Amounts of nuclear DNA in marine halophytes

The amount of nuclear DNA, expressed as the C-value, was estimated for 13 marine halophytic plant species from six families. Plant material was collected in the nature reserve of the Strunjan saltpan in the Northern Adriatic and comprised all halophytic species inside the investigated area. Reproductive region of the shoot or root tips of halophytes were dissected, nuclei were Feulgen stained and 2C-values were measured by DNA image cytometry as follows: Crithmum maritimum (4.38 pg DNA), Artemisia caerulescens (6.43 pg), Aster tripolium (21.43 pg), Inula crithmoides (3.63 pg), Atriplex portulacoides (1.83 pg), A. prostrata (1.51 pg), Salicornia europaea (2.75 pg), Salsola soda (2.62 pg), Sarcocornia fruticosa (5.91 pg), Suaeda maritima (2.11 pg), Limonium angustifolium (5.06 pg), Puccinellia palustris (8.15 pg) and Ruppia cirrhosa (4.65 pg). With the exception of the C-value estimate for A. caerulescens, which has been listed in the Plant DNA C-values Database, the C-values represent the first estimates for all the examined species. In addition, the C-value for R. cirrhosa is also the first report for the family Ruppiaceae. The investigated halophytes had a smaller genome size compared to other known C-values for species within a particular family and also when compared to the mean values of dicots and monocots. The study also showed that halophylic annuals have a smaller genome size (2.49 pg) than perennial ones (7.45 pg DNA).     

Genome size variation and polyploidy in the resurrection plant genus Ramonda: Cytogeography of living fossils

The genus Ramonda includes three preglacial paleoendemic species surviving as the rare resurrection angiosperms of the Northern hemisphere in refugia habitats in the Balkan (Ramonda nathaliae and Ramonda serbica) and Iberian Peninsulas (Ramonda myconi). This study focuses on: assessing genome size and base composition, determining chromosome number and ploidy level in several populations, evaluating inter- and intra-specific variations in DNA content and chromosome number as well as looking for the possible hybridization in the sympatric zones of Balkan species. R. nathaliae and R. myconi are diploid species (2n = 2x = 48) while R. serbica is hexaploid (2n = 6x = 144). The mean 2C DNA values ranged from 2.30 pg for R. nathaliae to 2.59 pg for R. myconi compared to 7.91 pg for R. serbica. The base composition for R. nathaliae was 42.1% GC, for R. myconi 39.9% and for R. serbica 41.2%. In one population of R. serbica the DNA content ranged from 2C = 7.65 to 11.82 pg, revealing different ploidy levels among its individuals. In sympatric populations genome size was intermediary (～5 pg) between the diploid and hexaploid classes which indicates the hybridization ability between R. serbica and R. nathaliae. It appears that polyploidization is the major evolutionary mechanism in the genus Ramonda.     

Plasma levels of intermedin (adrenomedullin-2) in healthy human volunteers and patients with heart failure

Intermedin/adrenomedullin-2 (IMD) is a member of the adrenomedullin/CGRP peptide family. Less is known about the distribution of IMD than for other family members within the mammalian cardiovascular system, particularly in humans. The aim was to evaluate plasma IMD levels in healthy subjects and patients with chronic heart failure. IMD and its precursor fragments, preproIMD_25–56 and preproIMD_57–92, were measured by radioimmunoassay in 75 healthy subjects and levels of IMD were also compared to those of adrenomedullin (AM) and mid-region proadrenomedullin_45–92 (MRproAM_45–92) in 19 patients with systolic heart failure (LVEF < 45%). In healthy subjects, plasma levels (mean + SE) of IMD (6.3 + 0.6 pg ml⁻¹) were lower than, but correlated with those of AM (25.8 + 1.8 pg ml⁻¹; r = 0.49, p < 0.001). Plasma preproIMD_25–56 (39.6 + 3.1 pg ml⁻¹), preproIMD_57–92 (25.9 + 3.8 pg ml⁻¹) and MRproAM_45–92 (200.2 + 6.7 pg ml⁻¹) were greater than their respective bioactive peptides. IMD levels correlated positively with BMI but not age, and were elevated in heart failure (9.8 + 1.3 pg ml⁻¹, p < 0.05), similarly to MRproAM_45–92 (329.5 + 41.9 pg ml⁻¹, p < 0.001) and AM (56.8 + 10.9 pg ml⁻¹, p < 0.01). IMD levels were greater in heart failure patients with concomitant renal impairment (11.3 + 1.8 pg ml⁻¹) than those without (6.5 + 1.0 pg ml⁻¹; p < 0.05). IMD and AM were greater in patients receiving submaximal compared with maximal heart failure drug therapy and were decreased after 6 months of cardiac resynchronization therapy. In conclusion, IMD is present in the plasma of healthy subjects less abundantly than AM, but is similarly correlated weakly with BMI. IMD levels are elevated in heart failure, especially with concomitant renal impairment, and tend to be reduced by high intensity drug or pacing therapy.     

