Allele Variants of Enterotoxigenic Escherichia coli Heat-Labile Toxin Are Globally Transmitted and Associated with Colonization Factors

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile toxin (LT) and heat-stable toxins (STp and STh), acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induces fluid secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of ETEC isolates from humans in Brazil reported the existence of natural LT variants. In the present study, analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms are associated with a geographically and temporally diverse set of 192 clinical ETEC strains and identified 12 novel LT variants. Twenty distinct LT amino acid variants were observed in the globally distributed strains, and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 allele variants were correlated with major ETEC lineages expressing CS1 + CS3 or CS2 + CS3, and the most prevalent LT2 allele variants were correlated with major ETEC lineages expressing CS5 + CS6 or CFA/I. LTB allele variants generally exhibited more-stringent amino acid sequence conservation (2 substitutions identified) than LTA allele variants (22 substitutions identified). The functional impact of LT1 and LT2 polymorphisms on virulence was investigated by measuring total-toxin production, secretion, and stability using GM1–enzyme-linked immunosorbent assays (GM1-ELISA) and in silico protein modeling. Our data show that LT2 strains produce 5-fold more toxin than LT1 strains (P < 0.001), which may suggest greater virulence potential for this genetic variant. Our data suggest that functionally distinct LT-CF variants with increased fitness have persisted during the evolution of ETEC and have spread globally.     

Role of glutamate in locomotor rhythm generating neuronal circuitry

Central pattern generators (CPGs) are defined as neuronal circuits capable of producing a rhythmic and coordinated output without the influence of sensory input. The locomotor and respiratory neuronal circuits are two of the better-characterized CPGs, although much work remains to fully understand how these networks operate. Glutamatergic neurons are involved in most neuronal circuits of the nervous system and considerable efforts have been made to study glutamate receptors in nervous system signaling using a variety of approaches. Because of the complexity of glutamate-mediated signaling and the variety of receptors triggered by glutamate, it has been difficult to pinpoint the role of glutamatergic neurons in neuronal circuits. In addition, glutamate is an amino acid used by every cell, which has hampered identification of glutamatergic neurons. Glutamatergic excitatory neurotransmission is dependent on the release from glutamate-filled presynaptic vesicles loaded by three members of the solute carrier family, Slc17a6-8, which function as vesicular glutamate transporters (VGLUTs). Recent data describe that Vglut2 (Slc17a6) null mutant mice die immediately after birth due to a complete loss of the stable autonomous respiratory rhythm generated by the pre-B?tzinger complex. Surprisingly, we found that basal rhythmic locomotor activity is not affected in Vglut2 null mutant embryos. With this perspective, we discuss data regarding presence of VGLUT1, VGLUT2 and VGLUT3 positive neuronal populations in the spinal cord.     

Endogenous prostaglandin E2 synthesis inhibits growth of polyoma virus-transformed 3T3 fibroblasts.

Indomethacin lowered the cellular content of adenosine 3 ′: 5 ′-monophosphate (cAMP) and stimulated growth of polyoma virus-transformed 3T3 fibroblasts. Exogenous prostaglandin E₂, at concentrations produced in the absence of inhibitor, reversed the effects of indomethacin on cAMP levels and cell proliferation. Therefore, endogenously produced prostaglandin E₂ decreases cell growth and raises the levels of cAMP in these cells.     

The phylogeographic architecture of the fucoid seaweed Ascophyllum nodosum: an intertidal ‘marine tree’ and survivor of more than one glacial–interglacial cycle

Aim Ascophyllum nodosum (L.) Le Jolis is a dominant fucoid seaweed occurring along sheltered, rocky shores throughout the North Atlantic (but not in the Pacific), where it is a foundational species of the intertidal community. Its large size and vulnerability to ice‐scour have led to the hypothesis that contemporary populations in the north‐west Atlantic may be the result of de novo recolonization from the north‐east Atlantic since the Last Glacial Maximum (LGM) (c. 20 ka). We tested this hypothesis. Location Temperate North Atlantic rocky intertidal between c. 42 and 65° N latitude. Methods More than 1300 individuals from 28 populations were sampled from across the entire range of A. nodosum and genotyped for six microsatellite loci, and > 500 individuals were genotyped for two mitochondrial loci, an intergenic spacer (IGS) and the tRNA (W) gene (trnW). Population structure and historical demography were analysed in a standard population genetics and coalescence framework. Results Based on the presence of private alleles and haplotypes, we found that A. nodosum has survived on both sides of the Atlantic (since before the LGM, dating back to at least the penultimate Eemian interglacial) with similar effective population sizes and divergence times (1.2 and 0.8 Ma). Dispersal has been predominantly from Europe to North America, and there is very weak present‐day population differentiation across the North Atlantic. Diversity measures provided additional support for determining the location of refugia. Main conclusions Ascophyllum nodosum was apparently little affected by the LGM, although contemporary climate change is likely to have major effects on its latitudinal distribution on both sides of the North Atlantic. It is a very long‐lived species, analogous in virtually all demographic aspects to a tree – resistant to extinction but vulnerable to catastrophic events. The Brittany peninsula is a hotspot of genetic diversity worthy of conservation.     

