miRNAs as biomarkers of autism spectrum disorder: a systematic review and meta-analysis.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with complex clinical manifestations that arise between 18 and 36 months of age. Social interaction deficiencies, a restricted range of interests, and repetitive stereotyped behaviors are characteristics which are sometimes difficult to detect early. Several studies show that microRNAs (miRs/miRNAs) are strongly implicated in the development of the disorder and affect the expression of genes related to different neurological pathways involved in ASD. The present systematic review and meta-analysis addresses the current status of miRNA studies in different body fluids and the most frequently dysregulated miRNAs in patients with ASD. We used a combined approach to summarize miRNA fold changes in different studies using the mean values. In addition, we summarized p values for differential miRNA expression using the Fisher method. Our literature search yielded a total of 133 relevant articles, 27 of which were selected for qualitative analysis based on the inclusion and exclusion criteria, and 16 studies evaluating miRNAs whose data were completely reported were ultimately included in the meta-analysis. The most frequently dysregulated miRNAs across the analyzed studies were miR-451a, miR-144-3p, miR-23b, miR-106b, miR150-5p, miR320a, miR92a-2-5p, and miR486-3p. Among the most dysregulated miRNAs in individuals with ASD, miR-451a is the most relevant to clinical practice and is associated with impaired social interaction. Other miRNAs, including miR19a-3p, miR-494, miR-142-3p, miR-3687, and miR-27a-3p, are differentially expressed in various tissues and body fluids of patients with ASD. Therefore, all these miRNAs can be considered candidates for ASD biomarkers. Saliva may be the optimal biological fluid for miRNA measurements, because it is easy to collect from children compared to other biological fluids.

Neurogenesis of medium spiny neurons in the nucleus accumbens continues into adulthood and is enhanced by pathological pain.

In mammals, most adult neural stem cells (NSCs) are located in the ventricular-subventricular zone (V-SVZ) along the wall of the lateral ventricles and they are the source of olfactory bulb interneurons. Adult NSCs exhibit an apico-basal polarity; they harbor a short apical process and a long basal process, reminiscent of radial glia morphology. In the adult mouse brain, we detected extremely long radial glia-like fibers that originate from the anterior-ventral V-SVZ and that are directed to the ventral striatum. Interestingly, a fraction of adult V-SVZ-derived neuroblasts dispersed in close association with the radial glia-like fibers in the nucleus accumbens (NAc). Using several in vivo mouse models, we show that newborn neurons integrate into preexisting circuits in the NAc where they mature as medium spiny neurons (MSNs), i.e., a type of projection neurons formerly believed to be generated only during embryonic development. Moreover, we found that the number of newborn neurons in the NAc is dynamically regulated by persistent pain, suggesting that adult neurogenesis of MSNs is an experience-modulated process.

Maternal inflammation has a profound effect on cortical interneuron development in a stage and subtype-specific manner.

Severe infections during pregnancy are one of the major risk factors for cognitive impairment in the offspring. It has been suggested that maternal inflammation leads to dysfunction of cortical GABAergic interneurons that in turn underlies cognitive impairment of the affected offspring. However, the evidence comes largely from studies of adult or mature brains and how the impairment of inhibitory circuits arises upon maternal inflammation is unknown. Here we show that maternal inflammation affects multiple steps of cortical GABAergic interneuron development, i.e., proliferation of precursor cells, migration and positioning of neuroblasts, as well as neuronal maturation. Importantly, the development of distinct subtypes of cortical GABAergic interneurons was discretely impaired as a result of maternal inflammation. This translated into a reduction in cell numbers, redistribution across cortical regions and layers, and changes in morphology and cellular properties. Furthermore, selective vulnerability of GABAergic interneuron subtypes was associated with the stage of brain development. Thus, we propose that maternally derived insults have developmental stage-dependent effects, which contribute to the complex etiology of cognitive impairment in the affected offspring.

Dynamic Changes in Ultrastructure of the Primary Cilium in Migrating Neuroblasts in the Postnatal Brain.

