Effect of ultrasound-assisted cold plasma pretreatment to obtain sea asparagus extract and its application in Italian salami.

The effect of the glow discharge cold plasma pretreatment (CPT) was investigated on the ultrasound-assisted extraction (UAE) of phenolic compounds from sea asparagus Salicornia neei. The extract was applied in dry fermented sausage (Italian salami). Thermal extraction methods demand a long processing time, are highly energy-consuming and cause irreversible nutrient losses. It was found that CPT (discharge power of 14 W for 5 min) prior to UAE increased the antioxidant activity by 22% and 19% measured by the DPPH and ABTS assays, respectively. The S. neei extract showed high antioxidant activity, low antimicrobial activity, and was added to salami formulations with reduced-sodium nitrite content and no addition of sodium erythorbate. Despite a slight color change, lipid oxidation and texture parameters were similar to the control at the end of ripening. Furthermore, higher antioxidant activity was observed in S. neei extract supplemented salami with no impact on its sensory overall acceptability, indicating its potential as a natural alternative to synthetic additives.

MiR-9-5p Down-Regulates PiT2, but not PiT1 in Human Embryonic Kidney 293 Cells.

PiT1 (SLC20A1) and PiT2 (SLC20A2) are members of the mammalian type-III inorganic phosphate transporters and recent studies linked SLC20A2 mutations with primary brain calcifications. MicroRNAs (miRNAs) are endogenous noncoding regulatory RNAs and MicroRNA-9 (miR-9) modulates neurogenesis but is also involved with different types of cancer. We evaluated possible interactions between miR-9 and the phosphate transporters (PiT1 and PiT2). SLC20A2, platelet-derived growth factor receptor beta (PDGFRB) and Fibrillin-2 (FBN2) showed binding sites with high affinity for mir-9, In silico. miR-9 mimic was transfected into HEK293 cells and expression was confirmed by RT-qPCR. Overexpression of miR-9 in these cells caused a significant reduction in PiT2 and FBN2. PDGFRB appeared to be decreased, but was not significantly down-regulated. PiT1 showed no significant difference relative to controls. The down-regulation of PiT2 protein by miR-9 was confirmed by western blotting. In conclusion, we showed that miR-9 can down-regulate PiT2, in HEK293 cells. [corrected].

Combined Genome-Wide CSF Aß-42's Associations and Simple Network Properties Highlight New Risk Factors for Alzheimer's Disease.

The abnormal deposition of amyloid-ß protein in the brain plays an important role in Alzheimer's disease (AD), being considered a potential clinical biomarker. To investigate genetic associations with amyloid-ß we used biomarker data and genome-wide variants from individuals with AD and mild cognitive impairment in the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. We used a standard linear model and retested the associations with a mixed linear model to correct the residual sample structure. Both methods' results showed two identical significant SNPs associated with the A ß-42 levels in CSF (rs2075650 at intron region TOMM40 with p-value ≥ 1 × 10-16 and rs439401 in the intergenic region of LOC100129500 and APOC1 with p-value ≥ 1 × 10-9) and highlighted APOC1 and TOMM40, which are well-known genes previously associated with AD. Extending our analysis, we considered possible candidate genes mapped to SNPs with p-value ≥ 1 × 10-6 to explore gene-set enrichment e gene-gene network analysis, which reveals genes related to synaptic transmission, transmission of nerve impulses, cell-cell signaling and neurological processes. These genes require fine mapping and replication studies to allow more detailed understanding of how they may contribute to the genetic architecture of AD.

XPR1: a Gene Linked to Primary Familial Brain Calcification Might Help Explain a Spectrum of Neuropsychiatric Disorders.

Primary familial brain calcifications (PFBC) compose a rare neurologic condition characterized by a bilateral pattern of hydroxyapatite deposits in basal ganglia, dentate nuclei, and thalamus. PFBC is identified through neuroimaging screenings such as computerized tomography. Patients with PFBC might present a wide variety of neurological symptoms such as mental and motor impairments, often misdiagnosed as Parkinson's disease, schizophrenia, Alzheimer's disease, and migraine. Four genes were confirmed as causative of PFBC: SLC20A2, PDGFB, PDGFRB, and XPR1. Curiously, other studies made occasional links between XPR1 variations or expression changes, in a few neuropsychiatric models. This letter is an assembly on XPR1 variants and expression change pattern data that were published in recent scientific reports, even before the current connection between that gene and brain calcification.

