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OBJECTIVE The excess of visceral adipose tissue might hinder and delay immune response. How people with abdominal obesity (AO) will respond to mRNA vaccines against SARS-CoV-2 is yet to be established. We evaluated SARS-CoV-2 specific antibody responses after the first and second dose of the BNT162b2 mRNA vaccine comparing the response of individuals with AO to those without, discerning between individuals with or without prior infection. METHODS IgG neutralizing antibodies against the Trimeric-complex (IgG-TrimericS) were measured at four time points: at baseline, at day 21 after vaccine dose 1, at one and three months after dose 2. Nucleocapsid antibodies were assessed to detect prior SARS-CoV-2 infection. Waist circumference was measured to determine AO. RESULTS Between the first and third month after vaccine dose 2, the drop in IgG-TrimericS levels was more remarkable in individuals with AO compared to those without AO (2.44 fold [95%CI: 2.22-2.63] vs 1.82 fold [95%CI: 1.69-1.92], respectively, p

Genetic frontotemporal lobar degeneration (FTLD) is characterized by heterogeneous phenotypic expression, with a disease onset highly variable even in patients carrying the same mutation. Herein we investigated if variants in lysosomal genes modulate the age of onset both in FTLD due to GRN null mutations and C9orf72 expansion. In a total of 127 subjects (n = 74 GRN mutations and n = 53 C9orf72 expansion carriers), we performed targeted sequencing of the top 98 genes belonging to the lysosomal pathway, selected based on their high expression in multiple brain regions. We described an earlier disease onset in GRN/C9orf72 pedigrees in subjects carrying the p.Asn521Thr variant (rs1043424) in PTEN-induced kinase 1 (PINK1), a gene that is already known to be involved in neurodegenerative diseases. We found that: (i) the PINK1 rs1043424 C allele is significantly associated with the age of onset; (ii) every risk C allele increases hazard by 2.11%; (iii) the estimated median age of onset in homozygous risk allele carriers is 10–12 years earlier than heterozygous/wild type homozygous subjects. A replication study in GRN/C9orf72 negative FTLD patients confirmed that the rs1043424 C allele was associated with earlier disease onset (−5.5 years in CC versus A carriers). Understanding the potential mechanisms behind the observed modulating effect of the PINK1 gene in FTLD might prove critical for identifying biomarkers and/or designing drugs to modify the age of onset, especially in GRN/C9orf72-driven disease.

Genetic frontotemporal lobar degeneration (FTLD) is characterized by heterogeneous phenotypic expression, with a disease onset highly variable even in patients carrying the same mutation. Herein we investigated if variants in lysosomal genes modulate the age of onset both in FTLD due to

Hypoxia-induced miR-210 is a crucial component of the tissue response to ischemia, stimulating angiogenesis and improving tissue regeneration. Previous analysis of miR-210 impact on the transcriptome in a mouse model of hindlimb ischemia showed that miR-210 regulated not only vascular regeneration functions, but also inflammation. To investigate this event, doxycycline-inducible miR-210 transgenic mice (Tg-210) and anti-miR-210 LNA-oligonucleotides were used. It was found that global miR-210 expression decreased inflammatory cells density and macrophages accumulation in the ischemic tissue. To dissect the underpinning cell mechanisms, Tg-210 mice were used in bone marrow (BM) transplantation experiments and chimeric mice underwent hindlimb ischemia. MiR-210 overexpression in the ischemic tissue was sufficient to increase capillary density and tissue repair, and to reduce inflammation in the presence of Wt-BM infiltrating cells. Conversely, when Tg-210-BM cells migrated in a Wt ischemic tissue, dysfunctional angiogenesis, inflammation, and impaired tissue repair, accompanied by fibrosis were observed. The fibrotic regions were positive for α-SMA, Vimentin, and Collagen V fibrotic markers and for phospho-Smad3, highlighting the activation of TGF-β1 pathway. Identification of Tg-210 cells by in situ hybridization showed that BM-derived cells contributed directly to fibrotic areas, where macrophages co-expressing fibrotic markers were observed. Cell cultures of Tg-210 BM-derived macrophages exhibited a pro-fibrotic phenotype and were enriched with myofibroblast-like cells, which expressed canonical fibrosis markers. Interestingly, inhibitors of TGF-β type-1-receptor completely abrogated this pro-fibrotic phenotype. In conclusion, a context-dependent regulation by miR-210 of the inflammatory response was identified. miR-210 expression in infiltrating macrophages is associated to improved angiogenesis and tissue repair when the ischemic recipient tissue also expresses high levels of miR-210. Conversely, when infiltrating an ischemic tissue with mismatched miR-210 levels, macrophages expressing high miR-210 levels display a pro-fibrotic phenotype, leading to impaired tissue repair, fibrosis, and dysfunctional angiogenesis. ispartof: CELL DEATH & DISEASE vol:12 issue:5 ispartof: location:England status: published

Recently, CITE-seq emerged as a multimodal single-cell technology capturing gene expression and surface protein information from the same single-cells, which allows unprecedented insights into disease mechanisms and heterogeneity, as well as immune cell profiling. Multiple single-cell profiling methods exist, but they are typically focussed on either gene expression or antibody analysis, not their combination. Moreover, existing software suites are not easily scalable to a multitude of samples. To this end, we designed gExcite, a start-to-end workflow that provides both gene and antibody expression analysis, as well as hashing deconvolution. Embedded in the Snakemake workflow manager, gExcite facilitates reproducible and scalable analyses. We showcase the output of gExcite on a study of different dissociation protocols on PBMC samples., Bioinformatics, 39 (5), ISSN:1367-4803, ISSN:1460-2059

