spp. Stable isotope ratios of POM and pelagic Sargassum spp. were significantly different from one another with δ¹³C values of POM depleted by 3–6‰ and δ¹⁵N values enriched by 2 relative to Sargassum spp. Stable isotope ratios were significantly different among the five pelagic fishes examined: blue marlin Makaira nigricans, dolphinfish Coryphaena hippurus, pompano dolphinfish Coryphaena equiselis, sailfish Istiophorus platypterus and swordfish Xiphias gladius. Mean δ¹³C values ranged almost 2 among fishes and were most depleted in I. platypterus. In addition, mean δ¹⁵N values ranged 4–5 with highest mean values found for both C. hippurus and C. equiselis and the lowest mean value for M. nigricans during both years. Increasing δ¹³C or δ¹⁵N with standard length suggested that shifts in trophic position and diet occurred during early life for several species examined. Results of a two‐source mixing model suggest approximately an equal contribution of organic matter by both sources (POM = 55%; pelagic Sargassum spp. = 45%) to the early life stages of pelagic fishes examined. Contribution of organic matter, however, varied among species, and sensitivity analyses indicated that organic source estimates changed from 2 to 13% for a δ¹³C fractionation change of ±0·25‰ or a δ¹⁵N fractionation change of ± 1·0‰ relative to original fractionation values.     

Influences of fish on food web structure and function in mountain lakes

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Synthesis,biological evaluation and molecular docking studies of chromone hydrazone derivatives as α-glucosidase inhibitors

A series of chromone hydrazone derivatives 4a–4p have been synthesized, characterized by ¹H NMR and ¹³C NMR and evaluated for their in vitro α-glucosidase inhibitory activity. Out of these tested compounds, six (4a, 4b, 4d, 4j, 4o and 4p) displayed potent α-glucosidase inhibitory activity with IC₅₀ values in the range of 20.1 ± 0.19 μM to 45.7 ± 0.23 μM, as compared to the standard drug acarbose (IC₅₀ = 817.38 ± 6.27 μM). Among this series, compound 4d (IC₅₀ = 20.1 ± 0.19 μM) with 4-sulfonamide substitution at phenyl part of hydrazide was found to be the most active compound. Lineweaver-Burk plot analysis indicated that compound 4d is a non-competitive inhibitor of α-glucosidase. The binding interactions of the most active analogs were confirmed through molecular docking studies. Docking studies showed 4d are interacting with the residues Glu-276, Asp-214, Asp-349 and Arg-439 through hydrogen bonds, arene-anion and arene-cation interactions. In summary, our studies shown that these chromone hydrazone derivatives are a new class of α-glucosidase inhibitors.     

Nitrogen isotopes in otoliths reconstruct ancient trophic position

The ratio of ¹⁵N/¹⁴N (δ¹⁵N) from consumer and prey tissue is commonly used in ecological studies to determine trophic level, food web structure, and mean trophic level in aquatic ecosystems. There is a predictable positive relationship between the δ¹⁵N values in tissue and trophic level, caused by the bioaccumulation of ¹⁵N in tissues of consumers with each step up the food chain. Reconstructing trophic structure or food chain length over time may provide resource managers with insights about ecosystem biodiversity and resilience. Yet, in many marine systems the absence of baseline information before anthropogenic disturbances makes comparative studies addressing ecosystem responses extremely difficult. Here we attempt to retrospectively reconstruct trophic position in four species of fish from the upper Gulf of California, Mexico before perturbations such as overfishing or the damming of the Colorado River. We first validated if otolith δ¹⁵N approximates the δ¹⁵N observed in fish tissue. We then used the δ¹⁵N encapsulated in ancient fish otoliths that are between 1,000 and 5,500 years old to define the food web structure. Our results suggested that δ¹⁵N in otoliths has slightly more positive δ¹⁵N than soft tissue. The δ¹⁵N values from ancient otoliths appropriately defined the fishes’ relative trophic position. We found significant differences in δ¹⁵N between functional groups, apex predator versus intermediate predators. Juveniles and adult fishes displayed trophic separation between functional groups. Our findings advocate the application of δ¹⁵N analysis of prehistoric otoliths for establishing pre-disturbance ecological benchmarks.     

