Benzyl Amino Purine and Gibberellic Acid Coupled to Nitrogen-Limited Stress Induce Fatty Acids,Biomass Accumulation,and Gene Expression in <i>Scenedesmus Obliquus</i>

The need for renewable energy sources makes microalgae an essential feedstock for biofuels production. The molecular aspects and the response to nitrogen (N)-limited conditions with a phytohormone stimulus in microalgae have been slightly explored. In this work, Scenedesmus obliquus was used as a study model to analyze the effect of benzyl amino purine (BAP) and gibberellic acid (GA) coupled to nitrogen limitation on cell growth, biomass and fatty acids. The selected 10^-5 M BAP increased the biomass by 1.44-fold, and 10^-6 M GA by 1.35-fold. The total lipids also increased by 2.8 and 1.11-fold, respectively. The 10^-5 M BAP and
10^-6 M GA addition to S. obliquus cultures at different initial nitrogen percentages (N-0, N-25, and N-50) showed a significant increase in cell growth and biomass productivity compared to the unstimulated cultures. BAP N-0 and GA N-0 produced the highest lipid yields with 55% and 50%, respectively. The lipid profile analysis revealed an increase, particularly in C18:1 and C16:0 fatty acids. Gene expression analysis showed an over-expression of acyl carrying protein (ACP), stearoyl-ACP desaturase (SAD), fatty acid acyl-ACP thioesterase (FATA), and diacylglycerol acyltransferase (DGAT) genes, which were mainly induced by nitrogen limitation. Furthermore, BAP and GA produced a significant over-expression on these genes in the N-replete cultures. This study shows that BAP and GA, coupled to N limitation stress, can be used to increase the biomass and lipid production in S. obliquus for sustainable biofuels.     

The gluconeogenic glucose-6-phosphatase gene is expressed during oxygen-limited conditions in the white shrimp Penaeus (Litopenaeus) vannamei: Molecular cloning,membrane protein modeling and transcript modulation in gills and hepatopancreas

Journal of Bioenergetics and Biomembranes - The white shrimp Penaeus (Litopenaeus) vannamei is the most economically important crustacean species cultivated in the Western Hemisphere. This...     

Uric acid best predicts metabolically unhealthy obesity with increased cardiovascular risk in youth and adults

Objective:

The obesity prevalence is growing worldwide and largely responsible for cardiovascular disease, the most common cause of death in the western world. The rationale of this study was to distinguish metabolically healthy from unhealthy overweight/obese young and adult patients as compared to healthy normal weight age matched controls by an extensive anthropometric, laboratory, and sonographic vascular assessment.

Design and Methods:

Three hundred fifty five young [8 to < 18 years, 299 overweight/obese(ow/ob), 56 normal weight (nw)] and 354 adult [>18‐60 years, 175 (ow/ob), 179 nw)] participants of the STYJOBS/EDECTA (STYrian Juvenile Obesity Study/Early DEteCTion of Atherosclerosis) cohort were analyzed. STYJOBS/EDECTA (NCT00482924) is a crossectional study to investigate metabolic/cardiovascular risk profiles in normal and ow/ob people free of disease except metabolic syndrome (MetS).

Results:

From 299 young ow/ob subjects (8‐< 18 years), 108 (36%), and from 175 adult ow/ob subjects (>18‐60 years), 79 (45%) had positive criteria for MetS. In both age groups, prevalence of MetS was greater among males. Overweight/obese subjects were divided into “healthy” (no MetS criterion except anthropometry fulfilled) and “unhealthy” (MetS positive). Although percentage body fat did not differ between “healthy” and “unhealthy” ow/ob, nuchal and visceral fat were significantly greater in the “unhealthy” group which had also significantly higher values of carotid intima media thickness (IMT). With MetS as the dependent variable, two logistic regressions including juveniles < 18 years or adults >18 years were performed. The potential predictor variables selected with the exception of age and gender by t test comparisons included IMT, ultrasensitive c‐reactive protein (US‐CRP), IL‐6, malondialdehyde (MDA), oxidized LDL, leptin, adiponectin, uric acid (UA), aldosterone, cortisol, transaminases, fibrinogen. In both groups, uric acid and in adults only, leptin and adiponectin, turned out as the best predictor.