Alexandrium pacificum Litaker sp. nov (Group IV): Resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia,Southern Mediterranean Sea)

A high spatial resolution sampling of Alexandrium pacificum cysts, along with sediment characteristics (% H₂O, % organic matter (OM), granulometry), vegetative cell abundance and environmental factors were investigated at 123 study stations in Bizerte Lagoon (Tunisia). Morphological examination and ribotyping of cells obtained from a culture called ABZ1 obtained from a cyst isolated in lagoon sediment confirmed that the species was A. pacificum. The toxin profile from the ABZ1 culture harvested during exponential growth phase was simple and composed of the N-sulfocarbamoyl toxins C1 (9.82 pg toxin cell⁻¹), the GTX6 (3.26 pg toxin cell⁻¹) and the carbamoyl toxin Neo-STX (0.38 pg toxin cell⁻¹). The latter represented only 2.8% of the total toxins in this strain.High abundance of A. pacificum cysts correlated with enhanced percentages of water and organic matter in the sediment. In addition, sediment fractions of less than 63 μm were examined as a favorable potential seedbed for initiation of future blooms and outbreaks of A. pacificum in the lagoon. A significant difference in the cyst distribution pattern was recorded among the lagoon's different zones, with the higher cyst abundance occurring in the inner waters. Also, no correlation due to the specific hydrodynamics of the lagoon was observed in the spatial distribution of A. pacificum cysts and vegetative cells.     

Growth and physiological characteristics of Fritillaria cirrhosa in response to high irradiance and shade in age-related growth phases

Fritillaria cirrhosa is a perennial and protected species (grade 3) inhabiting the Chuanxi altiplano of China. Unfortunately, large-scale introduction has not been successful. We hypothesized that excess irradiance is a key factor limiting its introduction, especially young phases. The current study compared responses to irradiance and shade in three age-related growth phases to determine whether shading was the primary limiting factor in cultivation. Polypropylene shading nets were used to mimic a natural shading rate under Rhododendron litangense boscages, the primary community type of F. cirrhosa. Growth and physiological characteristics were investigated. Results showed that shade could increase net assimilation of all phases significantly (p < 0.01). The net increased assimilation in YP (YiPiye, two years old) was mainly due to decreasing VPD (vapor pressure deficit) and increasing g_s (stomatal conductance). Net increased assimilation in SE (SuErzi, three years old) and DL (DengLonghua, four years old) resulted primarily from decreasing VPD and increasing WUE (water use efficiency). However, shade was not the only limiting factor for introduction. We also examined the importance of canopies to F. cirrhosa growth. Maximum photochemical efficiency (Fv/Fm) and non-photochemical quenching (qN) did not differ markedly in DL (p > 0.05), either in full light or under a shading net, which indicated that DL could grow well without canopies. Significant differences were observed (p < 0.01) in Fv/Fm of YP and SE; both values were lower than 0.80 in open sites. YP and SE only weakly subsisted without a canopy. This experiment provides original observations on the quantitative responses to irradiance and shade, introducing F. cirrhosa life history to a wider audience. Shading increased the rate of survival and the net assimilations of young F. cirrhosa (in the first three phases).     

Genome size stability among five subspecies of Pinus nigra Arnold s.l.