Design,synthesis and in vitro anticancer activity of novel quinoline and oxadiazole derivatives of ursolic acid

A series of new quinoline derivatives of ursolic acid were designed and synthesized in an attempt to develop potential anticancer agents. The structures of these compounds were identified by ¹H NMR, ¹³C NMR, IR and ESI-MS spectra analysis. The target compounds were evaluated for their in vitro cytotoxicity against three human cancer cell lines (MDA-MB-231, Hela and SMMC-7721). From the results, compounds 3a–d displayed significant antitumor activity against three cancer cell lines. Especially, compound 3b was found to be the most potent derivative with IC₅₀ values of 0.61 ± 0.07, 0.36 ± 0.05, 12.49 ± 0.08 μM against MDA-MB-231, HeLa and SMMC-7721 cells, respectively, stronger than positive control etoposide. Furthermore, the Annexin V-FITC/PI dual staining assay revealed that compound 3b could significantly induce the apoptosis of MDA-MB-231 cells in a dose-dependent manner. The cell cycle analysis also indicated that compound 3b could cause cell cycle arrest of MDA-MB-231 cells at G0/G1 phase.     

Effect of oregano essential oil and carvacrol on Cryptosporidium parvum infectivity in HCT-8 cells

Cryptosporidium parvum is the second leading cause of persistent diarrhea among children in low-resource settings. This study examined the effect of oregano essential oil (OEO) and carvacrol (CV) on inhibition of C. parvum infectivity in vitro. HCT-8 cells were seeded (1 × 10⁶) in 96-well microtiter plates until confluency. Cell viability and infectivity were assessed by seeding HCT-8 cell monolayers with C. parvum oocysts (1 × 10⁴) in two modalities: 1) 4 h co-culture with bioactive (0–250 μg/mL) followed by washing and incubation (48 h, 37 °C, 5% CO₂) in bioactive-free media; and 2) 4 h co-culture of C. parvum oocysts followed by washing and treatment with bioactive (0–250 μg/mL) during 48-h incubation. Cell viability was tested using Live/Dead? assay whereas infectivity was measured using C. parvum-specific antibody staining via immunofluorescence detection. Loss of cell viability was observed starting at 125 μg/mL and 60 μg/mL for OEO and CV, respectively. Neither OEO nor CV modulated the invasion of C. parvum sporozoites in HCT-8 cells. Treatment with bioactive after invasion reduced relative C. parvum infectivity in a dose-dependent manner to 55.6 ± 10.4% and 45.8 ± 4.1% at 60 and 30 μg/mL of OEO and CV, respectively. OEO and CV are potential bioactives to counteract C. parvum infection in children.     

Discriminating the endogenous and exogenous urinary estrogens in human by isotopic ratio mass spectrometry and its potential clinical value

Estrogens were prohibited in the food producing animals by European Union (96/22/EC directive) and added to the Report on Carcinogens in United States since 2002. Due to very low concentration in serum or urine (～pg/mL), the method of control its abuse had not been fully developed.The endogenous estrogens were separated from urines of 18 adult men and women. The exogenous estrogens were chemical reference standards and over the counter preparations. Two patients of dysfunctional uterine bleeding (DUB) administered exogenous estradiol and the urines were collected for 72 h. The urinary estrogens were separated by high-performance liquid chromatography (HPLC) and confirmed. The exogenous and exogenous estrogens were analyzed by gas chromatography combustion isotope ratio mass spectrometry (GC–C–IRMS) to determine the ¹³C/¹²C ratio (δ¹³C‰).The δ¹³C‰ values of reference standard of E1, E2, and E3 were ?29.36 ± 0.72, ?27.98 ± 0.35, ?27.62 ± 0.51, respectively. The δ¹³C‰ values of the endogenous E1, E2, and E3 were ?21.62 ± 1.07, ?22.14 ± 0.98, and ?21.88 ± 1.16, with P < 0.01 (t-test). Two DUB patients’ urinary estradiol δ¹³C‰ values was depleted to ?28.02 ± 0.33 after the administration. The progesterone, 17α-hydroxyprogesterone, pregnanediol, as well as desogestrel and ethinylestradiol from contraceptives were also determined.Stable carbon isotope analysis can distinguish the endogenous and exogenous urinary estrogen in human.     

