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A thermoplastic matrix composite flat panel stiffened by induction welded stringers, according to a typical aerospace configuration was tested. A Poly-Ether-Ether-Ketone (PEEK) matrix reinforced by carbon fibers was adopted. The aim of this study was to investigate the shear buckling, a load condition that is very common for the aerospace structures, of the panel obtained using a non-conventional approach for its fabrication, that is, induction welding. A finite element (FE) model capable to describe the buckling and the postbuckling behavior of the stiffened panel was also developed and compared with experimental results. Testing was performed using fixtures suitably designed to transmit a pure shear to the panel, and strain gauges were mounted on the external surfaces to measure the deformations.

Chronically stressing male mice can alter the behavior of their offspring across generations. This effect is thought to be mediated by stress-induced changes in the content of specific sperm miRNAs that modify embryo development after their delivery to oocytes at fertilization. A major problem with this hypothesis is that the levels of mouse sperm miRNAs are much lower than those present in preimplantation embryos. This makes it unclear how embryos could be significantly impacted without an amplification system to magnify changes in sperm miRNA content, like those present in lower organisms where transgenerational epigenetic inheritance is well established. Here, we describe such a system for Chronic Social Instability(CSI)stress that can explain how it reduces the levels of the miR-34b,c/449a,b family of miRNAs not only in sperm of exposed males but also in preimplantation embryos (PIEs) derived from their mating, as well as in sperm of male offspring. Sperm-derived miR-34c positively regulates expression of its own gene and that of miR-449 in PIEs. This feed forward, auto-amplification process is suppressed when CSI stress reduces sperm miR-34c levels. It is important for the transmission of traits to offspring because restoring miR-34c levels in PIEs from CSI stressed males, which also restores levels of miR-449 in them, suppresses elements of elevated anxiety and defective sociability normally found specifically in their female offspring, as well as reduced sperm miR-34 and miR-449 levels normally found in male offspring, who pass on these traits to their offspring. We previously published that the content of sperm miR-34/449 is also reduced in men raised in highly abusive and/or dysfunctional families. We show here that a similar miRNA auto-amplification system functions in human embryonic stem cells. This implies that PIEs in offspring of these men also display reduced levels of miR-34/449, which then promotes both the negative psychological effects these types of experiences are known to have on one’s children, in this case females, and reduced levels of miR-34/449 in sperm of male offspring that could produce this example of transgenerational epigenetic inheritance in humans.

It is currently unknown how many RNA transcripts are able to induce degradation of microRNAs (miRNA) via the mechanism known as target-directed miRNA degradation (TDMD). We developed TDMDfinder, a computational pipeline that identifies ‘high confidence’ TDMD interactions in the Human and Mouse transcriptomes by combining sequence alignment and feature selection approaches. Our predictions suggested that TDMD is widespread, with potentially every miRNA controlled by endogenous targets. We experimentally tested 37 TDMDfinder predictions, of which 17 showed TDMD effects as measured by RT-qPCR and small RNA sequencing, linking the miR-17, miR-19, miR-30, miR-221, miR-26 and miR-23 families to novel endogenous TDMDs. In some cases, TDMD was found to affect different members of the same miRNA family selectively. Features like complementarity to the miRNA 3′ region, bulge size and hybridization energy appeared to be the main factors determining sensitivity. Computational analyses performed using the multiomic TCGA platform substantiated the involvement of many TDMD transcripts in human cancer and highlighted 36 highly significant interactions, suggesting TDMD as a new potential oncogenic mechanism. In conclusion, TDMDfinder provides the first inventory of bona fide human and mouse TDMDs. Available as a free webtool, TDMDfinder allows users to search for any TDMD interaction of interest by customizing its selection criteria.

