Deregulation of microtubule organization and RNA metabolism in Arx models for lissencephaly and developmental epileptic encephalopathy.

X-linked lissencephaly with abnormal genitalia (XLAG) and developmental epileptic encephalopathy-1 (DEE1) are caused by mutations in the Aristaless-related homeobox (ARX) gene, which encodes a transcription factor responsible for brain development. It has been unknown whether the phenotypically diverse XLAG and DEE1 phenotypes may converge on shared pathways. To address this question, a label-free quantitative proteomic approach was applied to the neonatal brain of Arx knockout (ArxKO/Y) and knock-in polyalanine (Arx(GCG)7/Y) mice that are respectively models for XLAG and DEE1. Gene ontology and protein-protein interaction analysis revealed that cytoskeleton, protein synthesis and splicing control are deregulated in an allelic-dependent manner. Decreased α-tubulin content was observed both in Arx mice and Arx/alr-1(KO) Caenorhabditis elegans ,and a disorganized neurite network in murine primary neurons was consistent with an allelic-dependent secondary tubulinopathy. As distinct features of Arx(GCG)7/Y mice, we detected eIF4A2 overexpression and translational suppression in cortex and primary neurons. Allelic-dependent differences were also established in alternative splicing (AS) regulated by PUF60 and SAM68. Abnormal AS repertoires in Neurexin-1, a gene encoding multiple pre-synaptic organizers implicated in synaptic remodelling, were detected in Arx/alr-1(KO) animals and in Arx(GCG)7/Y epileptogenic brain areas and depolarized cortical neurons. Consistent with a conserved role of ARX in modulating AS, we propose that the allelic-dependent secondary synaptopathy results from an aberrant Neurexin-1 repertoire. Overall, our data reveal alterations mirroring the overlapping and variant effects caused by null and polyalanine expanded mutations in ARX. The identification of these effects can aid in the design of pathway-guided therapy for ARX endophenotypes and NDDs with overlapping comorbidities.

Emerging role of oncogenic long noncoding RNA as cancer biomarkers.

The view of long noncoding RNAs as nonfunctional "garbage" has been definitely outdated by the large body of evidence indicating this class of ncRNAs as "golden junk", especially in precision oncology. Indeed, in light of their oncogenic role and the higher expression in multiple cancer types compared with paired adjacent tissues, the clinical interest for lncRNAs as diagnostic and/or prognostic biomarkers has been rapidly increasing. The emergence of large-scale sequencing technologies, their subsequent diffusion even in small research and clinical centers, the technological advances for the detection of low-copy lncRNAs in body fluids, coupled to the huge reduction of operating costs, have nowadays made possible to rapidly and comprehensively profile them in multiple tumors and large cohorts. In this review, we first summarize some relevant data about the oncogenic role of well-studied lncRNAs having a clinical relevance. Then, we focus on the description of their potential use as diagnostic/prognostic biomarkers, including an updated overview about licensed patents or clinical trials on lncRNAs in oncology.

Targeting metabolism by B-raf inhibitors and diclofenac restrains the viability of BRAF-mutated thyroid carcinomas with Hif-1α-mediated glycolytic phenotype.

BACKGROUND: B-raf inhibitors (BRAFi) are effective for BRAF-mutated papillary (PTC) and anaplastic (ATC) thyroid carcinomas, although acquired resistance impairs tumour cells' sensitivity and/or limits drug efficacy. Targeting metabolic vulnerabilities is emerging as powerful approach in cancer. METHODS: In silico analyses identified metabolic gene signatures and Hif-1α as glycolysis regulator in PTC. BRAF-mutated PTC, ATC and control thyroid cell lines were exposed to HIF1A siRNAs or chemical/drug treatments (CoCl2, EGF, HGF, BRAFi, MEKi and diclofenac). Genes/proteins expression, glucose uptake, lactate quantification and viability assays were used to investigate the metabolic vulnerability of BRAF-mutated cells. RESULTS: A specific metabolic gene signature was identified as a hallmark of BRAF-mutated tumours, which display a glycolytic phenotype, characterised by enhanced glucose uptake, lactate efflux and increased expression of Hif-1α-modulated glycolytic genes. Indeed, Hif-1α stabilisation counteracts the inhibitory effects of BRAFi on these genes and on cell viability. Interestingly, targeting metabolic routes with BRAFi and diclofenac combination we could restrain the glycolytic phenotype and synergistically reduce tumour cells' viability. CONCLUSION: The identification of a metabolic vulnerability of BRAF-mutated carcinomas and the capacity BRAFi and diclofenac combination to target metabolism open new therapeutic perspectives in maximising drug efficacy and reducing the onset of secondary resistance and drug-related toxicity.