New neurons, referred to as neuroblasts, are continuously generated in the ventricular-subventricular zone of the brain throughout an animal's life. These neuroblasts are characterized by their unique potential for proliferation, formation of chain-like cell aggregates, and long-distance and high-speed migration through the rostral migratory stream (RMS) toward the olfactory bulb (OB), where they decelerate and differentiate into mature interneurons. The dynamic changes of ultrastructural features in postnatal-born neuroblasts during migration are not yet fully understood. Here we report the presence of a primary cilium, and its ultrastructural morphology and spatiotemporal dynamics, in migrating neuroblasts in the postnatal RMS and OB. The primary cilium was observed in migrating neuroblasts in the postnatal RMS and OB in male and female mice and zebrafish, and a male rhesus monkey. Inhibition of intraflagellar transport molecules in migrating neuroblasts impaired their ciliogenesis and rostral migration toward the OB. Serial section transmission electron microscopy revealed that each migrating neuroblast possesses either a pair of centrioles or a basal body with an immature or mature primary cilium. Using immunohistochemistry, live imaging, and serial block-face scanning electron microscopy, we demonstrate that the localization and orientation of the primary cilium are altered depending on the mitotic state, saltatory migration, and deceleration of neuroblasts. Together, our results highlight a close mutual relationship between spatiotemporal regulation of the primary cilium and efficient chain migration of neuroblasts in the postnatal brain.SIGNIFICANCE STATEMENT Immature neurons (neuroblasts) generated in the postnatal brain have a mitotic potential and migrate in chain-like cell aggregates toward the olfactory bulb. Here we report that migrating neuroblasts possess a tiny cellular protrusion called a primary cilium. Immunohistochemical studies with zebrafish, mouse, and monkey brains suggest that the presence of the primary cilium in migrating neuroblasts is evolutionarily conserved. Ciliogenesis in migrating neuroblasts in the rostral migratory stream is suppressed during mitosis and promoted after cell cycle exit. Moreover, live imaging and 3D electron microscopy revealed that ciliary localization and orientation change during saltatory movement of neuroblasts. Our results reveal highly organized dynamics in maturation and positioning of the primary cilium during neuroblast migration that underlie saltatory movement of postnatal-born neuroblasts.

Tracking Sensory Characteristics of Virgin Olive Oils During Storage: Interpretation of Their Changes from a Multiparametric Perspective.

Virgin olive oil is inevitably subject to an oxidation process during storage that can affect its stability and quality due to off-flavors that develop before the oil surpasses its 'best before' date. Many parameters are involved in the oxidation process at moderate conditions. Therefore, a multiparametric study is necessary to establish a link between physico-chemical changes and sensory quality degradation in a real storage experiment. In this context, a storage experiment of 27 months was performed for four monovarietal virgin olive oils, bottled in transparent 500-mL PET bottles and subjected to conditions close to a supermarket scenario. Volatile composition, quality parameters and phenolic compounds were determined monthly. Simultaneously, an accredited sensory panel assessed their sensory characteristics. The stability of the fresh samples was also studied with the oxidative stability index (OSI) and mesh cell-FTIR. (E)-2-hexenal, (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol were identified as markers of the fruity attribute. Hexanal and nonanal were also identified as compounds that were associated with the rise of median of defect during storage. Some disagreements were observed between the sensory assessment and the OSI analyzed by Rancimat. However, the increase of concentration of rancid markers agreed with the increase of aldehyde band measured with mesh cell-FTIR.

In House Validated UHPLC Protocol for the Determination of the Total Hydroxytyrosol and Tyrosol Content in Virgin Olive Oil Fit for the Purpose of the Health Claim Introduced by the EC Regulation 432/2012 for "Olive Oil Polyphenols".

