Vesicular Glutamate Release from Feeder-FreehiPSC-Derived Neurons

Human-induced pluripotent stem cells (hiPSCs) represent one of the main and powerful tools for the in vitro modeling of neurological diseases. Standard hiPSC-based protocols make use of animal-derived feeder systems to better support the neuronal differentiation process. Despite their efficiency, such protocols may not be appropriate to dissect neuronal specific properties or to avoid interspecies contaminations, hindering their future translation into clinical and drug discovery approaches. In this work, we focused on the optimization of a reproducible protocol in feeder-free conditions able to generate functional glutamatergic neurons. This protocol is based on a generation of neuroprecursor cells differentiated into human neurons with the administration in the culture medium of specific neurotrophins in a Geltrex-coated substrate. We confirmed the efficiency of this protocol through molecular analysis (upregulation of neuronal markers and neurotransmitter receptors assessed by gene expression profiling and expression of the neuronal markers at the protein level), morphological analysis, and immunfluorescence detection of pre-synaptic and post-synaptic markers at synaptic boutons. The hiPSC-derived neurons acquired Ca²⁺-dependent glutamate release properties as a hallmark of neuronal maturation. In conclusion, our study describes a new methodological approach to achieve feeder-free neuronal differentiation from hiPSC and adds a new tool for functional characterization of hiPSC-derived neurons.     

The Controversial Role of LPS in Platelet Activation In Vitro

Circulating platelets are responsible for hemostasis and thrombosis but are also primary sensors of pathogens and are involved in innate immunity, inflammation, and sepsis. Sepsis is commonly caused by an exaggerated immune response to bacterial, viral, and fungal infections, and leads to severe thrombotic complications. Among others, the endotoxin lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria is the most common trigger of sepsis. Since the discovery of the expression of the LPS receptor TLR4 in platelets, several studies have investigated the ability of LPS to induce platelet activation and to contribute to a prothrombotic phenotype, per se or in combination with plasma proteins and platelet agonists. This issue, however, is still controversial, as different sources, purity, and concentrations of LPS, different platelet-purification protocols, and different methods of analysis have been used in the past two decades, giving contradictory results. This review summarizes and critically analyzes past and recent publications about LPS-induced platelet activation in vitro. A methodological section illustrates the principal platelet preparation protocols and significant differences. The ability of various sources of LPS to elicit platelet activation in terms of aggregation, granule secretion, cytokine release, ROS production, and interaction with leukocytes and NET formation is discussed.     

Role of Biliverdin Reductase A in the Regulation of Insulin Signaling in Metabolic and Neurodegenerative Diseases: An Update

Insulin signaling is a conserved pathway that orchestrates glucose and lipid metabolism, energy balance, and inflammation, and its dysregulation compromises the homeostasis of multiple systems. Insulin resistance is a shared hallmark of several metabolic diseases, including obesity, metabolic syndrome, and type 2 diabetes, and has been associated with cognitive decline during aging and dementia. Numerous mechanisms promoting the development of peripheral and central insulin resistance have been described, although most of them were not completely clarified. In the last decades, several studies have highlighted that biliverdin reductase-A (BVR-A), over its canonical role in the degradation of heme, acts as a regulator of insulin signaling. Evidence from human and animal studies show that BVR-A alterations are associated with the aberrant activation of insulin signaling, metabolic syndrome, liver steatosis, and visceral adipose tissue inflammation in obese and diabetic individuals. In addition, recent findings demonstrated that reduced BVR-A levels or impaired BVR-A activation contribute to the development of brain insulin resistance and metabolic alterations in Alzheimer’s disease. In this narrative review, we will provide an overview on the literature by focusing on the role of BVR-A in the regulation of insulin signaling and how BVR-A alterations impact on cell dysfunctions in both metabolic and neurodegenerative disorders.     

Expanding the Molecular Spectrum of ANKRD11 Gene Defects in 33 Patients with a Clinical Presentation of KBG Syndrome

KBG syndrome (KBGS) is a neurodevelopmental disorder caused by the Ankyrin Repeat Domain 11 (ANKRD11) haploinsufficiency. Here, we report the molecular investigations performed on a cohort of 33 individuals with KBGS clinical suspicion. By using a multi-testing genomic approach, including gene sequencing, Chromosome Microarray Analysis (CMA), and RT-qPCR gene expression assay, we searched for pathogenic alterations in ANKRD11. A molecular diagnosis was obtained in 22 out of 33 patients (67%). ANKRD11 sequencing disclosed pathogenic or likely pathogenic variants in 18 out of 33 patients. CMA identified one full and one terminal ANKRD11 pathogenic deletions, and one partial duplication and one intronic microdeletion, with both possibly being pathogenic. The pathogenic effect was established by RT-qPCR, which confirmed ANKRD11 haploinsufficiency only for the three deletions. Moreover, RT-qPCR applied to six molecularly unsolved KBGS patients identified gene downregulation in a clinically typical patient with previous negative tests, and further molecular investigations revealed a cryptic deletion involving the gene promoter. In conclusion, ANKRD11 pathogenic variants could also involve the regulatory regions of the gene. Moreover, the application of a multi-test approach along with the innovative use of RT-qPCR improved the diagnostic yield in KBGS suspected patients.     

