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Purpose of the article: The aim of this multicenter observational study is to report data from real world on the use of bimekizumab in patients aged ≥ 65 years with moderate-to-severe plaque psoriasis. Elderly patients are poorly represented in clinical trials on bimekizumab for plaque psoriasis, and real-world studies are important to guide clinical choices.Materials and methods: A retrospective multicenter study was conducted in 33 dermatological outpatient clinics in Italy. Patients aged ≥ 65 years, with moderate-to-severe plaque psoriasis and treated with bimekizumab were enrolled. No exclusion criteria were applied. Bimekizumab was administered following the Italian Guidelines for the management of plaque psoriasis and according to the summary of product characteristics, in adult patients who were candidates for systemic treatments. Overall, 98 subjects were included, and received bimekizumab up to week 36. Clinical and demographic data were collected before the initiation of treatment with bimekizumab. At baseline and each dermatological examination (4, 16, and 36 weeks), clinical outcomes were measured by the following parameters: (1) PASI score; (2) site-specific (scalp, palmoplantar, genital, nail) Psoriasis Global Assessment (PGA). At each visit, the occurrence of any adverse events (AEs) was recorded, including serious AEs and AEs leading to bimekizumab discontinuation.Results: The mean PASI score was 16.6 ± 9.4 at baseline and significantly decreased to 4.3 ± 5.2 after 4 weeks (p

We examine the effect of higher-harmonic passive cavities (HHCs) on the beam response to rf noise. Upon invoking certain assumptions to make the problem tractable, we employ Vlasov methods to show that when the dipole approximation applies the HHCs have a generally limited impact. Beam loading in the main cavity is included in the analysis. We illustrate our results and the limitations of our model in application to the Lawrence Berkeley National Laboratory ALS (Advanced Light Source) and the future ALS Upgrade (ALS-U) offering validation against macroparticle simulations.

Abstract The MEG experiment, designed to search for the $${\mu ^+ \rightarrow \hbox {e}^+ \gamma }$$ μ+→e+γ decay, completed data-taking in 2013 reaching a sensitivity level of $${5.3\times 10^{-13}}$$ 5.3×10-13 for the branching ratio. In order to increase the sensitivity reach of the experiment by an order of magnitude to the level of $$6\times 10^{-14}$$ 6×10-14 , a total upgrade, involving substantial changes to the experiment, has been undertaken, known as MEG II. We present both the motivation for the upgrade and a detailed overview of the design of the experiment and of the expected detector performance.

The Advanced Light Source Upgrade will implement on axis single-train swap-out injection employing an accumulator between the booster and storage rings. The accumulator ring (AR) design is a twelve period triple-bend achromat that will be installed along the inner circumference of the storage-ring tunnel. A nonconventional injection scheme will be utilized for top-off off axis injection from the booster into the AR meant to accommodate a large ∼300 nm emittance beam into a vacuum-chamber with a limiting horizontal aperture radius as small as 8 mm. The scheme incorporates three dipole kickers distributed over three sectors, with two kickers perturbing the stored beam and the third affecting both the stored and the injected beam trajectories. This paper describes this “3DK” injection scheme and how it fits the AR’s particular requirements. We describe the design and optimization process, and how we evaluated its fitness as a solution for booster-to-accumulator ring injection.

The concept of using a single nonlinear kicker (NLK) to inject electron beams into a storage ring has been proposed and tested in several synchrotron radiation light source facilities. Different from pulsed dipole kicker magnets used in a conventional local-bump injection, the single nonlinear kicker provides a nonlinear distribution of magnetic fields which has a maximum value off axis where the injected beam arrives and a zero or near-zero value at the center where the stored beam passes by. Therefore, the injected beam will receive a kick from the NLK and lose its transverse momentum, and will be eventually captured by the storage ring. In the meantime the stored beam at the center will receive no kick or less kick, which significantly reduces the injection perturbations on the stored beam. In addition, the NLK injection requires less space for the kicker and removes the complications of synchronizing four pulsed kicker magnets. Because of these advantages, several light source facilities are either proposing or already using this NLK injection as a replacement of the conventional local-bump injection scheme. In this paper, we will discuss the working principal of this NLK injection, and use both Advanced Light Source and Advanced Light source Upgrade as examples to optimize the NLK injections. By optimizing the NLK design and injection conditions, we could achieve maximum injection efficiencies for both facilities with a large injected beam from the existing ALS booster.