An ongoing challenge in olive oil analytics is the development of a reliable procedure that can draw the consensus of all interested parties regarding the quantification of concentrations above the required minimum value of 5 mg of bioactive "olive oil polyphenols" per 20 g of the oil, to fulfill the health claim introduced by the European Commission (EC) Regulation 432/2012. An in-house validated ultra-high performance liquid chromatography (UHPLC) protocol fit for this purpose is proposed. It relies on quantification of the total hydroxytyrsol (Htyr) and tyrosol (Tyr) content in the virgin olive oil (VOO) polar fraction (PF) before and after acidic hydrolysis of their bound forms. PF extraction and hydrolysis conditions were as previously reported. The chromatographic run lasts ~1/3 of the time needed under high performance liquid chromatography (HPLC) conditions, this was also examined. Eluent consumption for the same piece of information was 6-fold less. Apart from being cost effective, a larger number of samples can be analyzed daily with less environmental impact. Two external curves, detection at 280 nm and correction factors for molecular weight difference are proposed. The method, which is fit for purpose, is selective, robust with satisfactory precision (percentage relative standard deviation (%RSD) values < 11%) and recoveries higher than 87.6% for the target analytes (Htyr, Tyr). Standard operational procedures are easy to apply in the olive oil sector.

Toward a Harmonized and Standardized Protocol for the Determination of Total Hydroxytyrosol and Tyrosol Content in Virgin Olive Oil (VOO). The Pros of a Fit for the Purpose Ultra High Performance Liquid Chromatography (UHPLC) Procedure.

Τoward a harmonized and standardized procedure for the determination of total hydroxytyrosol and tyrosol content in virgin olive oil (VOO), the pros of a recently published in house validated ultra high performance liquid chromatography (UHPLC) protocol are discussed comparatively with those of other procedures that determine directly or indirectly the compounds hosted under the health claim on "olive oil polyphenols" (EC regulation 432/2012). Authentic VOOs were analyzed with five different liquid chromatographic separation protocols and 1H-NMR one in five different laboratories with expertise in VOO phenol analysis within three months. Data comparison indicated differences in absolute values. Method comparison using appropriate tools (Passing-Bablok regression and Bland Altman analyses) for all protocols vs. the UHPLC one indicated slight or statistically significant differences. The results were also discussed in terms of cost effectiveness, detection means, standard requirements and ways to calculate the total hydroxytyrosol and tyrosol content. Findings point out that the in-house validated fit for the purpose UHPLC protocol presents certain pros that should be exploited by the interested parties. These are the simplicity of sample preparation, fast elution time that increase the number of samples analyzed per day and integration of well-resolved peaks with the aid of only two commercially available external standards. Importance of correction factors in the calculations is stressed.

The Management of Irrigation and Potassium Fertilization to Mitigate the Effect of Light Frosts on the Phenolic and Volatile Compounds in Virgin Olive Oils.

The frequency of early frosts has increased in recent years, which are injurious to olive growing, causing losses in the yield and quality of virgin olive oil. In this research, it was studied how the management of agronomic factors mitigates frost damage in Arbequina olives, minimizing the loss of phenols and volatiles in virgin olive oil, at different fruit ripening stages. A Box-Behnken design and multivariate analysis were performed, with three levels of irrigation, potassium fertilization, and foliar copper application (15 treatments). Virgin olive oil was extracted from fresh and frozen olives. Light frost caused a significant decrease in the total phenols and secoiridoid compounds in and the antioxidant capacity of the frost-affected oils, which were perceived as more pungent and had the slight defect of "frostbitten olives". According to the Box-Behnken design, an 86% reference evapotranspiration (ET0) or higher with 100 potassium oxide units (UK2O) and a 100% ET0 or higher with 250 UK2O would be required to minimize the effect of light frost on phenols and volatiles. Partial Least Squares Regression-Discriminant Analysis (PLS-DA) differentiated the virgin olive oils according to their ripening stage and fresh and frost conditions. Moreover, PLS-DA positively correlated a 75-100% ET0 and 0 Uk2O with the dialdehydic form of the decarboxymethyl ligstroside aglycone (p-HPEA-EDA), the dialdehydic form of the decarboxymethyl oleuropein aglycone (3,4-DHPEA-EDA), the dialdehydic form of the ligstroside aglycone (p-HPEA-EDA-DLA), and with fruity, pungent, and bitter attributes. Precision agronomic management based on the needs of the crop itself would avoid unnecessary stress on olive trees and oil damage.

The cysteine-rich region and the whey acidic protein domain are essential for anosmin-1 biological functions.