Acanthoscurrin fragment 101-132: total synthesis at 60 degrees C of a novel difficult sequence

Glycine-rich proteins (GRPs) serve a variety of biological functions. Acanthoscurrin is an antimicrobial GRP isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana. Aiming to contribute to the knowledge of the secondary structure and stepwise solid-phase synthesis of GRPs' glycine-rich domains, we attempted to prepare G(101)GGLGGGRGGGYG(113)GGGGYGGGYG(123) GGY(126)GGGKYK(132)-NH(2), acanthoscurrin C-terminal amidated fragment. Although a theoretical prediction did not indicate high aggregation potential for this peptide, repetitive incomplete aminoacylations were observed after incorporating Tyr(126) to the growing peptide-MBHA resin (Boc chemistry) at 60 degrees C. The problem was not solved by varying the coupling reagents or solvents, adding chaotropic salts to the reaction media or changing the resin/chemistry (Rink amide resin/Fmoc chemistry). Some improvement was made when CLEAR amide resin (Fmoc chemistry) was used, as it allowed for obtaining fragment G(113)-K(132). NIR-FT-Raman spectra collected for samples of the growing peptide-MBHA, -Rink amide resin and -CLEAR amide resin revealed the presence of beta-sheet structures. Only the combination of CLEAR-amide resin, 60 degrees C, Fmoc-(Fmoc-Hmb)Gly-OH and LiCl (the last two used alternately) was able to inhibit the phenomenon, as proven by NIR-FT-Raman analysis of the growing peptide-resin, allowing the total synthesis of desired fragment Gly(101)-K(132). In summary, this work describes a new difficult sequence, contributes to understanding stepwise solid-phase synthesis of this type of peptide and shows that, at least while protected and linked to a resin, this GRP's glycine-rich motif presents an early tendency to assume beta-sheet structures.     

Overweight in relation to tumour size and axillary lymph node involvement in postmenopausal breast cancer patients—Differences between women invited to vs. not invited to mammography in a randomized screening trial

Objectives: Overweight is associated with advanced stage at diagnosis in breast cancer patients. This could be explained by specific tumour characteristics or tumour promoting factors in the obese, but a diagnostic delay could also be of importance. Mammographic screening has caused a change towards diagnosis of less advanced tumours. This study investigates invitation to mammographic screening and the association between overweight and tumour size/axillary lymph node involvement at breast cancer diagnosis in postmenopausal women. Methods: In 1976 a randomized mammographic screening trial, inviting 50% of all women aged 45–69 was set up in Malmö, Sweden. The present analysis examined overweight (body mass index ≥25) as a determinant for large tumours (>20 mm) and axillary lymph node involvement in postmenopausal women. These associations were studied separately in patients diagnosed prior to the mammographic screening trial, in invited women and in non-invited subjects (controls). In all, 2478 postmenopausal women were diagnosed with invasive breast cancer in these groups between 1961 and 1991. Logistic regression analysis allowed adjustment for other potential determinants of tumours size and axillary lymph node involvement. Results: In women diagnosed before the onset of the screening trial and in women not invited to mammography in the trial (controls), overweight was positively associated with large tumour size and axillary node involvement. There was no statistically significant association between overweight and these factors in women invited to mammographic screening. Conclusion: Invitation to mammographic screening may be particularly important for overweight postmenopausal women in order to detect breast tumours early.     

Effects of deltamethrin on the reproduction of Trichogramma cordubensis (Hymenoptera: Trichogrammatidae)

The influence of deltamethrin on the reproduction of Trichogramma cordubensis, a thelytokous egg parasitoid, was investigated by studying egg maturation and daily fecundity of insecticide treated wasps and offspring emergence rates. The insecticide was applied to the parasitoids at the prepupal stage within its host eggs (Ephestia kuehniella). The total number of parasitized eggs per female during the first 7 days was not significantly influenced by the tested concentrations of deltamethrin. Prevalence of parasitism during this period had a similar pattern between deltamethrin treatments and the control. In addition, the mean number of mature eggs observed per female per day was significantly correlated to mean daily fecundity, regardless of the treatments. Offspring emergence was significantly influenced by the insecticide treatments experienced on their progenitors, decreasing significantly at 48 and 72 h for the highest tested concentration of deltamethrin (23.6 mg [a.i.]/L). Despite that, deltamethrin had no adverse effects on the reproduction of treated wasps, particularly when was applied at the concentration recommended by the manufacturer (12.5 mg [a.i.]/L).     

