Genotyping of pantophysin I (Pan I) of Atlantic cod (Gadus morhua L.) by allele‐specific PCR

The two main allelic variants of the Atlantic cod (Gadus morhua L.) pantophysin I (Pan I) locus have different frequencies within different cod stocks. The Dra I polymorphism which distinguishes the two alleles can thus be used for discrimination of coastal and offshore cod populations. We present a new method for Pan I genotyping using fluorescent allele‐specific duplex polymerase chain reaction (PCR). This method is more rapid, reliable and cost‐effective than the previously published method and it is not affected by DNA source and quality. This improvement is important for studies demanding high throughput and accuracy of Pan I genotyping     

Experimental design for the production of tensio-active agent by <Emphasis Type="Italic">Candida lipolytica</Emphasis>

The strategy of optimization using sequential factorial design was employed to enhance the tensio-active emulsifying agent produced by Candida lipolytica using soybean oil refinery residue as substrate. A full factorial design was used to evaluate the impact of three fermentation factors—amounts of refinery residue, glutamic acid and yeast extract. This allowed exclusion of the yeast extract. Full factorials designs were then sequentially used to optimize the levels of the residue and glutamic acid. The surface tension value was finally reduced to 25.29 mN/m. The maximum emulsifier activity using different substrates was within 40 h of cultivation. The surface tension of the cell-free broth containing the biosurfactant remained very stable during exposure to a wide range of pH (2–12), temperatures (0–120°C) and salinity (2–10% NaCl). The combination of an industrial waste and a cheap substrate therefore seems to be very promising for the low-cost production of potent biosurfactant.     

Expression and function of K(ATP) channels in normal and osteoarthritic human chondrocytes: Possible role in glucose sensing

ATP‐sensitive potassium [K(ATP)] channels sense intracellular ATP/ADP levels, being essential components of a glucose‐sensing apparatus in various cells that couples glucose metabolism, intracellular ATP/ADP levels and membrane potential. These channels are present in human chondrocytes, but their subunit composition and functions are unknown. This study aimed at elucidating the subunit composition of K(ATP) channels expressed in human chondrocytes and determining whether they play a role in regulating the abundance of major glucose transporters, GLUT‐1 and GLUT‐3, and glucose transport capacity. The results obtained show that human chondrocytes express the pore forming subunits, Kir6.1 and Kir6.2, at the mRNA and protein levels and the regulatory sulfonylurea receptor (SUR) subunits, SUR2A and SUR2B, but not SUR1. The expression of these subunits was no affected by culture under hyperglycemia‐like conditions. Functional impairment of the channel activity, using a SUR blocker (glibenclamide 10 or 20 nM), reduced the protein levels of GLUT‐1 and GLUT‐3 by approximately 30% in normal chondrocytes, while in cells from cartilage with increasing osteoarthritic (OA) grade no changes were observed. Glucose transport capacity, however, was not affected in normal or OA chondrocytes. These results show that K(ATP) channel activity regulates the abundance of GLUT‐1 and GLUT‐3, although other mechanisms are involved in regulating the overall glucose transport capacity of human chondrocytes. Therefore, K(ATP) channels are potential components of a broad glucose sensing apparatus that modulates glucose transporters and allows human chondrocytes to adjust to varying extracellular glucose concentrations. This function of K(ATP) channels seems to be impaired in OA chondrocytes. J. Cell. Biochem. 114: 1879–1889, 2013. © 2013 Wiley Periodicals, Inc.     

Harmonising Adult and Paediatric Reference Intervals in Australia and New Zealand: An Evidence-Based Approach for Establishing a First Panel of Chemistry Analytes

Scientific evidence supports the use of common reference intervals (RIs) for many general chemistry analytes, in particular those with sound calibration and traceability in place. Already the Nordic countries and United Kingdom have largely achieved harmonised RIs. Following a series of workshops organised by the Australasian Association of Clinical Biochemists (AACB) between 2012 and 2014 at which an evidence-based approach for determination of common intervals was developed, pathology organisations in Australia and New Zealand have reached a scientific consensus on what adult and paediatric intervals we should use across Australasia. The aim of this report is to describe the processes that the AACB and the Royal College of Pathologists of Australasia have taken towards recommending the implementation of a first panel of common RIs for use in Australasia.     

Resveratrol Modulates Cytokine-Induced JAK/STAT Activation More Efficiently than 5-Aminosalicylic Acid: An In Vitro Approach

Background

Many advances have been recently made focused on the valuable help of dietary polyphenols in chronic inflammatory diseases. On the other hand, current treatment options for intestinal bowel disease patients are unsatisfying and, for this reason, it is estimated that many patients use dietary supplements to achieve extra benefits.

Aim

The aim of this work was to analyze under a mechanistic perspective the anti-inflammatory potential of resveratrol, a natural polyphenolic compound, and to compare it with a pharmaceutical agent, 5-aminosalicylic acid, using the intestinal HT-29 cell line, as a cellular model.