The effect of rf harmonic cavities on the transverse mode-coupling instability (TMCI) is still not very well understood. We offer a fresh perspective on the problem by proposing a new numerical method for mode analysis and investigating a regime of potential interest to the new generation of light sources where resistive wall is the dominant source of transverse impedance. When the harmonic cavities are tuned for maximum flattening of the bunch profile we demonstrate that at vanishing chromaticities the transverse single-bunch motion is unstable at any current, with growth rate that in the relevant range scales as the 6th power of the current. With these assumptions and radiation damping included, we find that for machine parameters typical of 4th-generation light sources the presence of harmonic cavities could reduce the instability current threshold by more than a factor two.

The shot-noise driven microbunching instability can significantly degrade electron beam quality in x-ray free electron laser light sources. Experiments were carried out at the Linac Coherent Light Source (LCLS) to study this instability. In this paper, we present start-to-end simulations of the shot-noise driven microbunching instability experiment at the LCLS using the real number of electrons. The simulation results reproduce the measurements quite well. A microbunching self-heating mechanism is also illustrated in the simulation, which helps explain the experimental observation.

The need to operate an rf-gun-based electron photoinjector with a beam emitted away from the cathode center can occur under various circumstances. First, in some cases the cathode can be affected by ion back-bombardment that progressively reduces the quantum efficiency (QE) in its center, making off-axis operation mandatory; second, in some cases the drive laser intensity can be sufficiently high to generate QE depletion in the cathode area illuminated by the laser, forcing off-axis operation; last, in cathodes with nonuniform QE distribution it could be convenient to operate off axis to exploit a better QE. However, operation in this mode may lead to growth of the projected transverse beam emittances due to correlations between the transverse and longitudinal degrees of freedom that are introduced within the gun and downstream rf cavities. A strategy is described to mitigate this emittance growth by allowing the beam to propagate along a carefully tuned off-axis trajectory in downstream rf cavities to remove the time-dependent rf kicks introduced in the gun. Along this trajectory, short range wakefields do not degrade the emittance, and long range wakefields degrade the emittance for very high repetition rate only.

The buildup of low energy electrons in an accelerator, known as electron cloud, can be severely detrimental to machine performance. Under certain beam conditions, the beam can become resonant with the cloud dynamics, accelerating the buildup of electrons. This paper will examine two such effects: multipacting resonances, in which the cloud development time is resonant with the bunch spacing, and cyclotron resonances, in which the cyclotron period of electrons in a magnetic field is a multiple of bunch spacing. Both resonances have been studied directly in dipole fields using retarding field analyzers installed in the Cornell Electron Storage Ring. These measurements are supported by both analytical models and computer simulations.

Using a 1D steady-state free-space coherent synchrotron radiation (CSR) model, we identify a special design setting for a triple-bend isochronous achromat that yields vanishing emittance growth from CSR. When a more refined CSR model with transient effects is included in the analysis, numerical simulations show that the main effect of the transients is to shift the emittance growth minimum slightly, with the minimum changing only modestly.

We identify a new mechanism for generation of the microbunching instability that involves the transverse rather than the longitudinal component of the self-fields. We introduce an analytical model that captures the essential features of the effect and use the model to interpret results from recent LCLS-II design studies.

In this paper we report on start-to-end simulation of a next generation light source based on a high repetition rate free electron laser (FEL) driven by a CW superconducting linac. The simulation integrated the entire system in a seamless start-to-end model, including birth of photoelectrons, transport of electron beam through 600 m of the accelerator beam delivery system, and generation of coherent x-ray radiation in a two-stage self-seeding undulator beam line. The entire simulation used the real number of electrons (∼2 billion electrons/bunch) to capture the details of the physical shot noise without resorting to artificial filtering to suppress numerical noise. The simulation results shed light on several issues including the importance of space-charge effects near the laser heater and the reliability of x-ray radiation power predictions when using a smaller number of simulation particles. The results show that the microbunching instability in the linac can be controlled with 15 keV uncorrelated energy spread induced by a laser heater and demonstrate that high brightness and flux 1 nm x-ray radiation (∼10^{12} photons/pulse) with fully spatial and temporal coherence is achievable.

Free-electron lasers operating in the UV or x-ray radiation spectrum require peak beam currents that are generally higher than those obtainable by present electron sources, thus making bunch compression necessary. Compression, however, may heighten the effects of collective forces and degrade the beam quality. In this paper we provide a framework for investigating some of these effects in rf compressors by focusing on the longitudinal dynamics of small-amplitude density perturbations, which have the potential to cause the disruptive appearance of the so-called microbunching instability. We develop a linear theory valid for low-to-moderate compression factors under the assumption of a 1D impedance model of longitudinal space charge and provide validation against macroparticle simulations.