ObjectiveThe excess of visceral adipose tissue might hinder and delay the immune response. How people with abdominal obesity will respond to mRNA vaccines against SARS-CoV-2 is yet to be established. We evaluated SARS-CoV-2-specific antibody responses after the first and second dose of the BNT162b2 mRNA vaccine comparing the response of individuals affected by abdominal obesity (AO) to those without, discerning between individuals with or without prior infection.MethodsIgG neutralizing antibodies against the Trimeric complex (IgG-TrimericS) were measured at four time points: at baseline, at day 21 after vaccine dose-1, at one month and three months after dose-2. Nucleocapsid antibodies were assessed to detect prior SARS-CoV-2 infection. Waist circumference was measured to determine abdominal obesity.ResultsBetween the first and third month after vaccine dose-2, the drop in IgG-TrimericS levels was more remarkable in individuals with AO compared to those without AO (2.44 fold [95%CI: 2.22-2.63] vs 1.82 fold [95%CI: 1.69-1.92], respectively, pConclusionsOur findings highlight the need to extend the duration of serological monitoring of antibody levels in infection-naive individuals with abdominal obesity, a higher-risk population category in terms of possible weaker antibody response.

Dysfunctions in the endo-lysosomal system have been hypothesized to underlie neurodegeneration in major neurocognitive disorders due to Alzheimer's disease (AD), Frontotemporal Lobar Degeneration (FTLD), and Lewy body disease (DLB). The aim of this study is to investigate whether these diseases share genetic variability in the endo-lysosomal pathway. In AD, DLB, and FTLD patients and in controls (948 subjects), we performed a targeted sequencing of the top 50 genes belonging to the endo-lysosomal pathway. Genetic analyses revealed (i) four previously reported disease-associated variants in the SORL1 (p.N1246K, p.N371T, p.D2065V) and DNAJC6 genes (p.M133L) in AD, FTLD, and DLB, extending the previous knowledge attesting SORL1 and DNAJC6 as AD- and PD-related genes, respectively; (ii) three predicted null variants in AD patients in the SORL1 (p.R985X in early onset familial AD, p.R1207X) and PPT1 (p.R48X in early onset familial AD) genes, where loss of function is a known disease mechanism. A single variant and gene burden analysis revealed some nominally significant results of potential interest for SORL1 and DNAJC6 genes. Our data highlight that genes controlling key endo-lysosomal processes (i.e., protein sorting/transport, clathrin-coated vesicle uncoating, lysosomal enzymatic activity regulation) might be involved in AD, FTLD and DLB pathogenesis, thus suggesting an etiological link behind these diseases. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:22 issue:24 ispartof: location:Switzerland status: published

Background: Frontotemporal lobar degeneration (FTLD) designates a group of neurodegenerative diseases with remarkable clinical, pathological, and genetic heterogeneity. Mutations in progranulin gene (GRN) are among the most common causes of familial FTLD. The GRN C157KfsX97 mutation is the most frequent mutation occurring in Southern Italy and has been already described in a previous work. Objective: In this study, we reported on additional cases carrying the same mutation and performed a genetic study on the whole cohort, aiming at demonstrating the existence of a founder effect and estimating the age of this mutation. Methods/Results: Based on the haplotype sharing analysis, a founder effect was highly probable, while the age of the mutation, estimated by means of DMLE+ software, resulted in a range between 52 and 82 generations, with the highest frequency at about 62 generations, 1,550 years ago. Conclusion: This is the first study that reports the age estimation of the most recent common ancestor for the GRN C157KfsX97 mutation recurring in Southern Italy. Mutation dating in a geographically restricted population may be useful in order to plan genetic counseling and screening programs in the field of public health.

Circular RNAs are generated by back‐splicing of precursor‐mRNAs. Although they have been known for many years, only recently we have started to appreciate their widespread expression and their regulatory functions in a variety of biological processes. Not surprisingly, circular RNA dysregulation and participation in the pathogenic mechanisms have started to emerge in many instances, including cardiovascular diseases. Detection, differential expression analysis and validation are the three critical points for the characterization of any RNA, and circular RNAs are no exception. Their characteristics, however, generate several problems that are yet to be completely addressed, and literature still lacks comprehensive definitions of well‐defined best practices. We present a map of the current knowledge regarding circular RNAs and the critical issues limiting our understanding of their regulation and function. The goal was to provide the readers with the tools to critically decide which of the many approaches available is most suitable to their experimental plan. Although particularly focused on cardiovascular diseases, most critical issues concerning circular RNAs are common to many other fields of investigation.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by abnormally expanded stretches of CTG DNA triplets in the DMPK gene, leading to mutated-transcript RNA-toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that, after maturation, are loaded onto the RISC effector complex that destabilizes target mRNAs and represses their translation. In DM1 muscle biopsies not only the expression, but also the intracellular localization of specific miRNAs is disrupted, leading to the dysregulation of the relevant mRNA targets. To investigate the functional alterations of the miRNA/target interactions in DM1, we analyzed by RNA-sequencing the RISC-associated RNAs in skeletal muscle biopsies derived from DM1 patients and matched controls. The mRNAs found deregulated in DM1 biopsies were involved in pathways and functions relevant for the disease, such as energetic metabolism, calcium signaling, muscle contraction and p53-dependent apoptosis. Bioinformatic analysis of the miRNA/mRNA interactions based on the RISC enrichment profiles, identified 24 miRNA/mRNA correlations. Following validation in 21 independent samples, we focused on the couple miR-29c/ASB2 because of the role of miR-29c in fibrosis (a feature of late-stage DM1 patients) and of ASB2 in the regulation of muscle mass. Luciferase reporter assay confirmed the direct interaction between miR-29c and ASB2. Moreover, decreased miR-29c and increased ASB2 levels were verified also in immortalized myogenic cells and primary fibroblasts, derived from biopsies of DM1 patients and controls. CRISPR/Cas9-mediated deletion of CTG expansions rescued normal miR-29c and ASB2 levels, indicating a direct link between the mutant repeats and the miRNA/target expression. In conclusion, functionally relevant miRNA/mRNA interactions were identified in skeletal muscles of DM1 patients, highlighting the dysfunction of miR-29c and ASB2.