Despite the fact that genome size should be constant at species level, many reports of intraspecific variations exist. Thus, we carried out an analysis to determine the possible existence of nuclear DNA content variation in European black pine (Pinus nigra s.l.), a good model for such a study given its karyological uniformity, morphological polymorphism, broad geographical distribution, ecological plasticity and taxonomic heterogeneity. The panel comprised 20 populations across the natural range of P. nigra from Europe, Northwest Africa and Asia Minor including five subspecies: subsp. nigra, salzmanni, dalmatica, pallasiana and mauretanica. Mean 1C DNA content of the species was 23.62 pg (±0.209) assessed by flow cytometry. This converts to 23.1 G base pairs. The coefficients of variation within and between populations did not exceed 2.6%. Although we had already reported the existence of significant differences for three Black pine populations in our previous work on five Pinus spp. [Bogunic, F., Muratovic, E., Brown, S.C., Siljak-Yakovlev, S., 2003. Genome size of five Pinus from Balkan region. Plant Cell Rep. 22, 59–63], intraspecific variation was not confirmed in the present study dealing with many more populations. Subspecific divisions of Black pine were characterised with following mean values: subsp. pallasiana—23.80 pg, dalmatica—23.79 pg, nigra—23.65 pg, salzmanni—23.55 pg, and mauretanica—23.24 pg. A positive relationship between genome size and longitude was observed (r = 0.44, p < 0.05). We conclude that the diversification of populations of P. nigra has occurred without significant genome size changes throughout its wide geographical range from ecologically contrasting habitats. A clinal mode of genome size variation is present, in line with hypothesis of P. nigra spreading from south-western Asia towards European habitats.     

Morphology and molecular phylogeny of Nitzschia bizertensis sp. nov.—A new domoic acid-producer

A new toxin-producing marine diatom, Nitzschia bizertensis sp. nov., isolated from the Bizerte Lagoon (Tunisia, Southwest Mediterranean Sea) is, based on studies on eight different strains, characterized morphologically by light microscopy, transmission and scanning electron microscopy, and phylogenetically using the nuclear rDNA regions: SSU, ITS1, 5.8S, ITS2 and D1–D3 of the LSU. The species belongs to the sections Lanceolatae or Lineares as defined by Cleve and Grunow (1880). These sections are characterized by species having linear-lanceolate valves with an eccentric raphe where the fibulae does not extend into the valve, and are otherwise famous for the lack of characters useful for delineation of species. Nitzschia bizertensis differs from most other species in these sections by having a high density of interstriae. The morphological and phylogenetic studies and comparisons with previously described Nitzschia species showed Nitzschia bizertensis sp. nov. to be a new species. Batch culture experiments were conducted for estimations of maximum growth rate and production of domoic acid (DA). Maximum cellular DA content of the examined strains ranged from 2 × 10⁻⁴ to 3.6 × 10⁻² pg cells⁻¹. The total DA concentration (pg mL⁻¹) was high already in exponential growth phase maybe due to reinoculation of “old” stationary phase cells, and increased into stationary growth phase where it reached a stationary level varying among the strains from ca. 4500 to 9500 pg mL⁻¹. Nitzschia bizertensis represents a new domoic acid-producing diatom and is the second toxin producing Nitzschia species. The resolution of Nitzschia bizertensis and Nitzschia navis-varingica in different parts of the LSU phylogenetic tree, and the recovery of the Pseudo-nitzschia species phylogenetically distant from those two species suggests that the ability to produce DA either evolved multiple times independently or was lost multiple times.     

Molecular distinction of C × R hybrid (Coffea congensis × Coffea canephora) from morphologically resembling male parent using rbcL and matK gene sequences

Interspecific C × R hybrid (Coffea congensis × Coffea canephora) in India is cultivated as mixed population with male parent C. canephora as this species is an efficient pollen donor for enhanced yield. But distinction of C × R hybrid from C. canephora in old plantation is difficult due to varying plant sizes of C × R hybrid and often resembles with C. canephora. C × R hybrid cultivated under different agroclimatic conditions show distinct vegetative growth pattern with varying yields. Thus development of DNA marker for identification of C × R hybrid is important for clonal propagation and seed preparation from selective individuals. In this study, two DNA bar coding loci of chloroplast genome (rbcL and matK) of parents, F1 hybrid and its back cross progeny were partially sequenced to identify SNPs as DNA marker for distinction of C × R hybrid from C. canephora. Seven SNPs in the matK gene sequence and three nucleotides in the rbcL gene sequence were identified as DNA markers for the genetic identity of C. congensis. These SNPs were found in F1 and advanced progenies of C × R hybrid due to maternal inheritance. Large number of samples of C × R hybrids with varying morphological features revealed no polymorphism among C × R hybrid and C. congensis. Thus, the SNPs in C. congensis can be used as DNA markers for precise identification of C × R hybrid for production of clones besides tagging the chloroplast inheritance in advanced progenies.     

