Amygdalar neurotransmission alterations in the BTBR mice model of idiopathic autism.

Autism Spectrum Disorders (ASD) are principally diagnosed by three core behavioural symptoms, such as stereotyped repertoire, communication impairments and social dysfunctions. This complex pathology has been linked to abnormalities of corticostriatal and limbic circuits. Despite experimental efforts in elucidating the molecular mechanisms behind these abnormalities, a clear etiopathogenic hypothesis is still lacking. To this aim, preclinical studies can be really helpful to longitudinally study behavioural alterations resembling human symptoms and to investigate the underlying neurobiological correlates. In this regard, the BTBR T+ Itpr3tf/J (BTBR) mice are an inbred mouse strain that exhibits a pattern of behaviours well resembling human ASD-like behavioural features. In this study, the BTBR mice model was used to investigate neurochemical and biomolecular alterations, regarding Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF), together with GABAergic, glutamatergic, cholinergic, dopaminergic and noradrenergic neurotransmissions and their metabolites in four different brain areas, i.e. prefrontal cortex, hippocampus, amygdala and hypothalamus. In our results, BTBR strain reported decreased noradrenaline, acetylcholine and GABA levels in prefrontal cortex, while hippocampal measurements showed reduced NGF and BDNF expression levels, together with GABA levels. Concerning hypothalamus, no differences were retrieved. As regarding amygdala, we found reduced dopamine levels, accompanied by increased dopamine metabolites in BTBR mice, together with decreased acetylcholine, NGF and GABA levels and enhanced glutamate content. Taken together, our data showed that the BTBR ASD model, beyond its face validity, is a useful tool to untangle neurotransmission alterations that could be underpinned to the heterogeneous ASD-like behaviours, highlighting the crucial role played by amygdala.

Differential gene expression analysis pipelines and bioinformatic tools for the identification of specific biomarkers: A review.

In recent years, the role of bioinformatics and computational biology together with omics techniques and transcriptomics has gained tremendous importance in biomedicine and healthcare, particularly for the identification of biomarkers for precision medicine and drug discovery. Differential gene expression (DGE) analysis is one of the most used techniques for RNA-sequencing (RNA-seq) data analysis. This tool, which is typically used in various RNA-seq data processing applications, allows the identification of differentially expressed genes across two or more sample sets. Functional enrichment analyses can then be performed to annotate and contextualize the resulting gene lists. These studies provide valuable information about disease-causing biological processes and can help in identifying molecular targets for novel therapies. This review focuses on differential gene expression (DGE) analysis pipelines and bioinformatic techniques commonly used to identify specific biomarkers and discuss the advantages and disadvantages of these techniques.

Increased stress vulnerability in the offspring of socially isolated rats: Behavioural, neurochemical and redox dysfunctions.

Stressful events during pregnancy impact on the progeny neurodevelopment. However, little is known about preconceptional stress effects. The rat social isolation represents an animal model of chronic stress inducing a variety of dysfunctions. Moreover, social deprivation during adolescence interferes with key neurodevelopmental processes. Here, we investigated the development of behavioural, neurochemical and redox alterations in the male offspring of socially isolated female rats before pregnancy, reared in group (GRP) or in social isolation (ISO) from weaning until young-adulthood. To this aim, females were reared in GRP or in ISO conditions, from PND21 to PND70, when they were mated. Their male offspring was housed in GRP or ISO conditions through adolescence and until PND70, when passive avoidance-PA, novel object recognition-NOR and open field-OF tests were performed. Levels of noradrenaline (NA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), glutamate (GLU) and GABA were assessed in the prefrontal cortex (PFC). Moreover, cortical ROS levels were quantified, as well as NF-kB and the NADPH oxidase NOX2 expression, redox status (expressed as GSH:GSSG ratio) and SOD1 amount. A significant decrease of the latency time in the PA was observed in the offspring of ISO females. In the NOR test, while a significant increase in the exploratory activity towards the novel object was observed in the offspring of GRP females, no significant differences were found in the offspring of ISO females. No significant differences were found in the OF test among experimental groups. Theoffspring of ISO females showed increased NA and 5-HIAA levels, whereas in the offspring persistently housed in isolation condition from weaninguntil adulthood, we detected reduced 5-HT levels and ehnanced 5-HIAA amount. No significant changes in GLU concentrations were detected, while decreased GABA content was observed in the offspring of ISO females exposed to social isolation. Increased ROS levels as well as reduced NF-κB, NOX2 expression were detected in the offspring of ISO females. This was accompanied by reduced redox status and enhanced SOD1 levels. In conclusion, our results suggest that female exposure to chronic social stress before pregnancy might have a profound influence on the offspring neurodevelopment in terms of cognitive, neurochemical and redox-related alterations, identifying this specific time window for possible preventive and therapeutic strategies.