The protein anosmin-1, coded by the KAL1 gene responsible for the X-linked form of Kallmann syndrome (KS), exerts its biological effects mainly through the interaction with and signal modulation of fibroblast growth factor receptor 1 (FGFR1). We have previously shown the interaction of the third fibronectin-like type 3 (FnIII) domain and the N-terminal region of anosmin-1 with FGFR1. Here, we demonstrate that missense mutations reported in patients with KS, C172R and N267K did not alter or substantially reduce, respectively, the binding to FGFR1. These substitutions annulled the chemoattraction of the full-length protein over subventricular zone (SVZ) neuronal precursors (NPs), but they did not annul it in the N-terminal-truncated protein (A1Nt). We also show that although not essential for binding to FGFR1, the cysteine-rich (CR) region is necessary for anosmin-1 function and that FnIII.3 cannot substitute for FnIII.1 function. Truncated proteins recapitulating nonsense mutations found in KS patients did not show the chemotropic effect on SVZ NPs, suggesting that the presence behind FnIII.1 of any part of anosmin-1 produces an unstable protein incapable of action. We also identify the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway as necessary for the chemotropic effect exerted by FGF2 and anosmin-1 on rat SVZ NPs.

Volatile and amino acid profiling of dry cured hams from different swine breeds and processing methods.

The flavor of dry cured ham explains the high appreciation of this product and it determines consumer acceptance. Volatile compounds provide valuable information about the odor and sensory quality of dry cured hams. Since amino acids are the origin of some volatile compounds of dry cured ham, the volatile and amino acid compositions of forty-one dry cured hams from Spain and France were determined to establish associations between them. The samples included different pig breeds (non Iberian vs. Iberian), which were additionally affected by different maturation times and feeding types (acorn vs. fodder). Results showed that 20 volatile compounds were able to distinguish Iberian and non Iberian hams, and 16 of those had relevant sensory impact according to their odor activity values. 3-Methylbutanol, 2-heptanol and hexanal were among the most concentrated volatile compounds. In the case of non-volatile compounds, the concentrations of amino acids were generally higher in Iberian hams, and all the amino acids were able to distinguish Iberian from non Iberian hams with the exception of tryptophan and asparagine. A strong correlation of some amino acids with volatile compounds was found in the particular case of alcohols and aldehydes when only Iberian hams were considered. The high correlation values found in some cases proved that proteolysis plays an important role in aroma generation.

Gradual Changes of the Protective Effect of Phenols in Virgin Olive Oils Subjected to Storage and Controlled Stress by Mesh Cell Incubation.

The oxidation reactions that take place in virgin olive oil under moderate conditions involved the combined effect of antioxidant and prooxidant compounds. Given the complexity of oxidation processes of multicomponent matrices, there is still a need to develop new methods with a dynamic approach to study the persistence of the compounds with healthy properties. This work studied the joint evolution of them, including phenols and pheophytin a, modeling their tendency during a real storage. The regression equations performed with the total phenol concentration showed that around 2% of the concentration was lost every month. Simultaneously, the progress of oxidation was evaluated by mesh cell incubation and Fourier transform infrared analysis. This method pointed out that, in the presence of light, the prooxidant effect of pigments was able to mask the protective effect of phenols, until the pheophytin a concentration was lower than 1 mg/kg. The antioxidant effect of phenols was less remarkable when the concentration loss was 35% or more.

Contribution of specific volatile markers to green and ripe fruity attributes in extra virgin olive oils studied with three analytical methods.

An objective sensory evaluation of extra virgin olive oil (EVOO), involving the chemical characterization of positive attributes, is of interest. These attributes are objectively divided, according to fruitiness, into "green" and "ripe" fruity. This work studied the differentiation in the volatile profile of EVOOs into these two classes, obtained by three analytical methods, including different extraction techniques and detectors and two data processing strategies, and their relation with sensory results. According to the results, each method allowed the characterization of the two classes, providing information on different volatile compounds, which increased in number through PARADISe software (14 more than the conventional processing). Moreover, some volatile compounds showed significant differences between the two classes, 16 highlighted by the variables with importance in projection (VIP) for green fruity (e.g. (Z)-3-hexen-1-ol, methyl ether) and 23 for ripe fruity EVOOs (e.g. (Z)-2-hexen-1-ol), which could be considered as useful markers to complement quality assessment.