First report of a de novo mutation at SLC20A2 in a patient with brain calcification.

Primary familial brain calcification (PFBC) is identified by mineralization of the basal ganglia and other brain regions in the absence of known causes. The condition is often inherited in an autosomal dominant pattern and can manifest itself clinically with neuropsychiatric symptoms such as Parkinsonism, headaches, psychosis, and mood swings. Mutations in the SLC20A2 gene account for ~40% of inherited cases, and this gene encodes an inorganic phosphate transporter (PiT-2), a transmembrane protein associated with Pi homeostasis. The p.Y386X mutation in SLC20A2 was identified in a patient who presented migraines, brain calcification, and mild but chronic hypovitaminosis D. SLC20A2 c.1158C > G single-nucleotide heterozygous mutation results in a premature stop codon and a putative truncated protein of 385 amino acids. Proband parents do not present the mutation, which is also not present in major public SNP databases, suggesting a de novo sporadic trait. This study describes for the first time a de novo SLC20A2 mutation in a PFBC patient with migraine and mild hypovitaminosis D. This data further reinforces the pathogenic role of SLC20A2 mutations as causal factors in PFBC physiopathology.

Exaggerated blood pressure response during the exercise treadmill test as a risk factor for hypertension.

Exaggerated blood pressure response (EBPR) during the exercise treadmill test (ETT) has been considered to be a risk factor for hypertension. The relationship of polymorphisms of the renin-angiotensin system gene with hypertension has not been established. Our objective was to evaluate whether EBPR during exercise is a clinical marker for hypertension. The study concerned a historical cohort of normotensive individuals. The exposed individuals were those who presented EBPR. At the end of the observation period (41.7 months = 3.5 years), the development of hypertension was analyzed within the two groups. Genetic polymorphisms and blood pressure behavior were assessed as independent variables, together with the classical risk factors for hypertension. The I/D gene polymorphism of the angiotensin-converting enzyme and M235T of angiotensinogen were ruled out as risk factors for hypertension. EBPR during ETT is not an independent influence on the chances of developing hypertension. No differences were observed between the hypertensive and normotensive individuals regarding gender (P = 0.655), skin color (P = 0.636), family history of hypertension (P = 0.225), diabetes mellitus (P = 0.285), or hypertriglyceridemia (P = 0.734). The risk of developing hypertension increased with increasing body mass index (BMI) and advancing age. The risk factors, which independently influenced the development of hypertension, were age and BMI. EBPR did not constitute an independent risk factor for hypertension and is probably a preclinical phase in the spectrum of normotension and hypertension.

Analysis of gene expression pattern and neuroanatomical correlates for SLC20A2 (PiT-2) shows a molecular network with potential impact in idiopathic basal ganglia calcification ("Fahr's disease").

Familial idiopathic basal ganglia calcification (FIBGC), also known as "Fahr's disease," is a neuropsychiatric disorder with motor and cognitive symptoms. It is characterized pathologically by bilateral calcification most commonly in the basal ganglia and also in other brain regions such as the thalamus and cerebellum. A recent report by Wang et al. (2012) discovered multiple families with FIBGC carrying mutations in the SLC20A2 gene, encoding the inorganic phosphate transporter PiT-2, which segregated in an autosomal dominant pattern. To understand further the role of SLC20A2 in FIBGC brain pathology, here we described the gene expression pattern across the whole brain for SLC20A2, using the Allen Institute Human Brain Atlas database. Microarray analysis provided evidence that the neuroanatomical pattern of expression for SLC20A2 is highest in the regions most commonly affected in FIBGC. Neuroanatomical regions that demonstrated high correlation or anti-correlation with SLC20A2 expression also showed a molecular network with potential to explain the limited neuroanatomical distribution of calcifications in IBGC. Lastly, these co-expression networks suggest additional further candidate genes for FIBGC.