Critical limb ischemia is the most serious form of peripheral artery disease, characterized by severe functional consequences, difficult clinical management and reduced life expectancy. The goal of this study was to investigate the miR-210 role in the neo-angiogenic response after acute limb ischemia. Complementary approaches were used in a mouse model of hindlimb ischemia: miR-210 loss-of-function was obtained by administration of LNA-oligonucleotides anti-miR-210, for miR-210 gain-of-function, a doxycycline-inducible miR-210 transgenic mouse was used. We tested miR-210 ability to stimulate vascular regeneration following ischemia. We found that miR-210 was necessary and sufficient to stimulate blood perfusion recovery, as well as arteriolar and capillary density increase, in the ischemic muscle. To clarify the molecular events underpinning miR-210 pro-angiogenic action, the transcriptomic changes in ischemic muscles upon miR-210 blocking were analyzed. We found that miR-210 impacted the transcriptome significantly, regulating pathways and functions linked to vascular regeneration. In agreement with a pro-angiogenic role, miR-210 also improved cardiac function and left ventricular remodeling after myocardial infarction. Moreover, miR-210 blocking decreased capillary density in a Matrigel plug assay, indicating that miR-210 is necessary for angiogenesis independently of ischemia. Collectively, these data indicate that miR-210 plays a pivotal role in promoting vascular regeneration.

Frontotemporal dementia (FTD) is a common form of dementia among early-onset cases. Several genetic factors for FTD have been revealed, but a large proportion of FTD cases still have an unidentified genetic origin. Recent studies highlighted common pathobiological mechanisms among neurodegenerative diseases. In the present study, we investigated a panel of candidate genes, previously described to be associated with FTD and/or other neurodegenerative diseases by targeted next generation sequencing (NGS). We focused our study on sporadic FTD (sFTD), devoid of disease-causing mutations in GRN, MAPT and C9orf72. Since genetic factors have a substantially higher pathogenetic contribution in early onset patients than in late onset dementia, we selected patients with early onset (&lt, 65 years). Our study revealed that, in 50% of patients, rare missense potentially pathogenetic variants in genes previously associated with Alzheimer&rsquo, s disease, Parkinson disease, amyotrophic lateral sclerosis and Lewy body dementia (GBA, ABCA7, PARK7, FUS, SORL1, LRRK2, ALS2), confirming genetic pleiotropy in neurodegeneration. In parallel, a synergic genetic effect on FTD is suggested by the presence of variants in five different genes in one single patient. Further studies employing genome-wide approaches might highlight pathogenic variants in novel genes that explain the still missing heritability of FTD.

Aims Antisense long noncoding RNAs (ncRNAs) are transcripts emerging from the opposite strand of a coding-RNA region and their role in heart failure (HF) is largely unknown. Additionally, HF and Alzheimer's disease (AD) share several non-genetic effectors and risk factors. We investigated the regulation of the β-secretase-1 (BACE1) gene and of its antisense transcript BACE1-AS in ischaemic HF. Methods and results BACE1 and BACE1-AS expression was measured in left ventricle biopsies from 18 patients affected by non-end stage ischaemic HF and 17 matched controls. The levels of both transcripts were increased in HF patients. Likewise, both transcripts increased also in a mouse model of ischaemic HF, and their expression was directly correlated. BACE1-AS was expressed by all cardiac cell types and BACE1-AS up- or down-modulation in cultured cardiomyocytes and endothelial cells induced a concordant regulation of the cognate BACE1 transcript. Interestingly, BACE1 increase also induced the intracellular accumulation of its product β-amyloid. In keeping with these findings, higher BACE1 protein and β-amyloid peptide levels were also observed in HF. Moreover, increased β-amyloid 1-40 was also found in the plasma of HF patients. Transcriptomic changes of BACE1-AS overexpressing and β-amyloid 1-40 treated cells were largely overlapping and indicated changes of relevant biological process such as 'cell cycle and proliferation', 'apoptosis', and 'DNA repair' as well as 'TGFβ-, TNFα-, p38-, EGFR-signalling', suggesting a potential maladaptive role of the BACE1-AS/BACE1/β-amyloid axis. Accordingly, the administration of β-amyloid peptides decreased the cell viability in endothelial cells and in both human IPS-derived and mouse cardiomyocytes. Moreover, both β-amyloid treatment and BACE1-AS overexpression increased endothelial cell apoptosis, and this effect was prevented by BACE1 silencing. Conclusion Given the neurotoxic role of β-amyloid in AD, dysregulation of the BACE1/BACE1-AS/β-amyloid axis might be relevant in HF pathogenesis, further implicating ncRNAs in the complex scenario of proteotoxicity in cardiac dysfunction.

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponge for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by “back-splicing”, linking covalently 3’- and 5’-ends of exons. Thus, circRNA levels might be deregulated in conditions associated to altered RNA-splicing. Indeed, increasing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG-repeats in the DMPK gene, resulting in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of 9 tested, 4 transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03 and ZNF609. The circular fraction values correlated positively with skeletal muscle strength and Receiver-Operating-Characteristics curves showed that these four circRNAs allow to distinguish DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and plasma of DM1 patients, but they were not regulated significantly, indicating a tissue-selectivity of the identified modulations. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this proof-of-principle study identified circRNA dysregulation in DM1 patients.