The Chromatin-Oxygen Sensor Gene KDM5C Associates with Novel Hypoxia-Related Signatures in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a fatal brain tumor without effective drug treatment. In this study, we highlight, for the first time, the contribution of chromatin remodeling gene Lysine (K)-specific demethylase 5C (KDM5C) in GBM via an extensive analysis of clinical, expression, and functional data, integrated with publicly available omic datasets. The expression analysis on GBM samples (N = 37) revealed two informative subtypes, namely KDM5CHigh and KDM5CLow, displaying higher/lower KDM5C levels compared to the controls. The former subtype displays a strong downregulation of brain-derived neurotrophic factor (BDNF)-a negative KDM5C target-and a robust overexpression of hypoxia-inducible transcription factor-1A (HIF1A) gene, a KDM5C modulator. Additionally, a significant co-expression among the prognostic markers HIF1A, Survivin, and p75 was observed. These results, corroborated by KDM5C overexpression and hypoxia-related functional assays in T98G cells, suggest a role for the HIF1A-KDM5C axis in the hypoxic response in this tumor. Interestingly, fluorescence-guided surgery on GBM sections further revealed higher KDM5C and HIF1A levels in the tumor rim niche compared to the adjacent tumor margin, indicating a regionally restricted hyperactivity of this regulatory axis. Analyzing the TCGA expression and methylation data, we found methylation changes between the subtypes in the genes, accounting for the hypoxia response, stem cell differentiation, and inflammation. High NANOG and IL6 levels highlight a distinctive stem cell-like and proinflammatory signature in the KDM5CHigh subgroup and GBM niches. Taken together, our results indicate HIF1A-KDM5C as a new, relevant cancer axis in GBM, opening a new, interesting field of investigation based on KDM5C as a potential therapeutic target of the hypoxic microenvironment in GBM.

PPARγ and Diabetes: Beyond the Genome and Towards Personalized Medicine.

PURPOSE OF REVIEW: Full and partial synthetic agonists targeting the transcription factor PPARγ are contained in FDA-approved insulin-sensitizing drugs and used for the treatment of metabolic syndrome-related dysfunctions. Here, we discuss the association between PPARG genetic variants and drug efficacy, as well as the role of alternative splicing and post-translational modifications as contributors to the complexity of PPARγ signaling and to the effects of synthetic PPARγ ligands. RECENT FINDINGS: PPARγ regulates the transcription of several target genes governing adipocyte differentiation and glucose and lipid metabolism, as well as insulin sensitivity and inflammatory pathways. These pleiotropic functions confer great relevance to PPARγ in physiological regulation of whole-body metabolism, as well as in the etiology of metabolic disorders. Accordingly, PPARG gene mutations, nucleotide variations, and post-translational modifications have been associated with adipose tissue disorders and the related risk of insulin resistance and type 2 diabetes (T2D). Moreover, PPARγ alternative splicing isoforms-generating dominant-negative isoforms mainly expressed in human adipose tissue-have been related to impaired PPARγ activity and adipose tissue dysfunctions. Thus, multiple regulatory levels that contribute to PPARγ signaling complexity may account for the beneficial as well as adverse effects of PPARγ agonists. Further targeted analyses, taking into account all these aspects, are needed for better deciphering the role of PPARγ in human pathophysiology, especially in insulin resistance and T2D. The therapeutic potential of full and partial PPARγ synthetic agonists underlines the clinical significance of this nuclear receptor. PPARG mutations, polymorphisms, alternative splicing isoforms, and post-translational modifications may contribute to the pathogenesis of metabolic disorders, also influencing the responsiveness of pharmacological therapy. Therefore, in the context of the current evidence-based trend to personalized diabetes management, we highlight the need to decipher the intricate regulation of PPARγ signaling to pave the way to tailored therapies in patients with insulin resistance and T2D.