Conclusion:

Serum levels of UA are a significant predictor of unhealthy obesity in juveniles and adults.     

Contribution of FKBP5 Genetic Variation to Gemcitabine Treatment and Survival in Pancreatic Adenocarcinoma

Purpose

FKBP51, (FKBP5), is a negative regulator of Akt. Variability in FKBP5 expression level is a major factor contributing to variation in response to chemotherapeutic agents including gemcitabine, a first line treatment for pancreatic cancer. Genetic variation in FKBP5 could influence its function and, ultimately, treatment response of pancreatic cancer.

Experimental Design

We set out to comprehensively study the role of genetic variation in FKBP5 identified by Next Generation DNA resequencing on response to gemcitabine treatment of pancreatic cancer by utilizing both tumor and germline DNA samples from 43 pancreatic cancer patients, including 19 paired normal-tumor samples. Next, genotype-phenotype association studies were performed with overall survival as well as with FKBP5 gene expression in tumor using the same samples in which resequencing had been performed, followed by functional genomics studies.

Results

In-depth resequencing identified 404 FKBP5 single nucleotide polymorphisms (SNPs) in normal and tumor DNA. SNPs with the strongest associations with survival or FKBP5 expression were subjected to functional genomic study. Electromobility shift assay showed that the rs73748206 “A(T)” SNP altered DNA-protein binding patterns, consistent with significantly increased reporter gene activity, possibly through its increased binding to Glucocorticoid Receptor (GR). The effect of rs73748206 was confirmed on the basis of its association with FKBP5 expression by affecting the binding to GR in lymphoblastoid cell lines derived from the same patients for whom DNA was used for resequencing.

Conclusion

This comprehensive FKBP5 resequencing study provides insights into the role of genetic variation in variation of gemcitabine response.     

Modification of the Secretion Pattern of Proteases,Inflammatory Mediators,and Extracellular Matrix Proteins by Human Aortic Valve is Key in Severe Aortic Stenosis

One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Secretome analysis is particularly relevant in this search as it focuses on a subset of proteins released by a cell or tissue under certain conditions. The sample can be considered as a plasma subproteome and it provides a more direct approximation to the in vivo situation. Degenerative aortic stenosis is the most common worldwide cause of valve replacement. Using a proteomic analysis of the secretome from aortic stenosis valves we could identify candidate markers related to this pathology, which may facilitate early diagnosis and treatment. For this purpose, we have designed a method to validate the origin of secreted proteins, demonstrating their synthesis and release by the tissue and ruling out blood origin. The nLC-MS/MS analysis showed the labeling of 61 proteins, 82% of which incorporated the label in only one group. Western blot and selective reaction monitoring differential analysis, revealed a notable role of the extracellular matrix. Variation in particular proteins such as PEDF, cystatin and clusterin emphasizes the link between aortic stenosis and atherosclerosis. In particular, certain proteins variation in secretome levels correlates well, not only with label incorporation trend (only labeled in aortic stenosis group) but, more importantly, with alterations found in plasma from an independent cohort of samples, pointing to specific candidate markers to follow up in diagnosis, prognosis, and therapeutic intervention.Degenerative aortic stenosis (AS)¹ is currently the most common cause of valve replacement in Western countries, and a significant increase in its prevalence is expected in the future because of increasing longevity (1). At the same time that our population age increases it can be expected that cardiac valve disease, and AS in particular, will increase in parallel. More and more, clinicians are seeing patients with symptomatic severe AS who are very advanced in age and have severe comorbidities or significant frailty, making operative intervention either impossible or of very high risk in the eyes of the cardiac surgeon. For this reason, the need of new diagnostic and prognostic methods, together with the urgent need of new drugs for therapy, has increased making a correct diagnosis in the early stages of the disease thereby reducing the cost burden to society. Several lines of evidence have demonstrated that degenerative AS is an active process in which inflammation plays a key role. As such, preventative approaches similar to those used in coronary artery disease (CAD) may be also applicable to AS (2, 3, 4, 5, 6). However, recent studies reported no reduction in later states of AS disease using statins, which significantly benefit patients with atherosclerosis (7, 8). Hence, further research is required to elucidate the pathogenic mechanism of this prevalent disease and to identify the similarities and differences in relation to atherosclerosis.Proteomics has emerged as a particularly suitable platform for the nonbiased analysis of proteins involved in the pathogenesis of various diseases, such as AS (9, 10). This type of approach provided the basis for the development of biomarkers to detect patients at risk of developing degenerative AS. Plasma and serum have been the main source used in proteomic studies to identify candidate protein biomarkers, followed by tissue and cell samples. However, the use of plasma and serum is hampered by their complex nature and by the large dynamic range of protein concentrations, which may favor the detection of abundant proteins at the expense of those present at lower concentrations (11, 12). As such, the aortic valve secretome has emerged as an attractive target to further understand the AS pathogenic process. Tissue secretomes provide a more accurate model of the in vivo situation and, by minimizing serum contaminants, they facilitate the detection of low abundance proteins secreted into the blood (13).In the present study, aortic valves from AS patients and nonaffected control subjects were cultured and the secretome analyzed by nLC-MS/MS. The addition of labeled amino acids to the culture media enabled the origin of the secreted proteins to be validated (truly secreted versus serum contaminants) and provided with information related to the dynamics of their synthesis and release from tissue.     