The Hippo/YAP signaling pathway is a crucial regulator of tissue growth, stem cell activity, and tumorigenesis. However, the mechanism by which YAP controls transcription remains to be fully elucidated. Here, we utilize global chromatin occupancy analyses to demonstrate that robust YAP binding is restricted to a relatively small number of distal regulatory elements in the genome. YAP occupancy defines a subset of enhancers and superenhancers with the highest transcriptional outputs. YAP modulates transcription from these elements predominantly by regulating promoter-proximal polymerase II (Pol II) pause release. Mechanistically, YAP interacts and recruits the Mediator complex to enhancers, allowing the recruitment of the CDK9 elongating kinase. Genetic and chemical perturbation experiments demonstrate the requirement for Mediator and CDK9 in YAP-driven phenotypes of overgrowth and tumorigenesis. Our results here uncover the molecular mechanisms employed by YAP to exert its growth and oncogenic functions, and suggest strategies for intervention.

Background: Implantation of saphenous vein (SV) grafts into coronary position determines structural vessel wall remodeling and intimal hyperplasia. The role of the altered wall mechanics and cell-based mechanosensing has been recently implicated in the priming of this pathologic process. We investigated the effects of cyclic uniaxial strain on human saphenous vein progenitor cells (SVPs), a cell type endowed with pericyte stem cell characteristics resident in the adventitia. Methods: CD34 + CD31 - SVPs were isolated with immuno-magnetic sorting (MACS) from SVs of patients (age 58±12.6, Mean±SD) undergoing saphenectomy. Cells were subject to uniaxial strain (10% elongation; 1Hz) for 24 and 72 hrs using the FlexCell system. Cell orientation, immunofluorescence and western analyses were performed to assess the effects of strain on cell orientation/shape, cell cycle and activity of the YAP/Hippo-dependent mechanotransduction machinery. RNA-sequencing from control vs. strained SVPs was performed at both time points using RNA from cells of 5 independent donors. Results: Results indicated an increase in the expression of the cell cycle-associated markers Ki67 and pHH3 in mechanically stimulated vs. control SVPs at 24 hrs, followed by a significant reduction at 72 hrs of stimulation. Variations in cell shape was observed as verified by a significant change in the nuclei orientation in the strain field as well as in the cell shape index/spread areas at both time points. Immunofluorescence revealed a significant increase in cells showing a nuclear localization of the YAP transcription factor at 72hrs. In line with these findings Western analyses indicated a significant decrease in the ratio between phosphorylated/total YAP and its upstream kinase LATS in mechanically stimulated vs. control SVPs, suggesting an inhibition of the HIPPO kinase pathway by mechanosensing. RNASeq gene expression analyses showed a coherent modification of gene expression pathways and the upregulation of a specific HIPPO/YAP/TEAD gene expression signature in SVPs mechanically stimulated for 72hrs. Conclusions: These findings demonstrate the direct susceptibility of human SVPs to pathologic strain and identifies this cell population as mechano-perceptors in the vein wall.

The article reports on the design of a novel class of transducers for structural health monitoring and strain sensing designed using a Fourier-based approach. The design procedure formulates the problem considering an arbitrarily shaped distribution of the sensing surface. Interrogation of the sensors is based on the generation of guided and surface acoustic waves generated in the region surrounding the transducers. The representation of the distribution of the sensing material is analyzed and designed in the spatial Fourier domain, where the emission characteristics of the transducer in relation to the interrogating wave can be tailored to a specific application. For structural health monitoring, the sensing material distribution can be defined to provide the transducers with frequency-dependent directional properties, which can be employed as part of an interrogation scheme based on generation and processing of guided waves in the structure. For strain sensing, one-dimensional and two-dimensional gratin...

SummaryThe role of the Hippo pathway effector YAP1 in soft tissue sarcomas is poorly defined. Here we report that YAP1 activity is elevated in human embryonal rhabdomyosarcoma (ERMS). In mice, sustained YAP1 hyperactivity in activated, but not quiescent, satellite cells induces ERMS with high penetrance and short latency. Via its transcriptional program with TEAD1, YAP1 directly regulates several major hallmarks of ERMS. YAP1-TEAD1 upregulate pro-proliferative and oncogenic genes and maintain the ERMS differentiation block by interfering with MYOD1 and MEF2 pro-differentiation activities. Normalization of YAP1 expression reduces tumor burden in human ERMS xenografts and allows YAP1-driven ERMS to differentiate in situ. Collectively, our results identify YAP1 as a potent ERMS oncogenic driver and a promising target for differentiation therapy.

PRDM proteins belong to the SET domain protein family, which is involved in the regulation of gene expression. Although few PRDM members possess histone methyltransferase activity, the molecular mechanisms by which the other members exert transcriptional regulation remain to be delineated. In this study, we find that Prdm5 is highly expressed in mouse embryonic stem (mES) cells and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next-generation sequencing technologies, we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that although Prdm5 is dispensable for mES cell maintenance, it directly targets genomic regions involved in early embryonic development and affects the expression of a subset of developmental regulators during cell differentiation. Importantly, Prdm5 interacts with Ctcf, cohesin, and TFIIIC and cooccupies genomic loci. In summary, our data indicate how Prdm5 modulates transcription by interacting with factors involved in genome organization in mouse embryonic stem cells.