Exaggerated blood pressure response during the exercise treadmill test as a risk factor for hypertension

Exaggerated blood pressure response (EBPR) during the exercise treadmill test (ETT) has been considered to be a risk factor for hypertension. The relationship of polymorphisms of the renin-angiotensin system gene with hypertension has not been established. Our objective was to evaluate whether EBPR during exercise is a clinical marker for hypertension. The study concerned a historical cohort of normotensive individuals. The exposed individuals were those who presented EBPR. At the end of the observation period (41.7 months = 3.5 years), the development of hypertension was analyzed within the two groups. Genetic polymorphisms and blood pressure behavior were assessed as independent variables, together with the classical risk factors for hypertension. The I/D gene polymorphism of the angiotensin-converting enzyme and M235T of angiotensinogen were ruled out as risk factors for hypertension. EBPR during ETT is not an independent influence on the chances of developing hypertension. No differences were observed between the hypertensive and normotensive individuals regarding gender (P = 0.655), skin color (P = 0.636), family history of hypertension (P = 0.225), diabetes mellitus (P = 0.285), or hypertriglyceridemia (P = 0.734). The risk of developing hypertension increased with increasing body mass index (BMI) and advancing age. The risk factors, which independently influenced the development of hypertension, were age and BMI. EBPR did not constitute an independent risk factor for hypertension and is probably a preclinical phase in the spectrum of normotension and hypertension.

Revising the M235T polymorphism position for the AGT gene and reporting a modifying variant in the Brazilian population with potential cardiac and neural impact.

There is a growing need to curate the overwhelming amount of sequencing data which is available in many public databases. For instance, new information shows that the M235T polymorphism at the angiotensinogen gene (AGT) is actually positioned at the position corresponding to the amino acid 268 and not 235. This polymorphism is filled as rs699 in the NCBI SNP database and results in the synthesis of a threonine (T) instead of a methionine (M). It has been widely studied and associated as an important risk factor for several vascular and neuropsychiatric conditions. We faced this new situation during the targeted sequencing of 360 chromosomes from Brazilian subjects studied for the M235T polymorphism, leading to the identification of a novel variation (rs141900991). This report explores the potential impact of such a dinucleotide variation, which promotes the change of alanine (A) to serine (S) at the AGT protein structure (A237S). Considering the previous M268T variation at the four possible haplotypes combined (MA, MS, TA and TS), we performed a comparative hydrophobicity simulation, using the Kyte-Doolittle algorithm, available at the CLB Bio workbench, in the four possible haplotypes. Additional simulations were performed using the programs PolyPhen, I-Mutant and SIFT, in order to evaluate the pathogenicity of both mutations. The predicted hydrophobicity decreases of a similar magnitude, with both MS and TA haplotypes, but the presence of both variations induces a major decrease in hydrophobicity, suggesting a cumulative effect, with possible modifying effect since that this variation per se would limit the hydrophobicity range and the latter chances in finding significant phenotype differences. A better characterization of this kind of variant is particularly important because the current genome wide scan analyses in complex disorders with cardiac or neural etiology are not generating reliable findings, especially if we consider the huge investment with such approach. Additional and unknown variations like this one, with potential modifying effect, might be more common than previously expected.

Obstructive sleep apnea severity correlates with cellular and plasma oxidative stress parameters and affective symptoms.

Obstructive sleep apnea syndrome (OSAS) is considered a sleep-related respiratory disorder, characterized by repetitive episodes of complete (apnea) or partial (hypopnea) obstruction of airflow in the upper airway (UA) during sleep. The pathophysiology of upper airway obstruction in OSAS is multifactorial, leading to a chronic recurrent state of intermittent hypoxemia and reoxygenation during sleep, maintaining a state of oxidative stress, which seems to be the key to the pathophysiological manifestations of OSAS, and is associated with the development of a number of high morbidity-mortality systematic complications, such as obesity, type 2 diabetes, metabolic syndrome, and cardiovascular and neuropsychological diseases. This study is an open, cross-sectional, and comparative clinical trial, whose general objective was to assess the correlation between OSAS severity, oxidative stress markers, and the presence of affective symptoms (depressive and anxious) in OSAS patients. We studied 38 adult males, who had been diagnosed with OSAS by overnight polysomnography, between 18 and 60 years of age, divided into three groups: group 1-10 individuals with mild OSAS (AHI between 5 and 14.9/h), group 2-13 individuals with moderate OSAS (AHI between 15 and 30/h), and group 3-15 individuals with severe OSAS (AHI >30/h). All individuals were evaluated for level of subjective sleepiness using the Epworth Sleepiness Scale, for depressive and anxiety symptoms by the Hamilton Depression (HAM-D) and Anxiety (HAM-A) Scales, and for parameters of the oxidative stress state, measuring superoxide radical and serum nitrates and nitrites levels. There was a progressive and significant increase in the state of oxidative stress (p < 0.05), in the total score of depressive symptoms (p = 0.001) and in the overall score of anxiety symptoms (p = 0.004) directly proportional to the severity of apnea when comparing the mild group to the severe group. Positive correlations were identified between superoxide production and the apnea-hypopnea index (AHI) (r = 0.48), Epworth sleepiness score (r = 0.36), and Hamilton depression score (HAM-D) (r = 0.40); between serum nitrates and nitrites levels and SO(2) min (r = 0.44); and between the AHI and the HAM-D (r = 0.51) score and HAM-A (r = 0.40) score. Negative correlations were observed between the AHI and serum nitrates and nitrites levels (r = -0.42), between superoxide production and SO(2) min (r = -0.31), between serum nitrates and nitrites levels and HAM-D (r = -0.50) and HAM-A (-0.42) scores, and between SO(2) min and HAM-D (r = -0.48) and HAM-A (r = -0.40) scores. According to the results of this study, we can conclude that (1) individuals with OSAS show an increase in the production of superoxide radical and a decrease in serum nitrates and nitrites levels, which are objective signs of a state of oxidative stress. (2) The more severe the OSAS, the more fragmented the sleep and the greater the nocturnal hypoxemia, the more severe is the oxidative stress state and the greater is the incidence of daytime symptoms, especially sleepiness and depressive and anxiety symptoms. Future studies might explore the investigation of oxidative stress parameters as an alternative approach to anticipate symptoms, measure prognosis, and monitor OSAS progression or treatment response.