Case report: PIK3CA somatic mutation leading to Klippel Trenaunay Syndrome and multiple tumors.

We report a case of Klippel Trenaunay Syndrome that was monitored both clinically and molecularly over a period of 9 years. A somatic mosaic mutation of PIK3CA (p(E545G)) was identified using both cfDNA NGS liquid biopsy and tissue biopsy. At the age of 56, due to intervening clonal mutations in PIK3CA background, she developed a squamous cell carcinoma in the right affected leg which was treated surgically. Nine years later, lung bilateral adenocarcinoma arose on PIK3CA mutated tissues supported by different clonal mutations. One year later, the patient died from metastases led by a new FGFR3 clone unresponsive to standard-of-care, immunotherapy-based. Our results highlight the presence of a molecular hallmark underlying neoplastic transformation that occurs upon an angiodysplastic process and support the view that PIK3CA mutated tissues must be treated as precancerous lesions. Importantly, they remark the effectiveness of combining cfDNA NGS liquid and tissue biopsies to monitor disease evolution as well as to identify aggressive clones targetable by tailored therapy, which is more efficient than conventional protocols.

Different Liquid Biopsies for the Management of Non-Small Cell Lung Cancer in the Mutational Oncology Era.

In the last ten years, liquid biopsy has been slowly joining the traditional invasive techniques for the diagnosis and monitoring of tumors. Liquid biopsies allow easy repeated sampling of blood, reflect the tumor scenario, and make personalized therapy real for the patient. Liquid biopsies isolate and utilize different substrates present in patients' body fluids such as circulating tumor cells, circulating tumor DNA, tumor extracellular vesicles, etc. One of the most-used solid cancers in the development of the non-invasive liquid biopsy approach that has benefited from scientific advances is non-small cell lung cancer (NSCLC). Using liquid biopsy, it is possible to have more details on NSCLC staging, progression, heterogeneity, gene mutations and clonal evolution, etc., basing the treatment on precision medicine as well as on the screening of markers for therapeutic resistance. With this review, the authors propose a complete and current overview of all different liquid biopsies available to date, to understand how much has been carried out and how much remains to be completed for a better characterization of NSCLC.

Diagnostic accuracy of circulating free DNA testing for the detection of KRAS mutations in non-small cell lung cancer: A systematic review and meta-analysis.

Kirsten rat sarcoma viral oncogene homolog (KRAS) gene encodes a GTPase that acts as a molecular switch for intracellular signal transduction, promoting cell growth and proliferation. Mutations in the KRAS gene represent important biomarkers for NSCLC targeted therapy. However, detection of KRAS mutations in tissues has shown some limitations. During the last years, analyses of circulating free DNA (cfDNA) has emerged as an alternative and minimally invasive, approach to investigate tumor molecular changes. Here, we assessed the diagnostic performance of cfDNA analysis, compared to tissues through a meta-analysis and systematic review of existing literature. From 561 candidate papers, we finally identified 40 studies, including 2,805 NSCLC patients. We extracted values relating to the number of true-positive, false-positive, false-negative, and true-negative. Pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio, each with 95% CI, were calculated. A summary receiver operating characteristic curve and the area under curve (AUC) were used to evaluate the overall diagnostic performance. The pooled sensitivity was 0.71 (95% CI 0.68-0.74) and the specificity was 0.93 (95% CI 0.92-0.94). The diagnostic odds ratio was 35.24 (95% CI 24.88-49.91) and the area under the curve was 0.92 (SE = 0.094). These results provide evidence that detection of KRAS mutation using cfDNA testing is of adequate diagnostic accuracy thus offering to the clinicians a new promising screening test for NSCLC patients.