iNKT cells account for a relevant fraction of effector T-cells in the intestine and are considered an attractive platform for cancer immunotherapy. Although iNKT cells are cytotoxic lymphocytes, their functional role in colorectal cancer (CRC) is still controversial, limiting their therapeutic use. Thus, we examined the immune cell composition and iNKT cell phenotype of CRC lesions in patients (n = 118) and different murine models. High-dimensional single-cell flow-cytometry, metagenomics, and RNA sequencing experiments revealed that iNKT cells are enriched in tumor lesions. The tumor-associated pathobiont Fusobacterium nucleatum induces IL-17 and Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in iNKT cells without affecting their cytotoxic capability but promoting iNKT-mediated recruitment of neutrophils with polymorphonuclear myeloid-derived suppressor cells-like phenotype and functions. The lack of iNKT cells reduced the tumor burden and recruitment of immune suppressive neutrophils. iNKT cells in-vivo activation with α-galactosylceramide restored their anti-tumor function, suggesting that iNKT cells can be modulated to overcome CRC-associated immune evasion. Tumor co-infiltration by iNKT cells and neutrophils correlates with negative clinical outcomes, highlighting the importance of iNKT cells in the pathophysiology of CRC. Our results reveal a functional plasticity of iNKT cells in CRC, suggesting a pivotal role of iNKT cells in shaping the tumor microenvironment, with relevant implications for treatment.

A finite difference algorithm that evaluates the health conditions of a bonded joint is presented and discussed. The mathematical formulation of the problem is developed, paying particular attention to the singularity around the corners of the joint and implementing an original discretisation method of the partial differential equations governing the propagation of the elastic waves. The equations are solved under the only hypothesis of a bidimensional field. The algorithm is sensible to defects in the bonded joint and can be used as an effective structural health monitoring tool, as proven by the experiments that show close agreement with the numerical simulations.

Recent findings in human samples and animal models support the involvement of inflammation in the development of Parkinson’s disease. Nevertheless, it is currently unknown whether microglial activation constitutes a primary event in neurodegeneration. We generated a new mouse model by lentiviral-mediated selective α-synuclein (αSYN) accumulation in microglial cells. Surprisingly, these mice developed progressive degeneration of dopaminergic (DA) neurons without endogenous αSYN aggregation. Transcriptomics and functional assessment revealed that αSYN-accumulating microglial cells developed a strong reactive state with phagocytic exhaustion and excessive production of oxidative and proinflammatory molecules. This inflammatory state created a molecular feed-forward vicious cycle between microglia and IFNγ-secreting immune cells infiltrating the brain parenchyma. Pharmacological inhibition of oxidative and nitrosative molecule production was sufficient to attenuate neurodegeneration. These results suggest that αSYN accumulation in microglia induces selective DA neuronal degeneration by promoting phagocytic exhaustion, an excessively toxic environment and the selective recruitment of peripheral immune cells., Whether microglial activation constitutes a primary pathological event in synucleinopathies is not known. Here, the authors generated a mouse model over-expressing α-synuclein in microglial cells and found these mice developed progressive dopaminergic neuron degeneration and microglia developed a reactive, pro-inflammatory state with phagocytic exhaustion.

iNKT cells account for a relevant fraction of effector T-cells in the intestine. Although iNKT cells are cytotoxic lymphocytes, their role in colorectal cancer (CRC) remains controversial. From the analysis of colonic LPMCs of human and murine CRC specimens we report that tumor-infiltrating iNKT cells are characterized by an IL17/GM-CSF pro-tumorigenic phenotype, while maintaining cytotoxic properties in the adjacent non-tumoral tissue. Exposure of iNKT cells to the tumor-associated pathobiontFusobacterium nucleatumblunted their cytotoxic capability and enhanced iNKT cell-mediated neutrophils chemotaxis, which upregulated PMN-MDSC gene signatures and functions. Importantly,in vivostimulation of iNKT cells with αGalCer restored their anti-tumorigenic functions. Survival analyses demonstrated that human CRC co-infiltration by iNKT cells and tumor-associated neutrophils correlates with negative outcomes.Our results reveal a functional plasticity of human intestinal iNKT cells with pro- and anti-tumorigenic activities in CRC, suggesting an iNKT pivotal role in shaping the cancer developmental trajectory.