PRDM proteins are tissue-specific transcription factors often deregulated in diseases, particularly in cancer where different members have been found to act as oncogenes or tumor suppressors. PRDM5 is a poorly characterized member of the PRDM family for which several studies have reported a high frequency of promoter hypermethylation in cancer types of gastrointestinal origin. We report here the characterization of Prdm5 knockout mice in the context of intestinal carcinogenesis. We demonstrate that loss of Prdm5 increases the number of adenomas throughout the murine small intestine on an Apc(Min) background. By using the genome-wide ChIP-seq (chromatin immunoprecipitation (ChIP) followed by DNA sequencing) and transcriptome analyses we identify loci encoding proteins involved in metabolic processes as prominent PRDM5 targets and characterize monoacylglycerol lipase (Mgll) as a direct PRDM5 target in human colon cancer cells and in Prdm5 mutant mouse intestines. Moreover, we report the downregulation of PRDM5 protein expression in human colon neoplastic lesions. In summary, our data provide the first causal link between Prdm5 loss and intestinal carcinogenesis, and uncover an extensive and novel PRDM5 target repertoire likely facilitating the tumor-suppressive functions of PRDM5.

Diesel particulate matter poses a threat to human health, and in particular nitrated polycyclic aromatic hydrocarbons (NPAHs) found within and on the surface of these particles. Although diesel particulate filters (DPFs) have been designed and implemented to reduce these and other harmful diesel emissions, the particle loaded filters may act as a reaction chamber for the enhanced production of NPAHs from the nitration of PAHs with NO 2 . Focus is on the investigation of the heterogeneous reactions that occur on soot particles by exposing laboratory produced pyrene- or benzo(a)pyrene-coated spark discharge soot particles to varying concentrations of NO 2 and temperatures while following the formation of products over time. The sole nitration product that was observed throughout the experiments with pyrene-coated soot was 1-nitropyrene (1-NPYR), which increased linearly with reaction time for all NO 2 concentrations chosen (0.11, 1.0, 2.0, 4.0 ppm, m m −1 ). Resulting 1-NPYR formation rate increased exponentially with [NO 2 ]. Throughout the 3-h experiments less than 10% of pyrene has been converted to 1-NPYR and the partial reaction order with regard to [NO 2 ] was estimated to 1.52. Benzo(a)pyrene (BaP) was more reactive than pyrene. After 3 h reaction time almost 80% of the BaP has been converted to 6-NBaP. Highest 1-NPYR concentrations on particles were detected at 373 K, and at higher temperatures a considerable decrease in particulate 1-NPYR was observed. A similar trend was observed in a DPF simulation system (PM-Kat ® -like) with BaP-coated soot. In this case, highest 6-NBaP concentration on particles was detected at 423 K. Backed by corroborating results from separate gas/solid-phase partition experiments with 1-NPYR and 6-NBaP, it is likely that the newly formed 1-NPYR and 6-NBaP became transferred from particle to gas phase at higher temperatures. Results from this study confirm the presence of 1-NPYR and 6-NBaP in particulate and gas phase under conditions encountered in DPFs, especially when operated at low temperature situations of the aftertreatment system.

Micro-Raman spectroscopy (micro-RS) and Temperature Programmed Oxidation (TPO) combined with FTIR gas analysis have been used to determine structural changes and oxidation behavior in samples of spark discharge (GfG) and heavy duty engine (EURO IV) soot upon oxidation by oxygen in a temperature range between 293 K and 873 K. Raman spectra of soot and FTIR spectra of oxidation products have been recorded before and during the oxidation process. For micro-RS analysis spectral parameters have been determined by a five band curve fitting procedure (G, D1–D4). For GfG soot the relative intensity of D3 band is decreasing and the two observed Raman peaks are getting more separated during the TPO. This suggests a rapid preferential oxidation of highly reactive amorphous carbon. The decrease of the D1 band width indicates a decrease of chemical heterogeneity and an increase of structural order upon oxidation. Changes in Raman spectroscopic parameters are in good agreement with the behavior of soot during oxidation...

This work is focused on design and testing of a novel class of transducers for Structural Health Monitoring (SHM), able to perform directional interrogation of plate-like structures. These transducers leverage guided waves (GWs), and in particular Lamb waves, that have emerged as a very prominent option for assessing the state of a structure during operation. GW-SHM approaches greatly benefit from the use of transducers with controllable directional characteristics, so that selective scanning of a surface can be performed to locate damage, impacts, or cracks. In the concepts that we propose, continuous beam steering and directional actuation are achieved through proper selection of the excitation frequency. The design procedure takes advantage of the wavenumber representation of the device, and formulates the problem using a Fourier-based approach. The active layer of the transducer is made of piezoelectric fibers embedded into an epoxy matrix, allowing the device to be flexible, and thus suitable for application on non{ at surfaces. Proper shaping of the electrodes pattern through a compensation function allows taking into account the anisotropy level introduced by the active layer. The resulting spiral frequency steerable acoustic actuator is a configuration that features (i) enhanced performance, (ii) reduced complexity, and (iii) reduced hardware requirements of such devices.

In this work we explore efficient transformation of broadband wave energy into low-power electricity using patterned polymer piezoelectrics integrated with an Elliptical Acoustic Mirror (EAM) configuration. The mirror under consideration features a semi-elliptical continuous mirror with a rectangular arrangement of harvesting material overlapping the geometrical focus of the mirror. Spatial and temporal transformation of the wave propagation field into the frequency-wavenumber domain is performed in order to identify the wavenumber content inside the mirror region. A frequency-domain Root-Mean-Square (RMS) evaluation is then applied in order to guarantee broadband harvesting characteristics to the resulting Distributed Harvester (DH). Computational modeling and experimental testing are employed to quantify performance enhancement of the presented approach in the 20-120 kHz range, where broadband focusing characteristics of the continuous EAM are confirmed experimentally. Additionally the patterned configuration with proper wiring results in substantial power enhancement over 20-60 kHz, i.e. the neighborhood of the center frequency used in its Fourier transform-based design.

