Enhancing operational stability and exhibition of enzyme activity by removing water in the immobilized lipase‐catalyzed production of erythorbyl laurate

Erythorbyl laurate was continuously synthesized by esterification in a packed‐bed enzyme reactor with immobilized lipase from Candida antarctica. Response surface methodology based on a five‐level three‐factor central composite design was adopted to optimize conditions for the enzymatic esterification. The reaction variables, such as reaction temperature (10–70°C), substrate molar ratio ([lauric acid]/[erythorbic acid], 5–15), and residence time (8–40 min) were evaluated and their optimum conditions were found to be 56.2°C, 14.3, and 24.2 min, respectively. Under the optimum conditions, the molar conversion yield was 83.4%, which was not significantly different (P < 0.05) from the value predicted (84.4%). Especially, continuous water removal by adsorption on an ion‐exchange resin in a packed‐bed enzyme reactor improved operational stability, resulting in prolongation of half‐life (2.02 times longer compared to the control without water‐removal system). Furthermore, in the case of batch‐type reactor, it exhibited significant increase in initial velocity of molar conversion from 1.58% to 2.04%/min. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:882–889, 2013     

Stevens-Johnson Syndrome Associated with Drugs and Vaccines in Children: A Case-Control Study

Objective

Stevens-Johnson Syndrome (SJS) is one of the most severe muco-cutaneous diseases and its occurrence is often attributed to drug use. The aim of the present study is to quantify the risk of SJS in association with drug and vaccine use in children.

Methods

A multicenter surveillance of children hospitalized through the emergency departments for acute conditions of interest is currently ongoing in Italy. Cases with a diagnosis of SJS were retrieved from all admissions. Parents were interviewed on child’s use of drugs and vaccines preceding the onset of symptoms that led to the hospitalization. We compared the use of drugs and vaccines in cases with the corresponding use in a control group of children hospitalized for acute neurological conditions.

Results

Twenty-nine children with a diagnosis of SJS and 1,362 with neurological disorders were hospitalized between 1^st November 1999 and 31^st October 2012. Cases were more frequently exposed to drugs (79% vs 58% in the control group; adjusted OR 2.4; 95% CI 1.0–6.1). Anticonvulsants presented the highest adjusted OR: 26.8 (95% CI 8.4–86.0). Significantly elevated risks were also estimated for antibiotics use (adjusted OR 3.3; 95% CI 1.5–7.2), corticosteroids (adjusted OR 4.2; 95% CI 1.8–9.9) and paracetamol (adjusted OR 3.2; 95% CI 1.5–6.9). No increased risk was estimated for vaccines (adjusted OR: 0.9; 95% CI 0.3–2.8).

Discussion

Our study provides additional evidence on the etiologic role of drugs and vaccines in the occurrence of SJS in children.     

Stroma Cell-Derived Factor-1α Signaling Enhances Calcium Transients and Beating Frequency in Rat Neonatal Cardiomyocytes

Stroma cell-derived factor-1α (SDF-1α) is a cardioprotective chemokine, acting through its G-protein coupled receptor CXCR4. In experimental acute myocardial infarction, administration of SDF-1α induces an early improvement of systolic function which is difficult to explain solely by an anti-apoptotic and angiogenic effect. We wondered whether SDF-1α signaling might have direct effects on calcium transients and beating frequency.Primary rat neonatal cardiomyocytes were culture-expanded and characterized by immunofluorescence staining. Calcium sparks were studied by fluorescence microscopy after calcium loading with the Fluo-4 acetoxymethyl ester sensor. The cardiomyocyte enriched cellular suspension expressed troponin I and CXCR4 but was vimentin negative. Addition of SDF-1α in the medium increased cytoplasmic calcium release. The calcium response was completely abolished by using a neutralizing anti-CXCR4 antibody and partially suppressed and delayed by preincubation with an inositol triphosphate receptor (IP₃R) blocker, but not with a ryanodine receptor (RyR) antagonist. Calcium fluxes induced by caffeine, a RyR agonist, were decreased by an IP₃R blocker. Treatment with forskolin or SDF-1α increased cardiomyocyte beating frequency and their effects were additive. In vivo, treatment with SDF-1α increased left ventricular dP/dtmax.These results suggest that in rat neonatal cardiomyocytes, the SDF-1α/CXCR4 signaling increases calcium transients in an IP₃-gated fashion leading to a positive chronotropic and inotropic effect.     