Galdieria sulphuraria ACUF427 Freeze-Dried Biomass as Novel Biosorbent for Rare Earth Elements.

Rare earth elements (REEs) are essential components of modern technologies and are often challenging to acquire from natural resources. The demand for REEs is so high that there is a clear need to develop efficient and environmentally-friendly recycling methods. In the present study, freeze-dried cells of the extremophile Galdieria sulphuraria were employed to recover yttrium, cerium, europium, and terbium from quaternary-metal aqueous solutions. The biosorption capacity of G. sulphuraria freeze-dried algal biomass was tested at different pHs, contact times, and biosorbent dosages. All rare earths were biosorbed in a more efficient way by the lowest dose of biosorbent, at pH 4.5, within 30 min; the highest removal rate of cerium was recorded at acidic pH (2.5) and after a longer contact time, i.e., 360 min. This study confirms the potential of freeze-dried cells of G. sulphuraria as innovative ecological biosorbents in technological applications for sustainable recycling of metals from e-waste and wastewater.

In Vitro Model for the Evaluation of Innovative Transcatheter Debridement Device (TDD): Pericardium-Based Scaffold and Stem Cells to Reproduce Calcificated Valves.

Aortic valve stenosis has become the most common valvular disease in elderly patients. Several treatments are available such as surgical aortic valve replacement and transcatheter aortic valve implantation. To date, however, there is a need to discover alternative treatments that can delay the disease progression and, therefore, the implant of a prosthetic valve. In this regard, a decalcification procedure based on the use of ultrasonic waves could represent an innovative solution in transcatheter cardiovascular therapies. In this article, we describe an innovative transcatheter debridement device (TDD) that uses low-intensity ultrasound shock waves for calcium ablation from the native aortic valve and bioprosthetic valve. Mesenchymal stem cells were seeded onto pericardium-based scaffolds and committed into an osteogenic phenotype. After treatment with TDD, cell proliferation was analyzed, as well as lactate dehydrogenase release and cell morphology. The release of calcium and inflammation events were detected. The results confirmed that the TDD was able to induce a safe decalcification without any adverse inflammatory events.

Nosological and Theranostic Approach to Vascular Malformation through cfDNA NGS Liquid Biopsy.

Several different nosological classifications have been used over time for vascular malformations (VMs) since clinical and pathological signs are largely overlapping. In a large proportion of cases, VMs are generated by somatic mosaicism in key genes, belonging to a few different molecular pathways. Therefore, molecular characterization may help in the understanding of the biological mechanisms related to the development of pathology. Tissue biopsy is not routinely included in the diagnostic path because of the need for fresh tissue specimens and the risk of bleeding. Bypassing the need for bioptic samples, we took advantage of the possibility of isolating cell-free DNA likely released by the affected tissues, to molecularly characterize 53 patients by cfDNA-NGS liquid biopsy. We found a good match between the identified variant and the clinical presentation. PIK3CA variants were found in 67% of Klippel Trenaunay Syndrome individuals; KRAS variants in 60% of arteriovenous malformations; MET was mutated in 75% of lymphovenous malformations. Our results demonstrate the power of cfDNA-NGS liquid biopsy in VMs clinical classification, diagnosis, and treatment. Indeed, tailored repurposing of pre-existing cancer drugs, such as PIK3CA, KRAS, and MET inhibitors, can be envisaged as adjuvant treatment, in addition to surgery and/or endovascular treatment, in the above-defined VMs categories, respectively.

Bioremoval of Yttrium (III), Cerium (III), Europium (III), and Terbium (III) from Single and Quaternary Aqueous Solutions Using the Extremophile Galdieria sulphuraria (Galdieriaceae, Rhodophyta).