The role of a new class of long noncoding RNAs transcribed from ultraconserved regions in cancer.

Ultraconserved regions (UCRs) represent a relatively new class of non-coding genomic sequences highly conserved between human, rat and mouse genomes. These regions can reside within exons of protein-coding genes, despite the vast majority of them localizes within introns or intergenic regions. Several studies have undoubtedly demonstrated that most of these regions are actively transcribed in normal cells/tissues, where they contribute to regulate many cellular processes. Interestingly, these non-coding RNAs exhibit aberrant expression levels in human cancer cells and their expression profiles have been used as prognostic factors in human malignancies, as well as to unambiguously distinguish among distinct cancer types. In this review, we first describe their identification, then we provide some updated information about their genomic localization and classification. More importantly, we discuss about the available literature describing an overview of the mechanisms through which some transcribed UCRs (T-UCR) contribute to cancer progression or to the metastatic spread. To date, the interplay between T-UCRs and microRNAs is the most convincing evidence linking T-UCRs and tumorigenesis. The limitations of these studies and the future challenges to be addressed in order to understand the biological role of T-UCRs are also discussed herein. We envision that future efforts are needed to convincingly include this class of ncRNAs in the growing area of cancer therapeutics.

Intragenic Deletion in MACROD2: A Family with Complex Phenotypes Including Microcephaly, Intellectual Disability, Polydactyly, Renal and Pancreatic Malformations.

Diagnosing a complex genetic syndrome and correctly assigning the concomitant phenotypic traits to a well-defined clinical form is often a medical challenge. In this work, we report the analysis of a family with complex phenotypes, including microcephaly, intellectual disability, dysmorphic features, and polydactyly in the proband, with the aim of adding new aspects for obtaining a clear diagnosis. We performed array-comparative genomic hybridization and quantitative reverse transcriptase PCR (qRT-PCR) analyses. We identified a deletion of chromosome 20p12.1 involving the macrodomain containing 2/mono-ADP ribosylhydrolase 2 gene (MACROD2) in several members of the family. This gene is actually not associated with a specific syndrome but with congenital anomalies of multiple organs. qRT-PCR showed higher levels of a MACROD2 mRNA isoform in the individuals carrying the deletion. Our results, together with other data reported in the literature, support the hypothesis that the deletion in MACROD2 can affect correct embryonic development and that the presence of another associated event, such as epigenetic modifications at the MACROD2 locus, can influence the level of severity of the pathology.

Unique true predicted neoantigens (TPNAs) correlates with anti-tumor immune control in HCC patients.

BACKGROUND: A novel prediction algorithm is needed for the identification of effective tumor associated mutated neoantigens. Only those with no homology to self wild type antigens are true predicted neoantigens (TPNAs) and can elicit an antitumor T cell response, not attenuated by central tolerance. To this aim, the mutational landscape was evaluated in HCV-associated hepatocellular carcinoma. METHODS: Liver tumor biopsies and adjacent non-tumor liver tissues were obtained from 9 HCV-chronically infected subjects and subjected to RNA-Seq analysis. Mutant peptides were derived from single nucleotide variations and TPNAs were predicted using two prediction servers (e.g. NetTepi and NetMHCstabpan) by comparison with corresponding wild-type sequences, non-related self and pathogen-related antigens. Immunological confirmation was obtained in preclinical as well as clinical setting. RESULTS: The development of such an improved algorithm resulted in a handful of TPNAs despite the large number of predicted neoantigens. Furthermore, TPNAs may share homology to pathogen's antigens and be targeted by a pre-existing T cell immunity. Cross-reactivity between such antigens was confirmed in an experimental pre-clinical setting. Finally, TPNAs homologous to pathogen's antigens were found in the only HCC long-term survival patient, suggesting a correlation between the pre-existing T cell immunity specific for these TPNAs and the favourable clinical outcome. CONCLUSIONS: The new algorithm allowed the identification of the very few TPNAs in cancer cells, and those targeted by a pre-existing immunity strongly correlated with long-term survival. Only such TPNAs represent the optimal candidates for immunotherapy strategies.