Effect of Controlled Oxygen Supply during Crushing on Volatile and Phenol Compounds and Sensory Characteristics in Coratina and Ogliarola Virgin Olive Oils.

In virgin olive oil industries, the technological choices of the production plant affect the biochemical activities that take place in the olives being processed throughout the entire process, thereby affecting the quality of the final product. The lipoxygenase pool enzymes that operated their activity during the first phases of the process need the best conditions to work, especially concerning temperature and oxygen availability. In this study, a system was equipped to supply oxygen in the crusher at a controllable concentration in an industrial olive oil mill at pilot plant scale, and four oxygen concentrations and two cultivars, Coratina and Ogliarola, were tested. The best concentration for oxygen supply was 0.2 L/min at the working capacity of 0.64 Ton/h. Further, using this addition of oxygen, it was possible to increase the compound's concentration, which is responsible for the green, fruity aroma. The effect on volatile compounds was also confirmed by the sensory analyses. However, at the same time, it was possible to maintain the concentration of phenols in a good quality olive oil while also preserving all the antioxidant properties of the product due to the presence of phenols. This study corroborates the importance of controlling oxygen supply in the first step of the process for process management and quality improvement in virgin olive oil production.

Comparing the potential of IR-spectroscopic techniques to gas chromatography coupled to ion mobility spectrometry for classifying virgin olive oil categories.

Virgin olive oil (OO) can be classified into three different categories: extra virgin, virgin and lampante. The official method for this classification, based on physicochemical analysis and sensory tasting, is considered useful and effective, although it is a costly and time-consuming process. The aim of this study was to assess the potential of some analytical techniques for classifying and predicting different OO categories to support official methods and to provide olive oil companies with a rapid tool to assess product quality. Thus, mid and near infrared spectroscopies (MIR and NIR) have been compared by using different instruments and with head-space gas chromatography coupled to an ion mobility spectrometer (HS-GC-IMS). High classification success rates in validation models were obtained using IR spectrometers (>70% and > 80% in average for ternary and binary classifications, respectively), although HS-GC-IMS showed greater classification potential (>85% and > 90%).

Extracellular matrix protein anosmin-1 overexpression alters dopaminergic phenotype in the CNS and the PNS with no pathogenic consequences in a MPTP model of Parkinson's disease.

The development and survival of dopaminergic neurons are influenced by the fibroblast growth factor (FGF) pathway. Anosmin-1 (A1) is an extracellular matrix protein that acts as a major regulator of this signaling pathway, controlling FGF diffusion, and receptor interaction and shuttling. In particular, previous work showed that A1 overexpression results in more dopaminergic neurons in the olfactory bulb. Prompted by those intriguing results, in this study, we investigated the effects of A1 overexpression on different populations of catecholaminergic neurons in the central (CNS) and the peripheral nervous systems (PNS). We found that A1 overexpression increases the number of dopaminergic substantia nigra pars compacta (SNpc) neurons and alters the striosome/matrix organization of the striatum. Interestingly, these numerical and morphological changes in the nigrostriatal pathway of A1-mice did not confer an altered susceptibility to experimental MPTP-parkinsonism with respect to wild-type controls. Moreover, the study of the effects of A1 overexpression was extended to different dopaminergic tissues associated with the PNS, detecting a significant reduction in the number of dopaminergic chemosensitive carotid body glomus cells in A1-mice. Overall, our work shows that A1 regulates the development and survival of dopaminergic neurons in different nuclei of the mammalian nervous system.

Method for the analysis of volatile compounds in virgin olive oil by SPME-GC-MS or SPME-GC-FID.

During the course of the EU H2020 OLEUM project, a harmonized method was developed to quantify volatile markers of the aroma of virgin olive oil with the aim to support the work of sensory panel test to assess the quality grade. A peer validation of this method has been carried out, with good results in terms of analytical quality parameters. The method allows the quantification of volatile compounds by SPME-GC with two possible detectors, flame ionization detector and mass spectrometry, depending on the technical facilities of the labs applying this method. The method was optimized for the quantification of 18 volatile compounds that were selected as being markers responsible for positive attributes (e.g. fruity) and sensory defects (e.g. rancid and winey-vinegary). The quantification is carried out with calibration curves corrected by the internal standards. Additionally, a protocol is provided to prepare the calibration samples. This procedure enhances reproducibility between labs since one of the main sources of errors is the application of different procedures in calibration.