Promoter Hypermethylation of ARID1A Gene Is Responsible for Its Low mRNA Expression in Many Invasive Breast Cancers

ARID1A (AT-rich interactive domain 1A) has recently been identified as a tumor suppressor gene. Its mRNA expression is significantly low in many breast cancers; this is often associated with more aggressive phenotypes. However, the underlying molecular mechanism for its low expression has not been fully understood. This study was undertaken to evaluate the contribution of gene copy number variation, mutations, promoter methylation and histone modification to ARID1A’s low expression. 38 pairs of breast invasive ductal carcinomas and their normal breast tissue counterparts from the same patients were randomly selected for gene expression and copy number variation detection. Promoter methylation and histone modification levels were evaluated by MeDIP-qPCR and ChIP-qPCR, respectively. PCR product Sanger sequencing was carried out to detect the exon mutation rate. Twenty-two out of 38 invasive ductal carcinomas in the study (57.9%) revealed ARID1A mRNA low expression by realtime RT-PCR. The relative promoter methylation level was, significantly higher in ARID1A mRNA low expression group compared with its high expression group (p<0.001). In the low expression group, nineteen out of 22 invasive ductal carcinomas (86.4%) exhibited ARID1A promoter hypermthylation. In addition, the promoter hypermethylation was accompanied with repressive histone modification (H3K27Me3). Although five out of 38 invasive ductal carcinomas (13.2%) exhibited loss of ARID1A gene copy number by realtime PCR and nine exon novel mutations are seen from eight out of 33 invasive ductal carcinomas (24.2%), there was no statistically significant difference in both ARID1A mRNA low and high expression groups (p = 0.25,and p = 0.68, respectively). We demonstrate that promoter hypermethylation was the main culprit for ARID1A mRNA low expression in invasive ductal carcinomas. The influence of mutation and copy number variation on the expression were statistically insignificant at mRNA level, and were, therefore, not considered the main causes for ARID1A mRNA low expression in invasive breast cancer.     

Identifying SNP markers tightly associated with six major genes in peach [<Emphasis Type="Italic">Prunus persica</Emphasis> (L.) Batsch] using a high-density SNP array with an objective of marker-assisted selection (MAS)

One of the applications of genomics is to identify genetic markers linked to loci responsible for variation in phenotypic traits, which could be used in breeding programs to select individuals with favorable alleles, particularly at the seedling stage. With this aim, in the framework of the European project FruitBreedomics, we selected five main peach fruit characters and a resistance trait, controlled by major genes with Mendelian inheritance: fruit flesh color Y, fruit skin pubescence G, fruit shape S, sub-acid fruit D, stone adhesion-flesh texture F-M, and resistance to green peach aphid Rm2. They were all previously mapped in Prunus. We then selected three F₁ and three F₂ progenies segregating for these characters and developed genetic maps of the linkage groups including the major genes, using the single nucleotide polymorphism (SNP) genome-wide scans obtained with the International Peach SNP Consortium (IPSC) 9K SNP array v1. We identified SNPs co-segregating with the characters in all cases. Their positions were in agreement with the known positions of the major genes. The number of SNPs linked to each of these, as well as the size of the physical regions encompassing them, varied depending on the maps. As a result, the number of useful SNPs for marker-assisted selection varied accordingly. As a whole, this study establishes a sound basis for further development of MAS on these characters. Additionally, we also discussed some limitations that were observed regarding the SNP array efficiency.     

PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L‐associated PI3K activity

The Beclin1–VPS34 complex is recognized as a central node in regulating autophagy via interacting with diverse molecules such as ATG14L for autophagy initiation and UVRAG for autophagosome maturation. However, the underlying molecular mechanism that coordinates the timely activation of VPS34 complex is poorly understood. Here, we identify that PAQR3 governs the preferential formation and activation of ATG14L‐linked VPS34 complex for autophagy initiation via two levels of regulation. Firstly, PAQR3 functions as a scaffold protein that facilitates the formation of ATG14L‐ but not UVRAG‐linked VPS34 complex, leading to elevated capacity of PI(3)P generation ahead of starvation signals. Secondly, AMPK phosphorylates PAQR3 at threonine 32 and switches on PI(3)P production to initiate autophagosome formation swiftly after glucose starvation. Deletion of PAQR3 leads to reduction of exercise‐induced autophagy in mice, accompanied by a certain degree of disaggregation of ATG14L‐associated VPS34 complex. Together, this study uncovers that PAQR3 can not only enhance the capacity of pro‐autophagy class III PI3K due to its scaffold function, but also integrate AMPK signal to activation of ATG14L‐linked VPS34 complex upon glucose starvation.