Distinct Antigen Delivery Systems Induce Dendritic Cells' Divergent Transcriptional Response: New Insights from a Comparative and Reproducible Computational Analysis.

Vaccination is the most successful and cost-effective method to prevent infectious diseases. However, many vaccine antigens have poor in vivo immunogenic potential and need adjuvants to enhance immune response. The application of systems biology to immunity and vaccinology has yielded crucial insights about how vaccines and adjuvants work. We have previously characterized two safe and powerful delivery systems derived from non-pathogenic prokaryotic organisms: E2 and fd filamentous bacteriophage systems. They elicit an in vivo immune response inducing CD8+ T-cell responses, even in absence of adjuvants or stimuli for dendritic cells' maturation. Nonetheless, a systematic and comparative analysis of the complex gene expression network underlying such activation is missing. Therefore, we compared the transcriptomes of ex vivo isolated bone marrow-derived dendritic cells exposed to these antigen delivery systems. Significant differences emerged, especially for genes involved in innate immunity, co-stimulation, and cytokine production. Results indicate that E2 drives polarization toward the Th2 phenotype, mainly mediated by Irf4, Ccl17, and Ccr4 over-expression. Conversely, fd-scαDEC-205 triggers Th1 T cells' polarization through the induction of Il12b, Il12rb, Il6, and other molecules involved in its signal transduction. The data analysis was performed using RNASeqGUI, hence, addressing the increasing need of transparency and reproducibility of computational analysis.

Pan-Cancer Mutational and Transcriptional Analysis of the Integrator Complex.

The integrator complex has been recently identified as a key regulator of RNA Polymerase II-mediated transcription, with many functions including the processing of small nuclear RNAs, the pause-release and elongation of polymerase during the transcription of protein coding genes, and the biogenesis of enhancer derived transcripts. Moreover, some of its components also play a role in genome maintenance. Thus, it is reasonable to hypothesize that their functional impairment or altered expression can contribute to malignancies. Indeed, several studies have described the mutations or transcriptional alteration of some Integrator genes in different cancers. Here, to draw a comprehensive pan-cancer picture of the genomic and transcriptomic alterations for the members of the complex, we reanalyzed public data from The Cancer Genome Atlas. Somatic mutations affecting Integrator subunit genes and their transcriptional profiles have been investigated in about 11,000 patients and 31 tumor types. A general heterogeneity in the mutation frequencies was observed, mostly depending on tumor type. Despite the fact that we could not establish them as cancer drivers, INTS7 and INTS8 genes were highly mutated in specific cancers. A transcriptome analysis of paired (normal and tumor) samples revealed that the transcription of INTS7, INTS8, and INTS13 is significantly altered in several cancers. Experimental validation performed on primary tumors confirmed these findings.

E2 multimeric scaffold for vaccine formulation: immune response by intranasal delivery and transcriptome profile of E2-pulsed dendritic cells.

BACKGROUND: The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses. RESULTS: The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4(+) and CD8(+) cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. CONCLUSIONS: The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.

Computational Analysis of Single Nucleotide Polymorphisms Associated with Altered Drug Responsiveness in Type 2 Diabetes.