Total and Plant Protein Consumption: The Role of Inflammation and Risk of Non-Communicable Disease

Background: Inflammatory cytokine levels are associated with Non-Communicable Diseases (NCDs) and can be influenced by a person’s macronutrient profile. This work aims to evaluate the relationship between the compliance with the age-specific recommended protein intake and the levels of inflammatory markers related to the risk of NCDs. Methods: The study participants included 347 participants (119 men and 228 women), ages 18 to 86 years. Cardio-metabolic risk evaluations, including an assessment of the prevalence of Metabolic Syndrome, were performed. Leptin, IL-15, IL-6, and TNF-α levels were measured. Results: The adequacy of the total protein (TP) intake was lower in old people compared to individuals aged <60 years, and only few volunteers consumed the suggested 50% plant protein (PP) for a healthy and sustainable diet. A lower risk of NCDs with a PP consumption above at least 40% was observed only in old individuals. A differential effect on TNF-α and IL-6 was observed for both TP and PP intake by gender and age class, whereas for leptin and IL-15 only significant interactions among sex and the class of age were found. Conclusion: Although our data suggest that consuming more than 40% of PP can reduce the risk of NCDs, the effect of gender differences on cytokine levels should be considered in larger studies.     

Comparison of different soil organic matter fractionation methodologies: Evidences from ultrasensitive 14C measurements

Soils are studied with the aim to predict future climatic scenarios and find the best guidelines to manage terrestrial ecosystems for the mitigation of the atmospheric CO₂ rising. Carbon constituting soil organic matter (SOM) behaves as a cohort of different pools, characterized by a specific C turnover time. Both natural and anthropogenic occurring ¹⁴C reach the soil through plant littering, becoming a valid tool to trace SOM dynamics.In this study we present a series of Accelerator Mass Spectrometry (AMS) ¹⁴C measurements on SOM samples obtained by means of different laboratory protocols used for the isolation of soil pools from bulk soil (fractionation protocols). Radiocarbon signature of SOM fractions is used as a keyhole to look at the more effective fractionation procedure and comparison among measured ¹⁴C on SOM fractions revealed important indications for the proposal of a novel fractionation protocol. Our data put in evidence how particle size controls the recalcitrance of ancient SOM carbon pools.     

Adaptive systems in the era of the semantic and social web, a survey

In this paper we provide a classification of adaptive systems with respect to the kind of semantic technology they exploit to accomplish or improve specific adaptation and user modeling tasks. This classification is based on a distinction between strong semantic techniques and weak semantic techniques. The former are techniques based on the Semantic Web, while the latter regard technologies that, in different ways, annotate resources, enriching their meaning. This second category includes, in particular, Web 2.0 social annotations and mixed approaches between social annotations and Semantic Web techniques. While the impact of the Semantic Web on adaptive systems has been discussed in several survey papers, the potential of weak semantic technologies has, so far, received little attention. The aim of this analysis is to fill this gap. Therefore, we will discuss contributions and limits of both approaches, but we will focus special attention on weak semantic adaptive systems.     

Design of a holonic remote monitoring and diagnosis system for fleet management

The main challenges in sensor network based monitoring systems are the control and management of complex modules and elements interacting among them and with the environment. This work presents and validates a design model for monitoring and diagnosis systems based on sensor networks exploiting potentialities of the holonic approach. The holonic structure is designed with the final goal to guarantee the performances and the functionalities required by the application scenario of road and work vehicles rental companies. Actually they are interacting and geographically distributed systems. The capabilities of cooperation among modules, hierarchical organization, modularity and reliability of holonic design are exploited. Diagnosis of monitored events is coded and validated through an ad hoc ontology. The design does not give assumption on the physical architecture of holons but only on their capacities/properties, therefore it can be reused in different physical and hardware resources. The case studies confirm the capability of our design to be quickly transferred to programmers and coded in firmware and software.     

Frying performance of a sunflower/palm oil blend in comparison with pure palm oil

The aim of this study was to test the performance of a vegetable oil blend formulated as alternative to pure palm oil as frying medium. For this purpose, the evolution of many analytical parameters (free acidity, spectrophotometric indices, total polar components, fatty acid composition, short‐chain fatty acids, tocopherol and tocotrienol content and composition, color, flavor evaluated by means of an electronic nose) of the selected blend (sunflower/palm oil 65 : 35 vol/vol) has been monitored during a prolonged frying process (8 h discontinuous frying without oil replenishment) in comparison to pure palm oil. Sensory attributes of the fried food were also evaluated. The blend proved to keep qualitative parameters comparable to those shown by palm oil during the prolonged frying process. Even if some oxidation indices, such as spectrophotometric indices, short‐chain fatty acids and total polar components, increased faster in the blend, it showed a higher tocopherol content and a lower increment in free fatty acids as compared to pure palm oil. Chips fried in the two oils did not show significantly different sensory profiles.