The EU protected taxon <Emphasis Type="Italic">Morimus funereus</Emphasis> Mulsant, 1862 (Coleoptera: Cerambycidae) and its western Palaearctic allies: systematics and conservation outcomes

Morimus funereus is a large longhorn beetle included in the European Habitats Directive and in previous releases of the IUCN red list. It represents a flagship species of old-growth forest saproxylic communities in E and SE Europe. The morphologically based taxonomy of W Palaearctic Morimus is rather unstable due to high phenetic intrapopulational and geographic variability and different authors have attempted to recognise one to five different taxa of specific/subspecific rank. No previous molecular data are available for the genus Morimus. Here, for the first time, a molecular approach based on COI and ITS2 gene sequences was applied in European and Anatolian Morimus specimens. The genetic variability among Euro-Anatolian Morimus populations and the geographical structure suggest that they can not be ascribed to the currently accepted five W Palaearctic Morimus species and may actually represent a single, genetically and morphologically variable biological species (M. asper), highlighting the necessity of an extended taxonomical revision. In light of these results, a phylogeographical hypothesis of postglacial colonisation of the central Mediterranean area has been developed and the consequences of this new taxonomic arrangement regarding conservation strategies for “Morimus funereus” and allied taxa in Europe and Turkey are discussed.     

rs12512631 on the Group Specific Complement (Vitamin D-Binding Protein GC) Implicated in Melanoma Susceptibility

Background

Solar radiation should be avoided in melanoma patients. Nevertheless, this is the main means by which the body produces vitamin D. Evidence suggests a protective role against cancer for vitamin D. Since vitamin D performs its function by binding the receptor encoded by the vitamin D-receptor gene (VDR), most studies have focused on polymorphisms (SNPs) within this gene. However, the gene encoding the vitamin D-binding protein (GC) appears in recent studies as a major player in the role of a serum vitamin D level regulator and in Cutaneous Melanoma (CM) predisposition.

Methods

We performed a case-control study of 12 polymorphisms on GC and 9 on VDR among 530 cases and 314 controls from Spanish population.

Results

We found association between SNP rs12512631, located 3′downstream of GC, and risk of CM that seems to fit a dominant model (OR 1.63 95%CI 1.23–2.17 p-value 7×10⁻⁴). This association remained Bonferroni’s correction and after adjustment for potential confounders (p-value 3×10⁻³) and even after increasing the sample size to 1729 individuals (p-value 0.0129). Moreover, we confirmed evidence of an association between CM susceptibility and the linkage disequilibrium block marked by tag-SNP rs222016 (p-value 0.032). This block covers the GC intron 1 region, with probable regulatory functions.