Differential tolerance to combined salinity and O2 deficiency in the halophytic grasses Puccinellia ciliata and Thinopyrum ponticum: The importance of K+ retention in roots

Saline environments of terrestrial halophytes are often prone to waterlogging, yet the effects on halophytes of combined salinity and waterlogging have rarely been studied. Either salinity or hypoxia (low O₂) alone can interfere with K⁺ homeostasis, therefore the combination of salinity or hypoxia is expected to impact significantly on K⁺ retention in roots. We studied mechanisms of tolerance to the interaction of salinity with hypoxia in Puccinellia ciliata and Thinopyrum ponticum, halophytic grasses that differ in waterlogging tolerance. Plants were exposed to aerated and stagnant saline (250 mM NaCl) treatments with low (0.25 mM) and high (4 mM) K⁺ levels; growth, net ion fluxes and tissue ion concentrations were determined. P. ciliata was more tolerant than T. ponticum to stagnant-saline treatment, producing twice the biomass of adventitious roots, which accumulated high levels of Na⁺, and had lower shoot Na⁺. After 24 h of saline hypoxic treatment, MIFE measurements revealed a net uptake of K⁺ (∼40 nmol m⁻² s⁻¹) for P. ciliata, but a net loss of K⁺ (∼20 nmol m⁻² s⁻¹) for the more waterlogging sensitive T. ponticum. NaCl alone induced K⁺ efflux from roots of both species, with channel blocker tests implicating GORK-like channels. P. ciliata had constitutively a more negative root cell membrane potential than T. ponticum (−150 versus −115 mV). Tolerance to salinity and hypoxia in P. ciliata is related to increased production of adventitious roots, regulation of shoot K⁺/Na⁺, and a superior ability to maintain negative membrane potential in root cells, resulting in greater retention of K⁺.     

Uptake of paralytic shellfish poisoning and spirolide toxins by paddle crabs (Ovalipes catharus) via a bivalve vector

The uptake of paralytic shellfish poisoning (PSP) toxins and spirolides by the paddle crab (Ovalipes catharus) was investigated in two laboratory feeding trials using Greenshell? mussels (Perna canaliculus), which had been fed toxic strains of either Alexandrium catenella or A. ostenfeldii, as a vector. Toxin uptake by crabs occurred in both feeding trials and was limited to the visceral tissue; no toxins were detected in the body meat or the gills. The first trial utilized a strain of A. catenella that had high total PSP toxin content, 442.3 ± 91.6 fmol/cell, that was dominated by low toxicity N-sulfocarbamoyl toxins resulting in a low cellular toxicity, 5.5 ± 1.6 pg STXequiv./cell. In this trial, toxin accumulation in the crabs was highly variable and ranged from 3.8 to 221.5 μg STXequiv./100 g, with 3/4 of the crabs exceeding the regulatory limit of 80 μg STXequiv./100 g. Eight days after feeding on toxic mussels the crabs still retained high levels of toxin suggesting that depuration rates in this species may be slow. In the second feeding trial, the A. ostenfeldii strain fed to mussels produced low levels of both PSP toxins (52.0 ± 19.5 fmol/cell; 1.4 ± 0.3 pg STXequiv./cell) and spirolides (1.8 pg/cell) and, as a result, the concentration transferred to crabs via the mussels was very low-PSP toxins ranged from 2.5 to 6.8 μg STXequiv./100 g and spirolides from 6 to 7 μg/kg. The results of our study demonstrate that paddle crabs are capable of acquiring both PSP toxins and spirolides and suggest that this may occur in the wild during a toxic shellfish event. It also highlights the need to remove the viscera before consumption.     

Reproductive potential in Ruppia cirrhosa (Petagna) Grande in response to water permanence

We compared the reproductive effort of perennial and annual Ruppia cirrhosa (Petagna) Grande in a mediterranean coastal lagoon where the species develops in permanent waters as well as in shallow areas which dry up during some months. In permanently flooded areas, Ruppia cirrhosa was perennial with biomass maxima up to 800 g DW m⁻². Here, the reproductive period lasted 8–10 weeks (between April and June), the reproductive effort was low (with 4–8 fl g⁻¹ DW) and mature fruit development was practically absent. In temporarily flooded areas, in contrast, Ruppia cirrhosa developed in an annual cycle which was completed in less than 8 weeks (maximum biomass of 137 g DW m⁻²); reproductive activity began later in the year (June) and lasted 3–4 weeks, but led to more flowers (17 fl g⁻¹ DW) and a high fruit production (up to 590 fr m⁻²). We conclude that reproductive effort in Ruppia cirrhosa is highly plastic and interpret this as an adaptation to temporay flooding.     