Background RNAseq provides remarkable power in the area of biomarkers discovery and disease stratification. The main technical steps affecting the results of RNAseq experiments are Library Sample Preparation (LSP) and Bioinformatics Analysis (BA). At the best of our knowledge, a comparative evaluation of the combined effect of LSP and BA was never considered and it might represent a valuable knowledge to optimize alternative splicing detection, which is a challenging task due to moderate fold change differences to be detected within a complex isoforms background. Results Different LSPs (TruSeq unstranded/stranded, ScriptSeq, NuGEN) allow the detection of a large common set of isoforms. However, each LSP also detects a smaller set of isoforms, which are characterized both by lower coverage and lower FPKM than that observed for the common ones among LSPs. This characteristic is particularly critical in case of low input RNA NuGEN v2 LSP. The effect on statistical detection of alternative splicing considering low input LSP (NuGEN v2) with respect to high input LSP (TruSeq) on statistical detection of alternative splicing was studied using a benchmark dataset, in which both synthetic reads and reads generated from high (TruSeq) and low input (NuGEN) LSPs were spiked-in. Statistical detection of alternative splicing (AltDE) was done using prototypes of BA for isoform-reconstruction (Cuffdiff) and exon-level analysis (DEXSeq). Exon-level analysis performs slightly better than isoform-reconstruction approach although at most only 50% of the spiked-in transcripts are detected. Both isoform-reconstruction and exon-level analysis performances improve by rising the number of input reads. Conclusion Data, derived from NuGEN v2, are not the ideal input for AltDE, specifically when exon-level approach is used. It is notable that ribosomal depletion, with respect to polyA+ selection, reduces the amount of coding mappable reads resulting detrimental in the case of AltDE. Furthermore, we observed that both isoform-reconstruction and exon-level analysis performances are strongly dependent on the number of input reads.

Recent work has demonstrated efficient transformation of structure-borne propagating waves into low-power electricity using metamaterial-inspired mirror configurations. Elastoacoustic waves (i) originating from a point source and (ii) arriving as plane waves have been successfully focused on a piezoelectric energy harvester using elliptical and parabolic mirror concepts, respectively. Our present work investigates the spatial optimization of a piezoelectric energy harvester domain weakly coupled to a thin plate housing an elastoacoustic mirror (or lens). Mirrors considered include elliptical arrangements of periodic stubs, and an elliptical arrangement of continuous material. Spatial and temporal transformation of the wave propagation field into the frequency- wavenumber domain is performed in order to identify the wavenumber content inside the mirror. A frequency- domain root-mean-square (RMS) evaluation is then applied to the transformed field in order to extract the preferred propagation directions. Computational modeling and experimental testing are employed to quantify performance enhancement of the presented approach. Specifically, dramatic enhancement of the harvested power output is reported by patterned electroding of a rectangular PVDF harvester in the elliptical mirror domain.

The paper reports on the design of surface acoustic wave strain sensing devices based on a Fourier approach. A patterned sensing surface acts as a narrow band filter for surface acoustic waves. The center frequencies vary as functions of the amount of strain present in the sensing area, and can be thus used to estimate the surface strain. The design of the sensing pattern in the Fourier space allows the selection of multiple sensing frequencies and for their shifting characteristics to be related to the three components of strain, i.e. two normal and one shear. The result is a surface acoustic wave strain gauge that acts as a strain rosette. The design procedure formulates the problem in the wavenumber domain, whereby the radiation characteristics of the sensing surface in response to an incident broadband surface wave are selected to ensure sensitivity to all three strain components. The concept is first illustrated for a one-dimensional pattern, whose radiation characteristics are governed by a simple, scalar, grating equation. The design approach is then extended to a two-dimensional pattern to demonstrate the ability to simultaneously measure all three surface strain components. The rosette-like operation of the considered strain sensor is demonstrated through numerical simulations conducted on an elastic surface subjected to a pre-imposed strain state. Eventually, the multi-band filtering properties of the proposed sensor patterning are evaluated experimentally by means of a Scanning Laser Doppler Vibrometer (SLDV).

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 81% of all cases of lung cancer and they are often fatal because 60% of the patients are diagnosed at an advanced stage. Besides the need for earlier diagnosis, there is a high need for additional effective therapies. In this work, we investigated the feasibility of a lung cancer progression mouse model, mimicking features of human aggressive NSCLC, as biological reservoir for potential therapeutic targets and biomarkers.,RESULTS: We performed RNA-seq profiling on total RNA extracted from lungs of a 30 week-old K-ras(LA1)/p53(R172H?g) and wild type (WT) mice to detect fusion genes and gene/exon-level differential expression associated to the increase of tumor mass. Fusion events were not detected in K-ras(LA1)/p53(R172H?g) tumors. Differential expression at exon-level detected 33 genes with differential exon usage. Among them nine, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of more than 500 NSCLC RNA-seq transcriptomes. None of the genes showed a significant correlation between exon-level expression and disease prognosis. Differential expression at gene-level allowed the identification of 1513 genes with a significant increase in expression associated to tumor mass increase. 74 genes, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of two transcriptomics datasets of human NSCLC samples, encompassing more than 900 samples. SPP1 was the only molecule whose over-expression resulted statistically related to poor outcome regarding both survival and metastasis formation. Two other molecules showed over-expression associated to poor outcome due to metastasis formation: GM-CSF and ADORA3. GM-CSF is a secreted protein, and we confirmed its expression in the supernatant of a cell line derived by a K-ras(LA1)/p53(R172H?g) mouse tumor. ADORA3 is instead involved in the induction of p53-mediated apoptosis in lung cancer cell lines. Since in our model p53 is inactivated, ADORA3 does not negatively affect tumor growth but remains expressed on tumor cells. Thus, it could represent an interesting target for the development of antibody-targeted therapy on a subset of NSCLC, which are p53 null and ADORA3 positive.,CONCLUSIONS: Our study provided a complete transcription overview of the K-ras(LA1)/p53(R172H?g) mouse NSCLC model. This approach allowed the detection of ADORA3 as a potential target for antibody-based therapy in p53 mutated tumors.