Type 2 diabetes (T2D) is one of the most frequent mortality causes in western countries, with rapidly increasing prevalence. Anti-diabetic drugs are the first therapeutic approach, although many patients develop drug resistance. Most drug responsiveness variability can be explained by genetic causes. Inter-individual variability is principally due to single nucleotide polymorphisms, and differential drug responsiveness has been correlated to alteration in genes involved in drug metabolism (CYP2C9) or insulin signaling (IRS1, ABCC8, KCNJ11 and PPARG). However, most genome-wide association studies did not provide clues about the contribution of DNA variations to impaired drug responsiveness. Thus, characterizing T2D drug responsiveness variants is needed to guide clinicians toward tailored therapeutic approaches. Here, we extensively investigated polymorphisms associated with altered drug response in T2D, predicting their effects in silico. Combining different computational approaches, we focused on the expression pattern of genes correlated to drug resistance and inferred evolutionary conservation of polymorphic residues, computationally predicting the biochemical properties of polymorphic proteins. Using RNA-Sequencing followed by targeted validation, we identified and experimentally confirmed that two nucleotide variations in the CAPN10 gene-currently annotated as intronic-fall within two new transcripts in this locus. Additionally, we found that a Single Nucleotide Polymorphism (SNP), currently reported as intergenic, maps to the intron of a new transcript, harboring CAPN10 and GPR35 genes, which undergoes non-sense mediated decay. Finally, we analyzed variants that fall into non-coding regulatory regions of yet underestimated functional significance, predicting that some of them can potentially affect gene expression and/or post-transcriptional regulation of mRNAs affecting the splicing.

Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer.

Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells can manipulate alternative splicing patterns to modulate the expression of adhesion- and motility-related molecules, also at the isoform level. In this study, combining RNA-Sequencing on MCF-7 to targeted experimental validations-in human breast cell lines and breast tumor biopsies-we identified 12 new alternative splicing transcripts in genes encoding adhesion- and motility-related molecules, including semaphorins, their receptors and co-receptors. Among them, a new SEMA3F transcript is expressed in all breast cell lines and breast cancer biopsies, and is translated into a new semaphorin 3F isoform. In silico analysis predicted that most of the new putative proteins lack functional domains, potentially missing some functions and acquiring new ones. Our findings better describe the extent of alternative splicing in breast cancer and highlight the need to further investigate adhesion- and motility-related molecules to gain insights into breast cancer progression.

Novel transcription factor variants through RNA-sequencing: the importance of being "alternative".

Alternative splicing is a pervasive mechanism of RNA maturation in higher eukaryotes, which increases proteomic diversity and biological complexity. It has a key regulatory role in several physiological and pathological states. The diffusion of Next Generation Sequencing, particularly of RNA-Sequencing, has exponentially empowered the identification of novel transcripts revealing that more than 95% of human genes undergo alternative splicing. The highest rate of alternative splicing occurs in transcription factors encoding genes, mostly in Krüppel-associated box domains of zinc finger proteins. Since these molecules are responsible for gene expression, alternative splicing is a crucial mechanism to "regulate the regulators". Indeed, different transcription factors isoforms may have different or even opposite functions. In this work, through a targeted re-analysis of our previously published RNA-Sequencing datasets, we identified nine novel transcripts in seven transcription factors genes. In silico analysis, combined with RT-PCR, cloning and Sanger sequencing, allowed us to experimentally validate these new variants. Through computational approaches we also predicted their novel structural and functional properties. Our findings indicate that alternative splicing is a major determinant of transcription factor diversity, confirming that accurate analysis of RNA-Sequencing data can reliably lead to the identification of novel transcripts, with potentially new functions.

Analysis of SEMA6B gene expression in breast cancer: identification of a new isoform.