Cadmium tolerance in Thlaspi caerulescens: I. Growth parameters,metal accumulation and phytochelatin synthesis in response to cadmium

A population of the metallophyte, Thlaspi caerulescens, originating from a Cd–Pb–Zn old mining and smelter site at Plombières (Belgium) was studied. T. caerulescens was cultivated hydroponically to investigate Cd uptake and tolerance. Cd was added to Hoagland’s medium at concentration range from 5 to 500 μM. The plants could tolerate 500 μM Cd in the solution showing only minor visible symptoms of toxicity but with a 32% decrease in fresh weight. After 14 days at 500 μM, Cd content in roots and shoots was 707 and 602 mg kg⁻¹ of dry weight (d.w.), respectively. Application of Cd to hydroponically cultivated T. caerulescens induced the accumulation of PCs in plant roots and shoots. Buthionine sulfoximine (BSO) application almost completely reduced (by 98–100%) the accumulation of PCs without simultaneous increase in plants sensitivity to Cd. These results suggest a minor if any role of PCs in tolerance to Cd of the studied population of T. caerulescens in hydroponics. On the other hand, no PC accumulation was detected either in T. caerulescens plants growing in their natural environment at Plombierès or in plants growing in their native soil in a greenhouse. These results suggest that naturally selected tolerance in T. caerulescens population from Plombières is not associated with enhanced PCs synthesis.     

Stimuli responsive polymorphism of C12NO/DOPE/DNA complexes: Effect of pH,temperature and composition

N,N-dimethyldodecylamine-N-oxide (C₁₂NO) is a surfactant that may exist either in a neutral or cationic protonated form depending on the pH of aqueous solutions. Using small angle X-ray diffraction (SAXD) we observe the rich structural polymorphism of pH responsive complexes prepared due to DNA interaction with C₁₂NO/dioleoylphosphatidylethanolamine (DOPE) vesicles and discuss it in view of utilizing the surfactant for the gene delivery vector of a pH sensitive system. In neutral solutions, the DNA uptake is low, and a lamellar L_α phase formed by C₁₂NO/DOPE is prevailing in the complexes at 0.2 ≤ C₁₂NO/DOPE < 0.6 mol/mol. A maximum of ~ 30% of the total DNA volume in the sample is bound in a condensed lamellar phase L_α^C at C₁₂NO/DOPE = 1 mol/mol and pH 7.2. In acidic conditions, a condensed inverted hexagonal phase H_II^C was observed at C₁₂NO/DOPE = 0.2 mol/mol. Commensurate lattice parameters, a_HC ≈ d_LC, were detected at 0.3 ≤ C₁₂NO/DOPE ≤ 0.4 mol/mol and pH = 4.9–6.4 suggesting that L_α^C and H_II^C phases were epitaxially related. While at the same composition but pH ~ 7, the mixture forms a cubic phase (Pn3m) when the complexes were heated to 80 °C and cooled down to 20 °C. Finally, a large portion of the surfactant (C₁₂NO/DOPE > 0.5) stabilizes the L_α^C phase in C₁₂NO/DOPE/DNA complexes and the distance between DNA strands (d_DNA) is modulated by the pH value. Both the composition and pH affect the DNA binding in the complexes reaching up to ~ 95% of the DNA total amount at acidic conditions.     

Modification of the N-terminal sulfonyl residue in 3-amidinophenylalanine-based matriptase inhibitors

Replacement of the N-terminal β-alanyl-amide moiety in previously identified matriptase inhibitors by non-charged aryl groups caused a slightly decreased potency and partially reduced selectivity, especially towards thrombin. However, some of these analogues are still potent matriptase inhibitors with K_i-values <10 nM. In contrast, improved activity was observed for newly designed tribasic analogues, especially for compound 21, which inhibits matriptase with an K_i-value of 80 pM.     