Cardiac overexpression of locally acting muscle-restricted (m)IGF-1 and the consequent downstream activation of NAD+-dependent protein deacetylase sirtuin 1 (SIRT1) trigger potent cardiac antioxidative and antihypertrophic effects. Transient receptor potential (TRP) cation channel A1 (TRPA1) belongs to the TRP ion channel family of molecular detectors of thermal and chemical stimuli that activate sensory neurons to produce pain. Recently, it has been shown that TRPA1 activity influences blood pressure, but the significance of TRPA1 in the cardiovascular system remains elusive. In the present work, using genomic screening in mouse hearts, we found that TRPA1 is a target of mIGF-1/SIRT1 signaling. TRPA1 expression is increased in the heart of cardiac-restricted mIGF-1 transgenic (Tg) mice, both in cardiomyocytes and noncardiomyocytes. In wild-type mice, SIRT1 occupied the TRPA1 promoter, inhibiting its expression, whereas in the presence of the cardiac mIGF-1 transgene, SIRT1 was displaced from the TRPA1 promoter, leading to an increase in its expression. Cardiac-specific ablation of SIRT1 (cardiac-specific knockout) in mIGF-1 Tg mice paradoxically did not increase TRPA1 expression. We have recently reported a systemic “hormetic” effect in mIGF-1 Tg mice, mild hypertension, which was depleted upon cardiac-specific knockout of SIRT1. Administration of the selective TRPA1 antagonist HC-030031 to mIGF-1 Tg mice restored blood pressure to basal levels. We identified TRPA1 as a functional target of the cardiac mIGF-1/SIRT1 signaling pathway, which may have pharmacological implications for the management of cardiovascular stress.

Overexpression of the histone methyltransferase MMSET in t(4;14)+ multiple myeloma patients is believed to be the driving factor in the pathogenesis of this subtype of myeloma. MMSET catalyzes dimethylation of lysine 36 on histone H3 (H3K36me2), and its overexpression causes a global increase in H3K36me2, redistributing this mark in a broad, elevated level across the genome. Here, we demonstrate that an increased level of MMSET also induces a global reduction of lysine 27 trimethylation on histone H3 (H3K27me3). Despite the net decrease in H3K27 methylation, specific genomic loci exhibit enhanced recruitment of the EZH2 histone methyltransferase and become hypermethylated on this residue. These effects likely contribute to the myeloma phenotype since MMSET-overexpressing cells displayed increased sensitivity to EZH2 inhibition. Furthermore, we demonstrate that such MMSET-mediated epigenetic changes require a number of functional domains within the protein, including PHD domains that mediate MMSET recruitment to chromatin. In vivo, targeting of MMSET by an inducible shRNA reversed histone methylation changes and led to regression of established tumors in athymic mice. Together, our work elucidates previously unrecognized interplay between MMSET and EZH2 in myeloma oncogenesis and identifies domains to be considered when designing inhibitors of MMSET function., Author Summary Precise spatial and temporal gene expression is required for normal development, and aberrant regulation of gene expression is a common factor in many diseases, including cancer. Histone modifications contribute to the control of gene expression by altering chromatin structure and affecting the recruitment of transcriptional regulators. In this study, we demonstrate interplay between two oncogenic proteins, MMSET and EZH2, known to methylate histone H3 on lysine 36 (H3K36) and lysine 27 (H3K27), respectively. Overexpression of MMSET in myeloma cells increases global levels of H3K36 methylation, alters its normal distribution throughout the genome and decreases global levels of H3K27 methylation. We found that while the majority of the genome loses H3K27 methylation in the presence of MMSET, certain loci have augmented recruitment of EZH2 and enhanced H3K27 methylation, leading to transcriptional repression. Repression of these genes likely plays an important role in the disease because MMSET-overexpressing cells show higher sensitivity to small molecule inhibitors targeting EZH2-mediated methylation. Thus, our study suggests that the specific local changes may outweigh the gross global changes we frequently observe in cancer and implicates EZH2 as a novel therapeutic target in myeloma cells.

Stiffness and damping are conflicting requirements in many material systems. High stiffness is required in a wide range of structural components to provide sufficient robustness under demanding loading conditions. Simultaneously, a structure should be able to effectively mitigate shock and vibrations dynamically transmitted to it by the environment. While most conventional structures currently exhibit limited adaptability and damping capabilities, design strategies to simultaneously endow structural assemblies with high stiffness and high damping performance are proposed in this work. To this aim, a backbone structure suitable to meet stiffness requirements is combined with metamaterial inclusions able to provide fully-passive shock and vibration absorption. Viscoelastic resonant lattices with chiral topology are employed as inclusions, whose aim is to confine vibrational energy, pump it away from the backbone structure, and dissipate it through viscoelastic damping. The lattices are composed by an elastomeric matrix with the desired chiral shape, and stiff resonating inclusions are inserted at nodal locations. Both finite element simulations and experimental tests demonstrate that periodic chiral assemblies give rise to wide frequency bandgaps. Low-frequency tuning of the assembly for effective suppression of the first resonant mode of a backbone structure represented by an aluminum box-beam is demonstrated both numerically and experimentally. The considered lightweight inclusion is a chiral matrix realized with castable rubber, featuring graded cylinder mass insertions. The proposed design methodology can be flexibly tailored to various frequency ranges and is applicable to both existing and novel structural components at different scales.