Conclusion

To our knowledge, this is the first vitamin D pathway-related polymorphism study in melanoma risk conducted in the Spanish population. Furthermore, we show an association between polymorphisms in GC and melanoma risk, confirming recent studies in different populations.     

Deletion Genotypes Reduce Occlusion Body Potency but Increase Occlusion Body Production in a Colombian Spodoptera frugiperda Nucleopolyhedrovirus Population

A Colombian field isolate (SfCOL-wt) of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a mixture of different genotypes. To evaluate the insecticidal properties of the different genotypic variants, 83 plaque purified virus were characterized. Ten distinct genotypes were identified (named A through J). SfCOL-A was the most prevalent (71±2%; mean ± SE) showing a PstI restriction profile indistinguishable to that of SfCOL-wt. The remaining nine genotypes presented genomic deletions of 3.8 - 21.8 Kb located mainly between nucleotides 11,436 and 33,883 in the reference genome SfMNPV-B, affecting the region between open reading frames (ORFs) sf20 and sf33. The insecticidal activity of each genotype from SfCOL-wt and several mixtures of genotypes was compared to that of SfCOL-wt. The potency of SfCOL-A occlusion bodies (OBs) was 4.4-fold higher than SfCOL-wt OBs, whereas the speed of kill of SfCOL-A was similar to that of SfCOL-wt. Deletion genotype OBs were similarly or less potent than SfCOL-wt but six deletion genotypes were faster killing than SfCOL-wt. The potency of genotype mixtures co-occluded within OBs were consistently reduced in two-genotype mixtures involving equal proportions of SfCOL-A and one of three deletion genotypes (SfCOL-C, -D or -F). Speed of kill and OB production were improved only when the certain genotype mixtures were co-occluded, although OB production was higher in the SfCOL-wt isolate than in any of the component genotypes, or mixtures thereof. Deleted genotypes reduced OB potency but increased OB production of the SfCOL-wt population, which is structured to maximize the production of OBs in each infected host.     

Differential Expression of Apoptosis Related Genes in Selected Strains of Aedes aegypti with Different Susceptibilities to Dengue Virus

Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2) and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1) and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain.     

Microbiological and Molecular Characterization of Staphylococcus hominis Isolates from Blood

Background

Among Coagulase-Negative Staphylococci (CoNS), Staphylococcus hominis represents the third most common organism recoverable from the blood of immunocompromised patients. The aim of this study was to characterize biofilm formation, antibiotic resistance, define the SCCmec (Staphylococcal Chromosomal Cassette mec) type, and genetic relatedness of clinical S. hominis isolates.

Methodology

S. hominis blood isolates (n = 21) were screened for biofilm formation using crystal violet staining. Methicillin resistance was evaluated using the cefoxitin disk test and the mecA gene was detected by PCR. Antibiotic resistance was determined by the broth microdilution method. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) and SCCmec typed by multiplex PCR using two different methodologies described for Staphylococcus aureus.

Results

Of the S. hominis isolates screened, 47.6% (10/21) were categorized as strong biofilm producers and 23.8% (5/21) as weak producers. Furthermore, 81% (17/21) of the isolates were methicillin resistant and mecA gene carriers. Resistance to ampicillin, erythromycin, and trimethoprim was observed in >70% of isolates screened. Each isolate showed a different PFGE macrorestriction pattern with similarity ranging between 0–95%. Among mecA-positive isolates, 14 (82%) harbored a non-typeable SCCmec type: eight isolates were not positive for any ccr complex; four contained the mec complex A ccrAB1 and ccrC, one isolate contained mec complex A, ccrAB4 and ccrC, and one isolate contained the mec complex A, ccrAB1, ccrAB4, and ccrC. Two isolates harbored the association: mec complex A and ccrAB1. Only one strain was typeable as SCCmec III.

Conclusions

The S. hominis isolates analyzed were variable biofilm producers had a high prevalence of methicillin resistance and resistance to other antibiotics, and high genetic diversity. The results of this study strongly suggested that S. hominis isolates harbor new SCCmec structural elements and might be reservoirs of ccrC1 in addition to ccrAB1 and mec complex A.