Serum alpha-glutathione S-transferase as a sensitive marker of hepatocellular damage in patients with cystic fibrosis

The aim of the study was to evaluate serum a-glutathione S-transferase (s-GSTA) levels in patients with cystic fibrosis (CF) and to compare s-GSTA with other liver function tests and with a hepatic ultrasound scan (US). The cytosolic enzyme, alpha-glutathione S-transferase is predominantly found in the liver and is distributed uniformly in the liver tissue. In our study s-GSTA levels were measured in 37 CF patients aged 1 to 28 years (mean age 10.4 years, 24 males). The control group consisted of 27 patients aged 2 to 17 years (mean age 8.5 years, 18 males). The presence of hepatobiliary abnormalities was assessed by clinical examination, ultrasound scan, s-GSTA, and conventional liver enzymes: alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST) and gama-glutamyl transferase (GMT). The calculated 5-95 % range of s-GSTA for the control group was 0.098-2.54 microg/l, for the CF group 0.43-9.76 microg/l. Mean s-GSTA level in the control group was 1.55 microg/l (S.D.=1.57), and 2.05 micro/l (S.D.=2.60) in the CF group. In the group of CF patients, the serum levels were significantly higher than in the control group (P<0.01). No significant correlation existed in the CF group between s-GSTA and conventional liver tests (ALT, AST, ALP and GMT). Four patients in the CF group had hepatobiliary abnormalities detectable by conventional liver tests, s-GSTA and US. Four patients had abnormal s-GSTA, while conventional liver tests and US were normal. One other patient had abnormal hepatic US, but normal standard liver tests and s-GSTA. The study has suggested that a raised s-GSTA level might be a marker of possible pathological changes of the hepatobiliar system in CF patients. Serum GSTA seems to be a more sensitive marker than transaminases for the monitoring of hepatocellular integrity and as an early predictor of hepatic damage.     

Novel microsatellite markers suggest the mechanism of parthenogenesis in Extatosoma tiaratum is automixis with terminal fusion

Parthenogenetic reproduction is taxonomically widespread and occurs through various cytological mechanisms, which have different impact on the genetic variation of the offspring. Extatosoma tiaratum is a facultatively parthenogenetic Australian insect (Phasmatodea), in which females oviposit continuously throughout their adult lifespan irrespective of mating. Fertilized eggs produce sons and daughters through sexual reproduction and unfertilized eggs produce female offspring via parthenogenesis. Here, we developed novel microsatellite markers for E. tiaratum and characterized them by genotyping individuals from a natural population. We then used the microsatellite markers to infer the cytological mechanism of parthenogenesis in this species. We found evidence suggesting parthenogenesis in E. tiaratum occurs through automixis with terminal fusion, resulting in substantial loss of microsatellite heterozygosity in the offspring. Loss of microsatellite heterozygosity may be associated with loss of heterozygosity in fitness related loci. The mechanism of parthenogenetic reproduction can therefore affect fitness outcomes and needs to be considered when comparing costs and benefits of sex versus parthenogenesis.     

Carbon balances of bioenergy systems using biomass from forests managed with long rotations: bridging the gap between stand and landscape assessments

Studies report different findings concerning the climate benefits of bioenergy, in part due to varying scope and use of different approaches to define spatial and temporal system boundaries. We quantify carbon balances for bioenergy systems that use biomass from forests managed with long rotations, employing different approaches and boundary conditions. Two approaches to represent landscapes and quantify their carbon balances – expanding vs. constant spatial boundaries – are compared. We show that for a conceptual forest landscape, constructed by combining a series of time‐shifted forest stands, the two approaches sometimes yield different results. We argue that the approach that uses constant spatial boundaries is preferable because it captures all carbon flows in the landscape throughout the accounting period. The approach that uses expanding system boundaries fails to accurately describe the carbon fluxes in the landscape due to incomplete coverage of carbon flows and influence of the stand‐level dynamics, which in turn arise from the way temporal system boundaries are defined on the stand level. Modelling of profit‐driven forest management using location‐specific forest data shows that the implications for carbon balance of management changes across the landscape (which are partly neglected when expanding system boundaries are used) depend on many factors such as forest structure and forest owners’ expectations of market development for bioenergy and other wood products. Assessments should not consider forest‐based bioenergy in isolation but should ideally consider all forest products and how forest management planning as a whole is affected by bioenergy incentives – and how this in turn affects carbon balances in forest landscapes and forest product pools. Due to uncertainties, we modelled several alternative scenarios for forest products markets. We recommend that future work consider alternative scenarios for other critical factors, such as policy options and energy technology pathways.