Fire service instructor's undergarment choice to reduce Interleukin-6 and minimise physiological and perceptual strain

Fire Service Instructors frequently experience high levels of physiological and perceptual strain during live fire exposures. Instructors are also at risk of cardiovascular illnesses, with cardiac death being the greatest cause of fire fighter death. Current practice for UK instructors is to select undergarment type based on personal preference, between a boiler suit (BOILER) and a wicking base layer (WBL). Research suggests that shorts and t-shirt (SHORTS) may also be a beneficial alternative undergarment choice. The UK South East Fire Service requested an investigation to identify if undergarment selection can lessen the strain experienced by instructors, and reduce the acute inflammatory response to fire exposures. Eight males completed three 45 min sessions in a heat chamber (49.5±1.4 °C and 16.9±4.3% RH) whilst performing intermittent walking. At the end of heat exposure change in heart rate was not effected by garment type (p=0.061, ηp²=0.373). Change in rectal temperature was different between garments (p=0.009, η_p²=0.271), with trends suggesting that BOILER resulted in a greater change (1.03±0.60 °C) than SHORTS (0.76±0.37 °C, p=0.589, d=0.21) and WBL (0.72±0.33 °C, p=0.545, d=0.25). Interleukin-6 post exposure was greater for BOILER (6.96±0.28 pg mL⁻¹) than both SHORTS (6.59±0.30 pg mL⁻¹, p=0.043, d=0.42) and WBL (6.45±0.43 pg mL⁻¹, p=0.031, d=0.51). Overall, undergarment type had little impact on physiological or perceptual strain. However, wearing WBL or SHORTS may reduce the inflammatory response, and consequently decrease the risk of cardiovascular events.     

Seasonal variability of lipophilic toxins during a Dinophysis acuta bloom in Western Iberia: Differences between picked cells and plankton concentrates

This work describes and compares the seasonal variability of toxin profiles and content, estimated by LC–MS analyses, in picked cell of Dinophysis acuta Ehrenberg, in plankton concentrates rich in this species, and in extracellular lipophilic toxins collected by adsorbent resins during weekly sampling in a Galician ría (Western Iberia) from October 2005 to January 2006. Picked cells of D. acuta—which exhibited a fairly stable OA:DTX2 ratio, close to 3:2, but a variable okadaates:PTX2 ratio—showed a 9-fold variation in cell toxin quota, which was partly related to cellular volume, with maximum values (19 pg cell⁻¹) observed during the exponential decline of the population. Large differences in toxin profiles and content were observed between picked cells and plankton concentrates (up to 73 pg cell⁻¹ in the latter), that were most conspicuous after the bloom decline. The toxin profile of picked cells was more similar to that observed in the adsorbent resins than to the profiles of plankton concentrates. Their continued detection several weeks after the disappearance of Dinophysis spp. indicates that these toxins may take a long time to be degraded. It is concluded that analyses of picked-cells are essential to determine the contribution of each species of Dinophysis to a toxic outbreak. Estimates of cellular toxin content from plankton concentrates can lead to considerable overestimates after Dinophysis blooms decay due to extracellular toxins that persist in the water column, possibly bound to organic aggregates and detritus, and are retained (>0.22 μm) in the filters.     

A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax

This study aimed to develop a single-round multiplex PCR method for the identification of Anopheles minimus complex (An. minimus and Anopheles harrisoni) and Anopheles aconitus subgroup (An. aconitus and Anopheles varuna), and for the simultaneous detection of Plasmodium falciparum and Plasmodium vivax in these vectors. Five primers were created for a single-round multiplex PCR assay to identify four anopheline mosquitoes combined with three Plasmodium primers for the detection of P. falciparum and P. vivax in vectors. The four species of anopheline vectors and two Plasmodium species, P. falciparum and P. vivax, could be identified by the combination of eight primers in the single-round multiplex PCR assay. The amplified species-specific products were 380 bp for An. minimus, 180 bp for An. harrisoni, 150 bp for An. aconitus, 310 bp for An. varuna, 276 bp for P. falciparum, and 300 bp for P. vivax. The sensitivities were 0.5 pg/μl (25 sporozoites/μl) for P. falciparum DNA and between 0.5 and 5 pg/μl (25–250 sporozoites/μl) for P. vivax DNA. Furthermore, this developed method could be used to identify field caught An. minimus complex, An. aconitus subgroup from Thailand and Lao PDR. Also, it was successfully used to identify the species An. minimus, An. harrisoni, An. aconitus and An. varuna and to detect and identify P. falciparum and P. vivax in caught anopheline mosquitoes. The sensitivity of this method was high for simultaneous detection of P. falciparum and P. vivax in anopheline mosquitoes.     