SARS-CoV-2 is a positive single-stranded RNA virus that interacts with proteins of infected cells at different stages of its life cycle. These interactions are necessary for the host to recognize and block the replication of the virus. Yet, if cells fail to block SARS-CoV-2, host proteins are recruited to translate, transcribe and replicate the genetic material of the virus. To identify the host proteins that bind to SARS-CoV-2 RNA, we adopted the RNA-Protein Interaction Detection coupled to Mass Spectrometry (RaPID-MS) technology, which allows the purification and identification by MS-based proteomics of the proteins associated with a specific RNA of interest expressed in mammalian cells. We specifically investigated proteins associated with the 5′ and 3′ end regions of SARS-CoV-2 RNA. As associations might involve non-physical protein-RNA interactions, we defined a set of reliable protein-RNA interactions by exploiting the predictive power of the catRAPID algorithm that assesses the direct binding potential of proteins to a given RNA region. Among these specific SARS-CoV-2 RNA end interactors, we identified the pseudouridine synthase PUS7 that binds to both 5′ and 3′ ends of viral RNA, which harbor the canonical consensus sequence modified by PUS7. We corroborated our results through SARS-CoV-2 RNA analysis by nanopore direct RNA sequencing. Indeed, these PUS7 consensus regions were found highly modified on viral RNAs, as demonstrated by ionic current features that are significantly different compared to the unmodified in vitro transcribed RNA. Overall, our data map the specific host protein interactions of SARS-CoV-2 RNA and point to a role for cellular pseudouridine synthases and the post-transcriptional pseudouridine modifications in the viral life cycle.

MicroRNAs (miRNAs) are an evolutionarily conserved class of small (18-22 nucleotides) noncoding RNAs involved in the regulation of a variety of cellular and developmental processes. MiRNA expression is frequently altered in human cancers compared to normal tissues, potentially contributing to tumorigenesis. Generally, high-throughput profiles of miRNA expression levels are generated using bulk samples, from both normal and cancer tissues. However, cancer tissues are quite heterogeneous and might contain subpopulations critical for tumor development, i.e., cancer stem cells (CSCs) or tumor-initiating cells (TICs) with aberrant stem-like features, such as unlimited self-renewal potential. The isolation of these aberrant subpopulations from solid tumors is a relatively recent achievement, with breast cancer being one of the first solid human cancers in which CSCs have been identified and biologically characterized. Here, we describe a new methodology that can overcome the main challenge in dealing with rare cells such as SCs/CSCs, represented by the paucity of the starting material. Based on previously published protocols, used by both our and other research groups, we used the FACS-sorting approach to isolate mammary normal and cancer stem cells based on the amount of PKH26 fluorescent dye they retained. Depending on the number of SCs/CSCs isolated, we established two different protocols for the reliable and analytically sensitive detection of up to 384 miRNAs using the Taqman Low Density Array (TLDA) platform.

The transition from dividing progenitors to postmitotic motor neurons (MNs) is orchestrated by a series of events, which are mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. Here, we identify a post-transcriptional role of a MN-specific transcriptional unit (MN2) harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p) in this transition. Through the use of in vitro mESC differentiation and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects MN differentiation by sponging miR-466i-5p and upregulating its targets, including several factors involved in neuronal differentiation and function. In parallel, miR-325-3p and miR-384-5p, co-transcribed with lncMN2-203, act by repressing proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and exemplify additional layers of specificity regulation in MN differentiation.

The landscape of pervasive transcription in eukaryotic genomes has made space for the identification of thousands of transcripts that are difficult to frame in a specific functional category. A new class has been broadly named as long non-coding RNAs (lncRNAs) and shortly defined as transcripts that are longer than 200 nucleotides with no or limited coding potential. So far, about 19,000 lncRNAs genes have been annotated in the human genome (Gencode 41), nearly matching the number of protein-coding genes. A key scientific priority is the functional characterization of lncRNAs, a major challenge in molecular biology that has encouraged many high-throughput efforts. LncRNA studies have been stimulated by the enormous clinical potential that these molecules promise and have been based on the characterization of their expression and functional mechanisms. In this review, we illustrate some of these mechanisms as they have been pictured in the context of breast cancer.