Exploring the implications of INDELs in neuropsychiatric genetics: challenges and perspectives.

The decade passed after publishing the Human Genome first draft faced an enormous growth at the understanding of the genomic variation among different subjects, populations, and groups of patients. Single nucleotide polymorphisms (SNPs) and insertion or deletions (INDELs) have been increasingly recognized as a major type of genetic variations, with potential impact in protein activities and gene expression changes observed in complex genetic traits, like neuropsychiatric diseases. INDELs represent the second most common class of variations after SNPs, but there is still an important gap between the number of INDELs reported and the actual knowledge about the functional implications of such variations. There are approximately 10 million SNPs already reported, and the human populations are expected to collectively harbor at least 1.6-2.5 million INDELs. One of the major challenges is to find better platforms to screen for INDELs in a high throughput manner. The discordance in between the data from different studies might be explained by the diverse approaches employed to sequence the genomes with variable platforms. Short INDEL variations increased the scope of genetic markers in human genetic diseases, and various studies showed that common microdeletions and smaller INDELs might be highly associated with neuropsychiatric diseases such as schizophrenia, autism, mental retardation, and Alzheimer disease. The rapidly increasing amount of resequencing, genotyping, and personal genome data generated by large-scale genetic human projects require the development of integrated bioinformatics tools able to efficiently manage and analyze these genetic data. Our group is currently dealing with different approaches that might optimize sequencing and bioinformatics analyses of short INDELs to broaden our research capabilities of identifying those intriguing genetic variations. Hopefully, INDELs might become a new trend in association studies in neuropsychiatric genetics since so far the level of significant and positive associations with the standard SNPs reported presents limited predictive application.

Using ESTs database to predict and validate single polymorphisms at the HLA system.

We propose a bioinformatics pipeline in which we use an ESTs database to predict and validate single-nucleotide polymorphisms (SNPs) directly linked to gene-coding regions at the HLA class I genes (HLA-A, HLA-B and HLA-C). Annotation originated from our analysis revealed various classes of possible new variations that may indicate possible new alleles. Thus, bioinformatics pipelines seem to be useful approaches to help screening for novel genetic variations at the HLA panel, and further analysis will foster this aim to provide celerity at the massive analysis of data currently generated in large-scale high-throughput experiments.

In silico identification of new genetic variations as potential risk factors for Alzheimer's disease in a microarray-oriented simulation.