BACKGROUND: SEMA6B is a member of the semaphorins axon-guidance family. A growing body of evidence has been accumulated describing the role of semaphorin molecules in cancer development and the involvement of SEMA6B in cancer progression has recently been proposed. METHODS: Our analysis, based on real-time PCR, focused on the expression of SEMA6B in a panel of breast cancer tissues, compared to the normal counterpart. RESULTS: In cancer tissues we found a significantly strong down-modulation of this transcript. Moreover we identified and characterized a novel SEMA6B isoform, named SEMA6Ba. This isoform has a novel splice junction, created by the usage of alternative donor and acceptor splice sites internal to the exon 17. By in silico analysis we found that the new transcript 3' UTR lacks some highly-conserved miRNA binding sites, suggesting possible consequences on both spatial and temporal expression of SEMA6Ba. The translated sequence of SEMA6Ba lacks the cytoplasmic tail, crucial for triggering the reverse signaling described for the transmembrane semaphorins. We also demonstrated, by immunofluorescence analysis of endogenous and overexpressed SEMA6Ba, that the protein clearly localized to the endoplasmic reticulum and plasma membrane. In conclusion, SEMA6B gene products are strongly down modulated in breast cancer tissues and a new isoform named SEMA6Ba has been described and characterized. GENERAL SIGNIFICANCE: Our work states a clear relation among breast cancer and SEMA6B expression; moreover we describe for the first time the SEMA6Ba protein and report here the analysis of SEMA6Ba RNA messenger, the protein expression and the cellular localization.

Revolutionizing Cancer Treatment: Unveiling New Frontiers by Targeting the (Un)Usual Suspects.

This Special Issue includes original articles and reviews on both established and innovative approaches to cancer targeting, showcased at the 29th IGB Workshop titled "Targeting the (un)usual suspects in cancer" "https://29thigbworkshop [...].

GIPR expression is induced by thiazolidinediones in a PPARγ-independent manner and repressed by obesogenic stimuli.

Adipose tissue (AT) dysfunctions are associated with the onset of insulin resistance (IR) and type 2 diabetes mellitus (T2DM). Targeting glucose-dependent insulinotropic peptide receptor (GIPR) is a valid option to increase the efficacy of glucagon-like peptide 1 (GLP-1) receptor agonists in T2DM treatment. Nevertheless, the therapeutic potential of targeting the GIP/GIPR axis and its effect on the AT are controversial. In this work, we explored the expression and regulation of GIPR in precursor cells and mature adipocytes, investigating if and how obesogenic stimuli and thiazolidinediones perturb GIPR expression. Using publicly available gene expression datasets, we assessed that, among white adipose tissue (WAT) cells, adipocytes express lower levels of GIPR compared to cells of mesothelial origin, pericytes, dendritic and NK/T cells. However, we report that GIPR levels markedly increase during the in vitro differentiation of both murine and human adipocytes, from 3T3-L1 and human mesenchymal precursor cells (MSCs), respectively. Notably, we demonstrated that thiazolidinediones - ie. synthetic PPARγ agonists widely used as anti-diabetic drugs and contained in the adipogenic mix - markedly induce GIPR expression. Moreover, using multiple in vitro systems, we assessed that thiazolidinediones induce GIPR in a PPARγ-independent manner. Our results support the hypothesis that PPARγ synthetic agonists may be used to increase GIPR levels in AT, potentially affecting in turn the targeting of GIP system in patients with metabolic dysfunctions. Furthermore, we demonstrate in vitro and in vivo that proinflammatory stimuli, and especially the TNFα, represses GIPR both in human and murine adipocytes, even though discordant results were obtained between human and murine cellular systems for other cytokines. Finally, we demonstrated that GIPR is negatively affected also by the excessive lipid engulfment. Overall, we report that obesogenic stimuli - ie. pro-inflammatory cytokines and the increased lipid accumulation - and PPARγ synthetic ligands oppositely modulate GIPR expression, possibly influencing the effectiveness of GIP agonists.

Muscle Oxidative Stress Plays a Role in Hyperthyroidism-Linked Insulin Resistance.