In the last years, bistable composite structures are finding interest in several aeronautical applications such as power harvesting devices or morphing applications on very small aircraft/drones, not needing servo-activated control systems. Residual stresses, developed upon cooling after curing, leads to warped the composite laminates. Several batches of unsymmetrical and unbalanced [0°/90°] laminates were cured in an autoclave according to a standard temperature cycle, following the pre-preg supplier suggested curing cycle. In order to increase the thermal stresses (and hence the bistability phenomenon), these laminates were removed from the autoclave immediately after the curing reaction and rapidly cooled down at room temperature not applying the indicated cool rate between 2 and 5 °C min−1. During storage at room temperature, thermal stresses changed over time, indicating that asymptotic stress relaxation occurs. The first part of this work looks at residual stress characterization of bistable composite plates measuring the changes of shape observed during room temperature annealing. Rectangular plates were produced and the bistable geometric shapes were accurately assessed using a laser scanner system over several days, in order to monitor the curvature changes due to stress relaxation. Then phenomenological viscoelastic predicting models were proposed for a quick estimate for the strain/stress relaxation phenomenon. The loss of bistability was demonstrated with the help of numerical simulation and experimental testing. The final goal was to gain a better knowledge of the relation between processing and final shape of bistable laminates, in order to make them suitable for application on small air vehicles.

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5′ cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

The aim of this work is the study of the adhesion integrity of metallic Single Lap Joints (SLJs) through the assessment of the MUL2 CODE, software developed by the MUL2 Research Group - Department of Mechanical and Aerospace Engineering of Politecnico di Torino. The MUL2 CODE is implemented through the Carrera Unified Formulation (CUF) for 2D structures based on Hierarchical Legendre Expansion (HLE) polynomials. An efficient method for the Structural Health Monitoring (SHM) of bonded joints is simulated and verified by CUF approach, in order to reduce the computational cost of analyses: by using transient excitations (toneburst signals), the structural health of damaged SLJ can be numerically evaluated. The interaction mechanism between the waves traveling through the investigated specimens is numerically modeled with a simple Finite Elements (FE) model and it is solved via MUL2 CODE and commercial software Ansys Workbench, respectively. Experimental campaigns data are compared with CUF and Ansys results demonstrating the consistence of the MUL2 formulation that is computationally simpler, but very efficient for the joint analysis. The presented and discussed CUF application is able to quantify with a high accuracy the debonding extension in the damaged SLJ, simply tuning the excitation frequency of the SHM technique.

MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17–92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5′ cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

The present paper is focused on the analysis of the adhesion integrity of Single Lap Joints (SLJ). The technique adopted for the monitoring is based on the employment of vibration signals generated and received by piezo sensors attached on the specimens surfaces on the two opposite sides of the adhesive. Undamaged joints, when subjected to harmonic excitation, behave linearly exhibiting a spectral response including just the excitation frequency. As soon as the adhesive fails, harmonic excitation induces a rich variety of spectral components in the joints response, including subharmonics and superharmonics. A new approach is presented based on the beating resulting from the superposition of harmonic waves. The interpretation of the spectral content of the joint response is based on an analytical model of the single lap joint with different amounts of debondings associated with a nonlinear behaviour. Local Defect Resonance (LDR) conditions are promoted with the consequent superposition of defect with main structure vibration. Analytical results are compared with experiments and Finite Elements (FE) simulations demonstrating the consistence of the formulation. An efficient method for the Structural Health Monitoring (SHM) of the bonded single lap joints is provided, able to correlate the joint vibrational response to the debonding extension through the analysis of the response spectral content.