The lanthanides are among the rare earth elements (REEs), which are indispensable constituents of modern technologies and are often challenging to acquire from natural resources. The demand for REEs is so high that there is a clear need to develop efficient and environmentally-friendly recycling methods. In the present study, living cells of the extremophile Galdieria sulphuraria were used to remove four REEs, Yttrium, Cerium, Europium, and Terbium, from single- and quaternary-metal aqueous solutions. Two different strains, SAG 107.79 and ACUF 427, were exposed to solutions buffered at pH 2.5, 3.5, 4.5, and 5.5. Our data demonstrated that the removal performances were strain and pH dependent for all metal ions. At lower pH, ACUF 427 outperformed SAG 107.79 considerably. By increasing the pH of the solutions, there was a significant surge in the aqueous removal performance of both strains. The same trend was highlighted using quaternary-metal solutions, even if the quantities of metal removed were significantly lower. The present study provided the first insight into the comparative removal capacity of the Galdieria sulphuraria strains. The choice of the appropriate operational conditions such as the pH of the metal solutions is an essential step in developing efficient, rapid, and straightforward biological methods for recycling REEs.

Lithotripsy of Calcified Aortic Valve Leaflets by a Novel Ultrasound Transcatheter-Based Device.

The increasing incidence of calcific aortic valve disease necessitates the elaboration of new strategies to retard the progression of the pathology with an innovative solution. While the increasing diffusion of the transcatheter aortic valve replacements (TAVRs) allows a mini-invasive approach to aortic valve substitution as an alternative to conventional surgical replacement (SAVR) in an always larger patient population, TAVR implantation still has contraindications for young patients. In addition, it is liable to undergo calcification with the consequent necessity of re-intervention with conventional valve surgery or repeated implantation in the so-called TAVR-in-TAVR procedure. Inspired by applications for non-cardiac pathologies or for vascular decalcification before stenting (i.e., coronary lithotripsy), in the present study, we show the feasibility of human valve treatment with a mini-invasive device tailored to deliver shockwaves to the calcific leaflets. We provide evidence of efficient calcium deposit ruptures in human calcified leaflets treated ex vivo and the safety of the treatment in pigs. The use of this device could be helpful to perform shockwaves valvuloplasty as an option to retard TAVR/SAVR, or as a pretreatment to facilitate prosthesis implantation and minimize the occurrence of paravalvular leak.

1,25-dihydroxyvitamin D as Predictor of Renal Worsening Function in Chronic Kidney Disease. Results From the PASCaL-1,25D Study.

Background: Heterogeneous progression of chronic kidney disease (CKD) toward dialysis advocates improving in renal care management. Diagnosis and staging of CKD relies on estimated glomerular filtration rate (eGFR) and albuminuria. Tubular biomarkers emerged as new predictors of worsening renal function (WRF), due to partial inaccuracy of eGFR and existing WRF in non-proteinuric patients. Active vitamin D is synthesized in renal tubules and participates to mineral adaptation in CKD. Circulating 1,25-dihydroxyvitamin D [1,25(OH)2D] was poorly investigated as a biomarker of endocrine tubular function and predictor of WRF. Objective: Investigate capability of 1,25(OH)2D to predict parathormone (PTH) increase and WRF in CKD stage 3-4. Methods: PASCaL-1,25D was an observational, prospective, monocentric study. Primary outcomes were absolute and 20% increase in PTH, and WRF defined as 20% reduction in eGFR or dialysis initiation at 6 months. Results: Seventy-one patients completed follow up. Absolute increase in PTH (1-84) was independently predicted by lower 1,25(OH)2D levels (p = 0.0134). No association was detected between 1,25(OH)2D and iPTH increase. Higher 1,25(OH)2D was associated with reduced risk of WRF at univariate analysis [OR 0.89 (95% CI 0.86-0.93), p = 0.006]. The 1,25(OH)2D/PTH (1-84) ratio was associated with non-significant 84% risk reduction for WRF [OR 0.16 (95% CI 0.06-0.41), p = 0.05]. Low 1,25(OH)2D reached 100% sensitivity in predicting WRF in CKD stage 3 (AUC 9.909, p < 0.0001) and non-elderly patients (AUC 0.883, p < 0.0001). Machine learning models retained 1,25(OH)2D/PTH (1-84) as relevant predictor of WRF together with eGFR and albuminuria. Age influenced interaction between renal and mineral biomarkers. Conclusion: 1,25(OH)2D deserves attention as biomarker of tubular health, and sensible predictor of WRF on the short run among non-elderly patients affected by stage 3 CKD. The 1,25(OH)2D/PTH (1-84) ratio may represent a composite biomarker of tubular reserve/endocrine response to the transition from adaptive to maladaptive equilibrium in CKD-MBD.