Genomic and proteomic studies of neurodegenerative disorders require complementary approaches to integrate the massive amount of data generated in high throughput experimental procedures. We propose a Bioinformatics pipeline in which expression studies guide the selection of candidate genes that should be screened for potential new genetic variations from a public expressed site tags (ESTs) database. Motivated by the former interest of our group in genetic polymorphisms involved with the immune system, we selected five genes from a previous expression microarrays study of hippocampal cornu ammonis (CA1) area of Alzheimer's Disease subjects (AD). The CLCbio Workbench Combined version 3.6.2. was initially used to build ESTs and mRNA files retrieved respectively from the Goldenpath of University of California Santa Cruz (UCSC) and National Center for Biotechnology Information (NCBI) databases and latter to perform multiple batches of Smith-Waterman alignments. A total of 116 ESTs sequences were selected after proper stringent parameters were applied to the first set of mismatches. The annotation revealed various classes of variations, most of them deletions (176). Amongst this specific group, some were frameshift deletions (35) and the virtual translation of a few others (5) were predicted to induce no change other than a single aminoacid removal, with no subsequent repercussions at the protein sequence. In addition, the analysis identified transitions (three), transversions (52), synonymous (41), non-synonymous (12), and deletions in 36 ESTs located in Untranslated Regions -UTRs (Supplementary data). Deletions are often associated to major genetics syndromes with dysmorphic features. However, various recent studies show that common microdeletions might be highly associated with common neuropsychiatric disorders such as schizophrenia, autism, mental retardation, or even in various ethnicities, detected in whole genome sequencing experiments. A virtual validation confirmed that some of the variations identified were previously reported and confirmed in DNA samples, showing that this method is a feasible way to detect genetic variations that merit further exploration in AD genetic risk factor association studies.

The genetics of Alzheimer's disease in Brazil: 10 years of analysis in a unique population.

Alzheimer's Disease (AD) is the most common type of dementia among the elderly, with devastating consequences for the patient, their relatives, and caregivers. More than 300 genetic polymorphisms have been involved with AD, demonstrating that this condition is polygenic and with a complex pattern of inheritance. This paper aims to report and compare the results of AD genetics studies in case-control and familial analysis performed in Brazil since our first publication, 10 years ago. They include the following genes/markers: Apolipoprotein E (APOE), 5-hidroxytryptamine transporter length polymorphic region (5-HTTLPR), brain-derived neurotrophin factor (BDNF), monoamine oxidase A (MAO-A), and two simple-sequence tandem repeat polymorphisms (DXS1047 and D10S1423). Previously unpublished data of the interleukin-1alpha (IL-1alpha) and interleukin-1 beta (IL-1beta) genes are reported here briefly. Results from others Brazilian studies with AD patients are also reported at this short review. Four local families studied with various markers at the chromosome 21, 19, 14, and 1 are briefly reported for the first time. The importance of studying DNA samples from Brazil is highlighted because of the uniqueness of its population, which presents both intense ethnical miscegenation, mainly at the east coast, but also clusters with high inbreeding rates in rural areas at the countryside. We discuss the current stage of extending these studies using high-throughput methods of large-scale genotyping, such as single nucleotide polymorphism microarrays, associated with bioinformatics tools that allow the analysis of such extensive number of genetics variables, with different levels of penetrance. There is still a long way between the huge amount of data gathered so far and the actual application toward the full understanding of AD, but the final goal is to develop precise tools for diagnosis and prognosis, creating new strategies for better treatments based on genetic profile.

The study of genetic polymorphisms related to serotonin in Alzheimer's disease: a new perspective in a heterogenic disorder

Genetic and environmental factors have been implicated in the development of Alzheimer's disease (AD), the most common form of dementia in the elderly. Mutations in 3 genes mapped on chromosomes 21, 14 and 1 are related to the rare early onset forms of AD while the e4 allele of the apolipoprotein E (APOE) gene (on chromosome 19) is the major susceptibility locus for the most common late onset AD (LOAD). Serotonin (5-hydroxytryptamine or 5-HT) is a key neurotransmitter implicated in the control of mood, sleep, appetite and a variety of traits and behaviors. Recently, a polymorphism in the transcriptional control region upstream of the 5-HT transporter (5-HTT) gene has been studied in several psychiatric diseases and personality traits. It has been demonstrated that the short variant(s) of this 5-HTT gene-linked polymorphic region (5-HTTLPR) is associated with a different transcriptional efficiency of the 5-HTT gene promoter resulting in decreased 5-HTT expression and 5-HT uptake in lymphocytes. An increased frequency of this 5-HTTLPR short variant polymorphism in LOAD was recently reported. In addition, another common polymorphic variation in the 5-HT2A and 5-HT2C serotonin receptor genes previously analyzed in schizophrenic patients was associated with auditory and visual hallucinations in AD. These observations suggest that the involvement of the serotonin pathway might provide an explanation for some aspects of the affective symptoms commonly observed in AD patients. In summary, research on genetic polymorphisms related to AD and involved in receptors, transporter proteins and the enzymatic machinery of serotonin might enhance our understanding of this devastating neurodegenerative disorder.