In this paper we report on large-scale high resolution simulations of beam dynamics in electron linacs for the next-generation x-ray free electron lasers (FELs). We describe key features of a parallel macroparticle simulation code including three-dimensional (3D) space-charge effects, short-range structure wakefields, coherent synchrotron radiation (CSR) wakefields, and treatment of radio-frequency (rf) accelerating cavities using maps obtained from axial field profiles. We present a study of the microbunching instability causing severe electron beam fragmentation in the longitudinal phase space which is a critical issue for future FELs. Using parameters for a proposed FEL linac at Lawrence Berkeley National Laboratory (LBNL), we show that a large number of macroparticles (beyond 100 million) is generally needed to control the numerical macroparticle shot noise and avoid overestimating the microbunching instability. We explore the effect of the longitudinal grid on simulation results. We also study the effect of initial uncorrelated energy spread on the final uncorrelated energy spread of the beam for the FEL linac.

In the presence of rf focusing and a purely inductive impedance bunch, equilibria in the form of Haïssinski distributions—when they exist—are linearly stable. This is the case whether the potential well distortion associated with the impedance causes bunch lengthening or shortening. We provide a general proof of this fact using Hamiltonian methods and energy principles. In the presence of bunch shortening our analysis indicates that there is a critical current for linear stability. However, this threshold is identical to the critical current defining the condition for the very existence of a Haïssinski equilibrium.

Air-core printed-circuit (PC) quadrupoles and dipoles have been developed for the University of Maryland electron ring, currently under construction. The quadrupoles and dipoles are characterized by very small magnetic fields (about 15 G at the aperture edge) and small aspect ratios (length/diameter < 1). We review the theory behind the design of the PC lenses and bending elements, and present general expressions for estimating the values of integrated field and integrated field gradient as functions of design parameters. The new quadrupole magnet represents an improvement over an earlier version which was based on an empirical approach. Further, we summarize the results of multipole content of the magnet fields as measured with a rotating coil apparatus of special construction. The results are compared with calculations with an iron-free magnetics code and are related to different types of errors in the manufacture and assembly of the PC magnets.

We examine the transverse impedance of a periodic array of cavities in a beam pipe at high frequency. The calculation is an extension of a previous one for the longitudinal impedance of a periodic array of azimuthally symmetric pillboxes, for which only TM modes were needed. In the present case, we must include TE modes as well. In addition, we extend the applicability of the previous calculation by including an extra term in the coupling kernel so that the results are valid for all values of the ratio of the cavity length to the period of the structure (all values of the ratio of iris thickness to structure period). In spite of the presence of TE modes, we find that the high frequency limit of the transverse impedance is simply (2/ka^{2}) times the corresponding limit of the longitudinal impedance, just as it is for the resistive wall impedances, a relation which occurs frequently for azimuthally symmetric structures. Finally, we present numerical results as well as approximate expressions for the impedance per period, valid for all ratios of cavity length to structure period.

Cellular senescence is a permanent cell growth arrest that occurs in response to various intrinsic and extrinsic stimuli and is associated with cellular and molecular changes. Long non-coding RNAs (lncRNAs) are key regulators of cellular senescence by affecting the expression of many important genes involved in senescence-associated pathways and processes. Here, we evaluated a panel of lncRNAs associated with senescence for their differential expression between young and senescent human dermal fibroblasts (NHDFs) and studied the effect of a known senomorphic compound, resveratrol, on the expression of lncRNAs in senescent NHDFs. As markers of senescence, we evaluated cell growth, senescence-associated (SA)-β-Gal staining, and the expression of p21, Lamin B1 and γH2AX. We found that H19 and PURPL were the most altered lncRNAs in replicative, in doxorubicin (DOXO) and ionising radiation (IR)-induced senescence models. We then investigated the function of H19 and PURPL in cell senescence by siRNA-mediated silencing in young and senescent fibroblasts, respectively. Our results showed that H19 knockdown reduced cell viability and induced cell senescence and autophagy of NHDFs through the regulation of the PI3K/AKT/mTOR pathway; conversely, PURPL silencing reversed senescence by reducing (SA)-β-Gal staining, recovering cell proliferation with an increase of S-phase cells, and reducing the p53-dependent DNA damage response. Overall, our data highlighted the role of H19 and PURPL in the senescent phenotype and suggested that these lncRNAs may have important implications in senescence-related diseases., (© 2024. The Author(s).)