Rare variants in Toll-like receptor 7 results in functional impairment and downregulation of cytokine-mediated signaling in COVID-19 patients.

Toll-like receptors (TLR) are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the production of pro-inflammatory cytokines and type I and II interferons, which are responsible for innate antiviral responses. Among the different TLRs, TLR7 recognizes several single-stranded RNA viruses including SARS-CoV-2. We and others identified rare loss-of-function variants in X-chromosomal TLR7 in young men with severe COVID-19 and with no prior history of major chronic diseases, that were associated with impaired TLR7 signaling as well as type I and II IFN responses. Here, we performed RNA sequencing to investigate transcriptome variations following imiquimod stimulation of peripheral blood mononuclear cells isolated from patients carrying previously identified hypomorphic, hypofunctional, and loss-of-function TLR7 variants. Our investigation revealed a profound impairment of the TLR7 pathway in patients carrying loss-of-function variants. Of note, a failure in IFNγ upregulation following stimulation was also observed in cells harboring the hypofunctional and hypomorphic variants. We also identified new TLR7 variants in severely affected male patients for which a functional characterization of the TLR7 pathway was performed demonstrating a decrease in mRNA levels in the IFNα, IFNγ, RSAD2, ACOD1, IFIT2, and CXCL10 genes.

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor.

Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity.

The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management.

Extremophilic Microalgae Galdieria Gen. for Urban Wastewater Treatment: Current State, the Case of "POWER" System, and Future Prospects.

Over the past decades, wastewater research has increasingly focused on the use of microalgae as a tool to remove contaminants, entrapping nutrients, and whose biomass could provide both material and energy resources. This review covers the advances in the emerging research on the use in wastewater sector of thermoacidophilic, low-lipid microalgae of the genus Galdieria, which exhibit high content of protein, reserve carbohydrates, and other potentially extractable high-value compounds. The natural tolerance of Galdieria for high toxic environments and hot climates recently made it a key player in a single-step process for municipal wastewater treatment, biomass cultivation and production of energetic compounds using hydrothermal liquefaction. In this system developed in New Mexico, Galdieria proved to be a highly performing organism, able to restore the composition of the effluent to the standards required by the current legislation for the discharge of treated wastewater. Future research efforts should focus on the implementation, in the context of wastewater treatment, of more energetically efficient cultivation systems, potentially capable of generating water with increasingly higher purity levels.

Cyanidiophyceae (Rhodophyta) Tolerance to Precious Metals: Metabolic Response to Palladium and Gold.

Polyextremophilic red algae, which belong to the class Cyanidiophyceae, are adapted to live in geothermal and volcanic sites. These sites often have very high concentrations of heavy and precious metals. In this study, we assessed the capacity of three strains of Galdieria (G. maxima, G. sulphuraria, and G. phlegrea) and one strain of Cyanidiumcaldarium to tolerate different concentrations of precious metals, such as palladium (Cl4K2Pd) and gold (AuCl4K) by monitoring algal growths in cultures exposed to metals, and we investigated the algae potential oxidative stress induced by the metals. This work provides further understanding of metals responses in the Cyanidiophyceae, as this taxonomic class is developed as a biological refinement tool.

Corrigendum: Exome Sequencing in BRCA1-2 Candidate Familias: The Contribution of Other Cancer Susceptibility Genes.

[This corrects the article DOI: 10.3389/fonc.2021.649435.].