In this paper, we investigate the scattering behavior of defects in composite plates. Scattering coefficients are of great importance for the estimation of wave-damage interaction, for the interpretation of recorded Lamb wave signals, and for the development of novel signal processing strategies. The anisotropy of composite laminates makes modeling of wave propagation and the numerical estimation of scattering coefficients particularly challenging. The paper studies numerical models of wave propagation in composites, and evaluates their predictive ability. These evaluations rely of full wave field measurements on selected composite plate specimens, through which information on dispersion and directionality of propagation are conveniently extracted. In addition, filtering in the frequency-wavenumber domain allows the extraction of scattered wave fields and the estimation of the scattering coefficients. Comparisons between numerical and experimental data highlight modeling challenges, illustrate mesh-driven directional propagation, and suggest an effective strategy for the estimation of the scattering coefficients through tests and FE modeling.

Background. Gene fusions arising from chromosomal translocations have been implicated in cancer. RNA-seq has the potential to discover such rearrangements generating functional proteins (chimera/fusion). Recently, many methods for chimeras detection have been published. However, specificity and sensitivity of those tools were not extensively investigated in a comparative way.Results. We tested eight fusion-detection tools (FusionHunter, FusionMap, FusionFinder, MapSplice, deFuse, Bellerophontes, ChimeraScan, and TopHat-fusion) to detect fusion events using synthetic and real datasets encompassing chimeras. The comparison analysis run only on synthetic data could generate misleading results since we found no counterpart on real dataset. Furthermore, most tools report a very high number of false positive chimeras. In particular, the most sensitive tool, ChimeraScan, reports a large number of false positives that we were able to significantly reduce by devising and applying two filters to remove fusions not supported by fusion junction-spanning reads or encompassing large intronic regions.Conclusions. The discordant results obtained using synthetic and real datasets suggest that synthetic datasets encompassing fusion events may not fully catch the complexity of RNA-seq experiment. Moreover, fusion detection tools are still limited in sensitivity or specificity; thus, there is space for further improvement in the fusion-finder algorithms.

Introduction Fusion genes, also known as chimeras, play important roles in tumorigenesis and cancer progression. Then, their role becomes crucial in the areas of biomarkers and therapeutic targets investigation. High-throughput sequencing technologies combined with sophisticated bioinformatics tools might facilitate the discovery of such aberrations. A significant number of bioinformatics algorithms have been developed to detect fusion genes. Detection strategies are quite variegated. In this review, we inspect the strategy of 18 fusion-finder algorithms to understand how these tools call chimeras. Materials and methods In this review, we considered 18 tools which, to the best of our knowledge, are the current state-of-the-art chimera detection tools. Results The considered tools can be classified according to their alignment strategies into four different macro-groups as follows: whole paired-end, paired-end + fragmentation, direct fragmentation and statistical read distribution. The first two techniques require pairedend reads because they exploit encompassing reads during the first alignment phase, while the last two can be applied on both the read formats. Conclusion There is still some work to be done in the area of chimeras detection, especially concerning the definition of common benchmarks and increased specificity.

Enhancement of structure-borne wave energy harvesting is investigated by exploiting metamaterial-based and metamaterial-inspired electroelastic systems. The concepts of wave focusing, funneling, and localization are leveraged to establish novel Metamaterial–Energy Harvester (MEH) configurations. The MEH system transforms the incoming structure-borne wave energy into electrical energy by coupling the metamaterial and electroelastic domains. The energy harvesting component of the work employs piezoelectric transduction due to the high power density and ease of application offered by piezoelectric materials. Therefore, in all MEH configurations studied in this work, the metamaterial system is combined with piezoelectric energy harvesting for enhanced electricity generation from waves propagating in elastic structures. Experiments are conducted to validate the dramatic performance enhancement in MEH systems as compared to using the same volume of piezoelectric patch in the absence of the metamaterial component. It is shown that MEH systems can be used for both broadband and tuned wave energy harvesting. Examples include (1) wave guiding using an acoustic funnel, (2) wave focusing using a metamaterial-inspired elliptical acoustic mirror (both for broadband energy harvesting), and (3) energy localization using an imperfection in a 2-D lattice structure (for tuned energy harvesting).

RNA-seq has the potential to discover genes created by complex chromosomal rearrangements. 'Fusion' genes formed by the breakage and re-joining of two different chromosomes have repeatedly been implicated in the development of cancer. However, although RNA-seq can detect these fusion events, normal cells are also characterized by read-through fusions across adjacent genes in the genome, or by transcription-induced chimeras (TICs). In this paper, we detected TICs events in normal tissue samples. The information derived from our analysis indicates the presence of a significant number of TICs in normal tissues. Some of these TICs can be associated with cancer development. In some cases TIC expression is corroborated by a large number of reads and their expression is significantly spread over different tissues. Our data highlight that TICs could be erroneously associated with cancer aberrations if RNA-seq analysis is only evaluated in tumor samples without considering the parallel analysis of normal tissue samples associated with the tumor.