Necrotic cell death and suppression of T-cell immunity characterized acute liver failure due to drug-induced liver injury

Background & aims: The aim of this study was to investigate the clinical characteristics and pathophysiology of drug-induced liver injury (DILI) – acute liver failure (ALF). Methods: The patients with acute liver injury (ALI) including ALF from 2009 to 2014 were analyzed. The hepatic encephalopathy (HE) development rate was compared with the findings from a national survey in Japan. The serum cytokines levels and the findings of a liver function test were evaluated in the DILI patients. Results: The HE development rate substantially decreased for autoimmune hepatitis (AIH) – and undetermined cause-induced ALI owing to the early prediction system, but not in DILI-ALI. Among the DILI-ALF and AIH-ALF cases, the CK-18 fragment (1480.1 U/L, 3945.4 U/L), IL-8 (82.9 pg/mL, 207.5 pg/mL), IP-10 (1379.6 pg/mL, 3731.2 pg/mL) and MIP-1β (1017.7 pg/mL, 2273.3 pg/mL) levels were lower in the DILI-ALF cases. Among the DILI-ALI and DILI-ALF cases, IL-4 (19.8 pg/mL, 25.4 pg/mL) and RANTES (14028.0 pg/mL, 17804.7 pg/mL) were higher in DILI-ALI, and HMGB-1 (397.1 pg/μL, 326.2 pg/μL) and HGF (2.41 ng/mL, 0.55 ng/mL) were higher in DILI-ALF. We observed that HGF independently associated with DLI-ALF development. Conclusions: Despite the low grade apoptosis and inflammation, DILI patients progressed to ALF comparable with that of the AIH patients.     

Optimization of Cd2+ removal by the cyanobacterium Synechocystis pevalekii using the response surface methodology

Response surface methodology (RSM) has been used to optimize the critical parameters responsible for higher Cd²⁺ removal by a unicellular cyanobacterium Synechocystis pevalekii. A three-level Box–Behnken factorial design was used to optimize pH, biomass and metal concentration for Cd²⁺ removal. A coefficient of determination (R²) value (0.99), model F-value (86.40) and its low p-value (F < 0.0001) along with lower value of coefficient of variation (5.61%) indicated the fitness of response surface quadratic model during the present study. At optimum pH (6.48), biomass concentration (0.25 mg protein ml^?1) and metal concentration (5 μg ml^?1) the model predicted 4.29 μg ml^?1 Cd²⁺ removal and experimentally, 4.27 μg ml^?1 Cd²⁺ removal was obtained.     

Regulation of spiroimine neurotoxins and hemolytic activity in laboratory cultures of the dinoflagellate Alexandrium peruvianum (Balech & Mendiola) Balech & Tangen

Defined experimental regimes were used to determine the effects of nutrient limitation on the toxicity of Alexandrium peruvianum in batch culture. Subsamples for cell counts and spiroimine analysis at six day intervals were used to investigate the concentrations and composition of these compounds throughout growth. An erythrocyte lysis assay for hemolytic activity was performed on cell pellets and supernatants also collected every six days over the entire growth period from all treatments. From the data, growth rates, cellular spiroimine quotas and effective concentration-fifty (EC₅₀s) for cellular and supernatant associated hemolytic activity were calculated. Phosphate limitation was identified as a key regulator of toxicity in this species, yielding maximum values of 54.1 pg cell⁻¹ for 13-desmethyl spirolide C, 96.4 pg cell⁻¹ for 12-methylgymnodimine and a potent hemolytic EC₅₀ value of 7.1 × 10³ cells. The concentrations of spiroimines detected in A. peruvianum among various treatments, in addition to a unique profile of paralytic shellfish poisoning toxins, is unique in the body of microalgal literature. Because of the multiple toxin arsenal produced by this organism, the evaluation of a single toxin clearly would have underestimated the potential virulence and significance of this clone. This study provides the first evidence that growth and toxin production of A. peruvianum are influenced by altered nutrient ratios.