While a low level of ROS plays a role in cellular regulatory processes, a high level can lead to oxidative stress and cellular dysfunction. Insulin resistance (IR) is one of the dysfunctions in which oxidative stress occurs and, until now, the factors underlying the correlation between oxidative stress and IR were unclear and incomplete. This study aims to explore this correlation in skeletal muscle, a tissue relevant to insulin-mediated glucose disposal, using the hyperthyroid rat as a model of oxidative stress. The development of IR in the liver from hyperthyroid animals has been widely reported, whereas data concerning the muscle are quite controversial. Thus, we investigated whether hyperthyroidism induces IR in skeletal muscle and the role of oxidative stress in this process. Particularly, we compared the effects of hyperthyroidism on IR both in the absence and presence of vitamin E (Vit E), acting as an antioxidant. Putative correlations between ROS production, oxidative stress markers, antioxidant capacity and changes in intracellular signalling pathways related to insulin action (AKT) and cellular stress response (EIF2α; JNK; PGC1α; BIP; and NRF1) were investigated. Moreover, we assessed the effects of hyperthyroidism and Vit E on the expression levels of genes encoding for glucose transporters (Slc2a1; Slc2a4), factors involved in lipid homeostasis and insulin signalling (Pparg; Ppara, Cd36), as well as for one of the IR-related inflammatory factors, i.e., interleukin 1b (Il1b). Our results suggest that hyperthyroidism-linked oxidative stress plays a role in IR development in muscle and that an adequate antioxidant status, obtained by vitamin E supplementation, that mitigates oxidative stress, may prevent IR development.

Functional characterization of the RNA chaperone Hfq in the opportunistic human pathogen Stenotrophomonas maltophilia.

Hfq is an RNA-binding protein known to regulate a variety of cellular processes by interacting with small RNAs (sRNAs) and mRNAs in prokaryotes. Stenotrophomonas maltophilia is an important opportunistic pathogen affecting primarily hospitalized and immunocompromised hosts. We constructed an hfq deletion mutant (Δhfq) of S. maltophilia and compared the behaviors of wild-type and Δhfq S. maltophilia cells in a variety of assays. This revealed that S. maltophilia Hfq plays a role in biofilm formation and cell motility, as well as susceptibility to antimicrobial agents. Moreover, Hfq is crucial for adhesion to bronchial epithelial cells and is required for the replication of S. maltophilia in macrophages. Differential RNA sequencing analysis (dRNA-seq) of RNA isolated from S. maltophilia wild-type and Δhfq strains showed that Hfq regulates the expression of genes encoding flagellar and fimbrial components, transmembrane proteins, and enzymes involved in different metabolic pathways. Moreover, we analyzed the expression of several sRNAs identified by dRNA-seq in wild-type and Δhfq S. maltophilia cells grown in different conditions on Northern blots. The accumulation of two sRNAs was strongly reduced in the absence of Hfq. Furthermore, based on our dRNA-seq analysis we provide a genome-wide map of transcriptional start sites in S. maltophilia.

Diabetic Retinopathy: Are lncRNAs New Molecular Players and Targets?

The growing incidence of diabetes mellitus worldwide implies the increasing prevalence of several related macro- (e.g., hypertension and atherosclerosis) and micro-vascular (e.g., nephropathy and retinopathy) complications. Notably, diabetic retinopathy (DR) is the leading cause of blindness in older diabetic patients and can occur with different degrees of severity. Chronic hyperglycemia is the main determinant of the functional damage of retinal cells. The oxidative stress, inflammatory factors and vascular endothelial growth factor signaling have been widely reported as contributors of DR onset and progression, and an emerging role has been described for different classes of non-coding RNA, including several long non-coding RNAs (lncRNAs). Here, we report the main results of all research articles (i.e., 150) listed on PubMed database from 2014 to 2022 regarding the putative role of lncRNAs in DR, including small nucleolar RNA host genes (SNHGs). Particularly, in this review we describe all lncRNAs and SNHGs with altered expression in DR and related contexts, discussing their association with DR outcomes, their mechanism of action related to DR, the molecular/functional effects, as well as the biological and experimental contexts. Thus, herein we provide an overview of the current state of knowledge regarding the putative involvement of 50 lncRNAs and SNHGs in the pathogenesis of DR, highlighting their potential as therapeutic targets or biomarkers for improving the clinical management of DR.