An International Survey on Olive Oils Quality and Traceability: Opinions from the Involved Actors.

A survey was launched to understand the current problems and sensitivities of the olive oil market through a series of questions clustered around topics related to quality, traceability, regulation, standard methods and other issues. The questions were selected after a series of interviews with different actors to identify those aspects where some disagreement or different points of view may exist. These questions were grouped in topics such as geographical traceability, consumer perception and quality management. The survey was addressed to eight different olive oil actors independently: producers, retailers, importers, exporters, analysts, workers at regulatory bodies, and consumers. Approximately half of the respondents (67.0% for consumers and 56.0% for the rest of olive oil actors) claimed to understand the importance of the protected designation of origin. In fact, the traceability objectives that were selected as the most relevant were those related with geographical traceability (19.3%) followed by the detection of adulteration (15.6%). Most of the respondents (80%) would agree to share data for a common database; however, some concerns exist about the use of these data and the issue of paying to have access to this database. The respondents mostly expressed an affirmative answer concerning the efficiency of panel test (74%) and a negative answer (90%) concerning the proposal of removing from regulation, although 42% agree with their revision for improvement. The opinions on "best before" date and their relationship with quality and the willingness to apply non-targeted methods were also surveyed.

The Impact of Mild Frost Occurring at Different Harvesting Times on the Volatile and Phenolic Composition of Virgin Olive Oil.

The organoleptic characteristics of virgin olive oil (VOO), together with its nutritional and health properties, have led the olive tree to be cultivated beyond the Mediterranean basin, reaching latitudes with colder climates, with minimum temperatures below 0 °C and with a higher probability of early frosts. The freezing of olives generates destruction within the tissues and promotes degradation reactions. In this study, the impact of mild frost occurring at different harvesting times on the composition of volatiles and phenolic compounds in VOO were investigated. Arbequina variety olives were harvested at different stages of ripening. Half of the olives were subjected to oil extraction and the other half were frozen at -3 ± 1 °C for 12 h prior to oil extraction. A significant decrease of phenolic compounds with harvesting time was observed in both types of oils (fresh and frozen olives). Oils from frozen olives presented a slightly higher content of total phenols, except in the advanced ripening stage (September), and a slightly lower content of volatile compounds at all harvesting times. In addition, a higher content of 3,4-DHPEA-EDA was observed in oils from frozen olives, which is attributed to an early action of the endogenous ß-glucosidase enzyme on oleuropein in freeze-damaged olive fruits. Principal component analysis and Discriminant Partial Least Square Regression allowed the oils to be classified according to the type of fruit (fresh and frozen) and the month of harvest. This study would indicate that mild frost would have a low impact on the chemical composition of virgin olive oil, although, this depends on the ripening stage.

Upper cortical layer-driven network impairment in schizophrenia.

Schizophrenia is one of the most widespread and complex mental disorders. To characterize the impact of schizophrenia, we performed single-nucleus RNA sequencing (snRNA-seq) of >220,000 neurons from the dorsolateral prefrontal cortex of patients with schizophrenia and matched controls. In addition, >115,000 neurons were analyzed topographically by immunohistochemistry. Compositional analysis of snRNA-seq data revealed a reduction in abundance of GABAergic neurons and a concomitant increase in principal neurons, most pronounced for upper cortical layer subtypes, which was substantiated by histological analysis. Many neuronal subtypes showed extensive transcriptomic changes, the most marked in upper-layer GABAergic neurons, including down-regulation in energy metabolism and up-regulation in neurotransmission. Transcription factor network analysis demonstrated a developmental origin of transcriptomic changes. Last, Visium spatial transcriptomics further corroborated upper-layer neuron vulnerability in schizophrenia. Overall, our results point toward general network impairment within upper cortical layers as a core substrate associated with schizophrenia symptomatology.