Microbiological and chemical quality of a traditional salted-fermented fish (Hout-Kasef) product of Jazan Region,Saudi Arabia

The Hout-Kasef is traditional salted fermented fish product of natural fermentation of salted mullet fish of coastal area of Jazan region of Saudi Arabia. The present study was carried out to investigate the microbiological and chemical characteristic of Hout-Kasef. A total of twenty-four salted fish samples were purchased from fish market in Jazan and Abu-Arish at different times of the year. The microbial studies of salted-fermented fish revealed a total bacterial count ranging from 2.81 to 4.72 Log₁₀ CFU/g, yeast and mold counts ranging from 0.48 to 3.14 Log₁₀ CFU/g, total staphylococci count 2.71–3.85 Log₁₀ CFU/g, halophile bacteria count 3.26–5.14 Log₁₀ CFU/g, and coliforms count <1 Log₁₀ CFU/g. However, pathogenic bacteria such as Listeria monocytogenes, Vibrio spp., Campylobacter spp. and Yersinia species were not detected. The major bacteria species isolated and identified from the salted fermented fish were Bacillus Subtilus, Bacillus mycoides, Bacillus licheniformis, Bacillus pumilus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus xylosus, Staphylococcus saprophticus and Staphylococcus cahnii subsp cahnii. The chemical analysis of salted fermented fish showed high content of moisture (47.96%), protein (25.71%), ash (19.6%) and salt (15.19%) but low contents of lipid (7.25%). The salted-fermented fish also showed high level of total volatile basic nitrogen (78.86 mg/100 gm sample) and thiobarbutric acid number (32.32 mg malonaldehyde/kg) with a pH value of pH 6.3. Finally, this study showed the presence of gram positive and gram negative bacteria in the fish product. The predominant microorganisms found were Bacillus and Staphylococcus spp. The fish product had high content of salt and TVB-N levels.     

Radiosynthesis and evaluation of new PET ligands for peripheral cannabinoid receptor type 1 imaging

Cannabinoid receptor type 1 (CB1) is mainly expressed in the brain, as well as being expressed in functional relevant concentrations in various peripheral tissues. 1-(4-Chlorophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea (PSNCBAM-1, 1) was developed as a potent allosteric antagonist for CB1 and its oral administration led to reductions in the appetite and body weight of rats. Several analogs of 1 (compounds 2 and 3) were recently identified through a series of structure-activity relationship studies. Herein, we report the synthesis of radiolabeled analogs of these compounds using [¹¹C]COCl₂ and an evaluation of their potential as PET ligands for CB1 imaging using in vitro and in vivo techniques. [¹¹C]2 and [¹¹C]3 were successfully synthesized in two steps using [¹¹C]COCl₂. The radiochemical yields of [¹¹C]2 and [¹¹C]3 were 17 ± 8% and 20 ± 9% (decay-corrected to the end of bombardment, based on [¹¹C]CO₂). The specific activities of [¹¹C]2 and [¹¹C]3 were 42 ± 36 and 37 ± 13 GBq/μmol, respectively. The results of an in vitro binding assay using brown adipose tissue (BAT) homogenate showed that the binding affinity of 2 for CB1 (K_D = 15.3 µM) was much higher than that of 3 (K_D = 26.0 µM). PET studies with [¹¹C]2 showed a high uptake of radioactivity in BAT, which decreased in animals pretreated with AM281 (a selective antagonist for CB1). In conclusion, [¹¹C]2 may be a useful PET ligand for imaging peripheral CB1 in BAT.