In the present paper a method for the Structural Health Monitoring (SHM) of bonded lap joints is presented. This method is based on the interference of elastic waves generated by piezo sensors and travelling along thin bonded plates through the adhesion area. Tone bursts of different extension were generated and, when they encountered the debonded area, the wave speed changed. This affected the wave reflection at the boundary of the disbond and the subsequent interference of the reflected wave with the main wave travelling along the joint. Destructive interference conditions were promoted when the adhesive was partially debonded and this was related to the length of the disbond. In the present study numerical simulations based on finite elements were performed and compared with a simplified analytical model describing the Lamb waves propagation through the joint. The simulation results were subsequently compared with the experimental data and a good agreement was found: the proposed method was simple, straightforward and its application on thin single lap joints provided reliable results.

Functional RNAs, such as microRNAs, are emerging as innovative tools in the treatment of aggressive and incurable cancers. In this study, we explore the potential of silica dioxide nanoparticles (SiO2NPs) in the delivery of biologically active miRNAs. Focusing on the tumor-suppressor miR-34a, we evaluated miRNAs delivery by SiO2NPs into the mammary gland, using in vitro as well as in vivo model systems. We showed that silica nanoparticles can efficiently deliver miR-34a into normal and cancer epithelial cells grown in culture without major signs of toxicity. Delivered miRNA retained the ability to silence artificial as well endogenous targets and can reduce the growth of mammospheres in 3D culture. Finally, miR-34a delivery through intra-tumor administration of SiO2NPs leads to a reduced mammary tumor growth. In conclusion, our studies suggest that silica nanoparticles can mediate the delivery of miR-34a directly into mammary tumors while preserving its molecular and biological activity.

Tordonato et al. reveal miRNA-146 as a specific marker for breast stem cells and for cancer stem cells. miR-146 maintains the stem cell identity and coordinates a transcriptional and metabolic program, distinct from bulk cells, connected to the refractoriness to antifolate drugs., Although ectopic overexpression of miRNAs can influence mammary normal and cancer stem cells (SCs/CSCs), their physiological relevance remains uncertain. Here, we show that miR-146 is relevant for SC/CSC activity. MiR-146a/b expression is high in SCs/CSCs from human/mouse primary mammary tissues, correlates with the basal-like breast cancer subtype, which typically has a high CSC content, and specifically distinguishes cells with SC/CSC identity. Loss of miR-146 reduces SC/CSC self-renewal in vitro and compromises patient-derived xenograft tumor growth in vivo, decreasing the number of tumor-initiating cells, thus supporting its pro-oncogenic function. Transcriptional analysis in mammary SC-like cells revealed that miR-146 has pleiotropic effects, reducing adaptive response mechanisms and activating the exit from quiescent state, through a complex network of finely regulated miRNA targets related to quiescence, transcription, and one-carbon pool metabolism. Consistent with these findings, SCs/CSCs display innate resistance to anti-folate chemotherapies either in vitro or in vivo that can be reversed by miR-146 depletion, unmasking a “hidden vulnerability” exploitable for the development of anti-CSC therapies.

Carbon fiber-epoxy thin laminates, manufactured adopting a [0/90] non-symmetric lay-up, are characterized by two stable curved shapes, so being bistable. These composite thin laminates are attracting great interest for their potential application as novel morphing structures and power harvesting devices in many engineering fields. The curvature changes of rectangular bistable plates were monitored at room temperature by a laser scanner system over a time span of 10 d. These changes were ascribed to the relaxation of thermal stresses over time. Anisotropic stress relaxation behavior was then studied looking at the matrix dominated properties, such as the transverse modulus in accelerated high temperature experiments and the moisture absorption. Then, a simple viscoelastic model was proposed together with a finite element analysis of the distortion arising from laminate cooling at the end of high temperature cure process. The experimental and numerical results showed that the transverse elastic modulus and the hygroscopic effect are the main factors affecting the shape relaxation of bistable laminates.

Friedreich’s ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons., Friedreich’s ataxia (FRDA) is an autosomal-recessive disorder. Here the authors describe a DRG organoid from patient derived-neurons and co-culture with muscle cells to mimic the disorder in vitro and demonstrate potential correction of the phenotype by CRISPR based editing.