Methodology and Principal Findings

In the present study, HT-29 colon epithelial cells were pre-treated with 25 µM resveratrol and/or 500 µM 5-aminosalicylic acid and then exposed to a combination of cytokines (IL-1α, TNF-α, IFN-γ) for a certain period of time. Our data showed that resveratrol, used in a concentration 20 times lower than 5-aminosalicylic acid, was able to significantly reduce NO and PGE₂ production, iNOS and COX-2 expression and reactive oxidant species formation induced by the cytokine challenge. However, as already verified with 5-aminosalicylic acid, in spite of not exhibiting any effect on IkB-α degradation, resveratrol down-regulated JAK-STAT pathway, decreasing the levels of activated STAT1 in the nucleus. Additionally, resveratrol decreased the cytokine-stimulated activation of SAPK/JNK pathway but did not counteract the cytokine-triggered negative feedback mechanism of STAT1, through p38 MAPK.

Conclusion/Significance

Taken together, our results show that resveratrol may be considered a future nutraceutical approach, promoting remission periods, limiting the inflammatory process and preventing colorectal cancer, which is common in these patients.     

Mutation Prevalence of Cerebral Cavernous Malformation Genes in Spanish Patients

Objective

To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients.

Methods

We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing.

Results

A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported.

Conclusions

Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.     

Behavioural evidence for a female sex pheromone in Cotesia flavipes (Hymenoptera: Braconidae)

Abstract.  The mating behaviour of Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae) was observed under laboratory conditions. The results demonstrate that chemical stimuli play an important role in bringing males and females together for mating. Females from this species release chemical compounds to attract males, which exhibit a distinct courtship behaviour divided into three main steps: (i) antennation, (ii) lateral bouncing and (iii) wing fluttering. Ethological tests using hexane extracts from two different parts of the female's body (head plus thorax and abdomen) demonstrate that males are more attracted and also display a courtship behaviour when a filter paper containing hexane extracts from the abdomen is presented to them, indicating that the abdomen is the source of the sex pheromone of this species. Naive males of this species respond to hexane extracts of this gland by displaying courtship behaviour. Furthermore, additional tests show that C. flavipes females mate only once, whereas virgin males do not show any preference for virgin or mated females, suggesting that mated females of this species continue to produce sex attractants after mating.     

Revisiting the Iberian honey bee (Apis mellifera iberiensis) contact zone: maternal and genome‐wide nuclear variations provide support for secondary contact from historical refugia

Dissecting diversity patterns of organisms endemic to Iberia has been truly challenging for a variety of taxa, and the Iberian honey bee is no exception. Surveys of genetic variation in the Iberian honey bee are among the most extensive for any honey bee subspecies. From these, differential and complex patterns of diversity have emerged, which have yet to be fully resolved. Here, we used a genome‐wide data set of 309 neutrally tested single nucleotide polymorphisms (SNPs), scattered across the 16 honey bee chromosomes, which were genotyped in 711 haploid males. These SNPs were analysed along with an intergenic locus of the mtDNA, to reveal historical patterns of population structure across the entire range of the Iberian honey bee. Overall, patterns of population structure inferred from nuclear loci by multiple clustering approaches and geographic cline analysis were consistent with two major clusters forming a well‐defined cline that bisects Iberia along a northeastern–southwestern axis, a pattern that remarkably parallels that of the mtDNA. While a mechanism of primary intergradation or isolation by distance could explain the observed clinal variation, our results are more consistent with an alternative model of secondary contact between divergent populations previously isolated in glacial refugia, as proposed for a growing list of other Iberian taxa. Despite current intense honey bee management, human‐mediated processes have seemingly played a minor role in shaping Iberian honey bee genetic structure. This study highlights the complexity of the Iberian honey bee patterns and reinforces the importance of Iberia as a reservoir of Apis mellifera diversity.     

Characterisation of the chemical profiles of Brazilian and Andean morphotypes belonging to the Anastrepha fraterculus complex (Diptera,Tephritidae)

Fruit fly sexual behaviour is directly influenced by chemical and non-chemical cues that play important roles in reproductive isolation. The chemical profiles of pheromones and cuticular hydrocarbons (CHs) of eight fruit fly populations of the Andean, Brazilian-1 and Brazilian-3 morphotypes of the Anastrepha fraterculus cryptic species complex originating from Colombia (four populations) and Brazil (four populations) were analysed using two-dimensional gas chromatography with mass spectrometric detection. The resulting chemical diversity data were studied using principal component analyses. Andean morphotypes could be discriminated from the Brazilian-1 and Brazilian-3 morphotypes by means of male-borne pheromones and/or male and female CH profiles. The Brazilian-1 and Brazilian-3 morphotypes were found to be monophyletic. The use of chemical profiles as species- and sex-specific signatures for cryptic species separations is discussed.     

Efficient use of reactive nitrogen for cultivation of bioenergy: less is more

A further increase in nitrogen (N) intensive biomass supplies to substitute fossil carbon sources implies inclusion of additional reactive nitrogen (Nr) into the biosphere. A Danish model study compared low‐intensity managed seminatural beech forest and a winter wheat system with respect to N losses and greenhouse gas (GHG) emissions. Losses of reactive N to air and groundwater per unit of energy produced were four to six times higher for the winter wheat system. The energy efficiency was an order of magnitude higher in the forest system, whereas the related GHG emission reduction by fossil coal substitution differed by <25%. The question is whether a low or a high intensity of cultivation yields the best overall ecosystem service performance? Given the detrimental effect of excess reactive N on natural ecosystems, we suggest that bioenergy production from unfertilized forest with seminatural structure and function should be preferred over N‐intensive crop production.