Influence of yeast immobilization on fermentation and aldehyde reduction during the production of alcohol-free beer

Production of alcohol-free beer by limited fermentation is optimally performed in a packed-bed reactor. This highly controllable system combines short contact times between yeast and wort with the reduction of off-flavors to concentrations below threshold values. In the present study, the influence of immobilization of yeast to DEAE-cellulose on sugar fermentation and aldehyde reduction was monitored. Immobilized cells showed higher activities of hexokinase and pyruvate decarboxylase compared to cells grown in batch culture. In addition, a higher glucose flux was observed, with enhanced excretion of main fermentation products, indicating a reduction in the flux of sugar used for biomass production. ADH activity was higher in immobilized cells compared to that in suspended cells. However, during prolonged production a decrease was observed in NAD-specific ADH activity, whereas NADP-specific activity increased in the immobilized cells. The shifts in enzyme activities and glucose flux correlate with a higher in vivo reduction capacity of the immobilized cells.     

High levels of population differentiation for mitochondrial DNA haplotypes inPinus radiata,muricata, andattenuata

We analyzed mitochondrial (mt) DNA restriction fragment length polymorphisms (RFLPs) associated with cytochrome oxidase, subunit I (coxI)-related gene sequences in 268 trees derived from 19 natural populations of three species of pines from California (USA): Monterey pine (Pinus radiata D. Don), bishop pine (P. Muricata D. Don), and knobcone pine (P. attenuata Lemm.). Total genomic DNA was digested with four restriction endonucleases and probed with a 750-bp fragment of the mitochondrialcoxI gene amplified fromP. attenuata via the polymerase chain reaction (PCR). ThecoxI gene is repeated at least 4 times in some populations, and all variants that we observed resulted from complex rearrangements rather than from point mutations. There was limited intrapopulation variation, but strong differentiation among populations. When applied to haplotype frequencies, Nei's gene diversity within populations (H_s) averaged 7% (±3), and G_st varied from 75% forP. Radiata to 96% forP. muricata. The high degree of population differentiation for mtDNA suggests that it can be a powerful marker of population differences, but its rapid rate of structural evolution appears to result from recombination among a limited number of repetitive elements-giving frequent homoplasious fragment phenotypes. The phylogenetic trees disagreed with results from chloroplast DNA, nuclear gene, and morphological studies.     

DNA methylation pattern of CALCA in preterm neonates with bacterial sepsis as a putative epigenetic biomarker

Diagnosis of bacterial sepsis in preterm neonates can be difficult when using serum markers that rely on physiological changes because these changes may not necessarily be the result of bacterial infections alone. This retrospective investigation explores the potential use of the DNA methylation pattern of CpG sites in the promoter region of the calcitonin-related polypeptide α (CALCA) gene as an epigenetic biomarker for bacterial sepsis in preterm newborns. Four novel changes in the DNA methylation of eight CpG sites were detected in this gene and are present only in neonates with bacterial sepsis: (1) partial methylation at -769 CpG in gram-negative or gram-positive early onset sepsis (EOS) and late onset sepsis (LOS) episodes; (2) demethylation of 8 CpGs in gram-negative EOS followed by LOS (ELS) and in gram-negative EOS; (3) demethylation of 7 CpGs in gram-positive ELS and gram-positive EOS; (4) -771 C:G > T:A; 5′ de novo -778 CpG mutation on both alleles in EOS. These changes were not detected in birth weight and gestational age matched controls or in newborns with isolated infections. Our results indicate that the DNA methylation pattern of the promoter region of the CALCA gene varies in different types of bacterial preterm sepsis, thus suggesting a potential use as an epigenetic biomarker. A prospective confirmation of these results is essential.     

Physiological and biochemical characterization of NERICA‐L‐44: a novel source of heat tolerance at the vegetative and reproductive stages in rice

The predicted increase in the frequency and magnitude of extreme heat spikes under future climate can reduce rice yields significantly. Rice sensitivity to high temperatures during the reproductive stage is well documented while the same during the vegetative stage is more speculative. Hence, to identify and characterize novel heat‐tolerant donors for both the vegetative and reproductive stages, 71 rice accessions, including approximately 75% New Rice for Africa (NERICAs), were phenotyped across field experiments during summer seasons in Delhi, India, and in a controlled environment study at International Rice Research Institute , Philippines. NERICA‐L‐44 (NL‐44) recorded high seedling survival (52%) and superior growth and greater reproductive success exposed to 42.2°C (sd ± 2.3) under field conditions. NL‐44 and the heat‐tolerant check N22 consistently displayed lower membrane damage and higher antioxidant enzymes activity across leaves and spikelets. NL‐44 recorded 50–60% spikelet fertility, while N22 recorded 67–79% under controlled environment temperature of 38°C (sd ±1.17), although both had about 87% fertility under extremely hot field conditions. N22 and NL‐44, exposed to heat stress (38°C), had similar pollen germination percent and number of pollen tubes reaching the ovary. NL‐44 maintained low hydrogen peroxide production and non‐photochemical quenching (NPQ) with high photosynthesis while N22 avoided photosystem II damage through high NPQ under high‐temperature stress. NL‐44 with its reproductive stage resilience to extreme heat stress, better antioxidant scavenging ability in both vegetative tissue and spikelets and superior yield and grain quality is identified as a novel donor for increasing heat tolerance at both the vegetative and reproductive stages in rice.     

Spatially explicit and multi-sourced genetic information is critical for conservation of an endangered plant species,San Diego thornmint (<Emphasis Type="Italic">Acanthomintha ilicifolia</Emphasis>)

San Diego thornmint Acanthomintha ilicifolia (Gray) Gray (Lamiaceae) is a winter herb restricted to San Diego county in the United States and Baja California Norte in Mexico. Historic records document 80 occurrences of this species, with 55 extant occurrences in San Diego County currently known. We compared three measures of genetic variation to inform ongoing conservation efforts: putatively neutral genetic structure revealed from isozyme markers, apparent cytogenetic variation confirmed using flow cytometry, and potentially adaptive morphological variation quantified in a common-garden study. Together, these data indicated that this rare endemic is genetically complex, revealing significant differentiation of neutral and potentially adaptive genetic variation among populations, and possessing at least two cytotypes, sometimes even within the same population. While additional study is required to resolve the extent of potential local adaptation in this species, conservation plans should limit the movement of germplasm among occurrences and monitor populations in order to limit potential long-term impacts to population viability. Given that these findings challenge the canonical model of genetic structure in rare plants (low genetic variation and limited genetic structure), we recommend guidelines to apply genetic information to conservation strategies.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.