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized neuropathologically by TDP-43 proteinopathy with loss of TDP-43 nuclear splicing activity and formation of cytoplasmic TDP-43 aggregates. The lack of suitable experimental models of TDP-43 proteinopathy has hampered the discovery of effective therapies. We already showed that chronic and mild oxidative insult by sodium arsenite (ARS) triggered TDP-43 cytoplasmic aggregation and stress granules (SGs) formation in ALS patient-derived fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs). However, whether this insult induces a reduction of TDP-43 splicing activity in the nucleus, thus recapitulating both gain and loss of function pathomechanisms, still remains to be determined. In this study we first showed that chronic ARS in human neuroblastoma cells triggered TDP-43 cytoplasmic mislocalization, SGs formation and defective splicing of TDP-43 target genes UNC13A and POLDIP3 as functional readouts of TDP-43 proteinopathy. Additionally, a dysregulation of autophagy and senescence markers was observed in this condition. In a preliminary drug screening approach with autophagy-promoting drugs, namely rapamycin, lithium carbonate and metformin, only rapamycin prevented ARS-induced loss of TDP-43 splicing activity. We then demonstrated that, in addition to TDP-43 cytoplasmic aggregation, chronic ARS triggered TDP-43 loss of splicing activity also in ALS patient-derived primary fibroblasts and iPSC-MNs and that rapamycin was beneficial to reduce these TDP-43 pathological features. By switching to a neuro-glial 3D in vitro model, we observed that treatment of ALS iPSC-brain organoids with chronic ARS also induced a defective TDP-43 splicing activity which was prevented by rapamycin. Collectively, we established different human cell models of TDP-43 proteinopathy which recapitulate TDP-43 gain and loss of function, prevented by rapamycin administration. Human neuroblastoma cells and patient-derived fibroblasts and 2D- and 3D-iPSC models exposed to chronic oxidative stress represent therefore suitable in vitro platforms for future drug screening approaches in ALS., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Moebius syndrome (MBS) is a rare congenital disorder characterized by non-progressive facial palsy and ocular abduction paralysis. Most cases are sporadic, but also rare familial cases with autosomal dominant transmission and incomplete penetrance/variable expressivity have been described. The genetic etiology of MBS is still unclear: de novo pathogenic variants in REV3L and PLXND1 are reported in only a minority of cases, suggesting the involvement of additional causative genes. With the aim to uncover the molecular causative defect and identify a potential genetic basis of this condition, we performed trio-WES on a cohort of 37 MBS and MBS-like patients. No de novo variants emerged in REV3L and PLXND1 . We then proceeded with a cohort analysis to identify possible common causative genes among all patients and a trio-based analysis using an in silico panel of candidate genes. However, identified variants emerging from both approaches were considered unlikely to be causative of MBS, mainly due to the lack of clinical overlap. In conclusion, despite this large cohort, WES failed to identify mutations possibly associated with MBS, further supporting the heterogeneity of this syndrome, and suggesting the need for integrated omics approaches to identify the molecular causes underlying MBS development.

Cellular senescence is characterized by proliferation and migration exhaustion, senescence-associated secretory phenotype (SASP), and oxidative stress. Senescent vascular smooth muscle cells (VSMCs) contribute to cardiovascular diseases and atherosclerotic plaque instability. Since there are no unanimously agreed senescence markers in human VSMCs, to improve our knowledge, we looked for new possible senescence markers. To this end, we first established and characterized a model of replicative senescence (RS) in human aortic VSMCs. Old cells displayed several established senescence-associated markers. They stained positive for the senescence-associated β-galactosidase, showed a deranged proliferation rate, a dramatically reduced expression of PCNA, an altered migratory activity, increased levels of TP53 and cell-cycle inhibitors p21/p16, and accumulated in the G1 phase. Old cells showed an altered cellular and nuclear morphology, downregulation of the expression of LMNB1 and HMGB1, and increased expression of SASP molecules (IL1β, IL6, IL8, and MMP3). In these senescent VSMCs, among a set of 12 manually selected long non-coding RNAs (lncRNAs), we detected significant upregulation of PURPL and NEAT1. We observed also, for the first time, increased levels of RRAD mRNA. The detection of modulated levels of RRAD, PURPL, and NEAT1 during VSMC senescence could be helpful for future studies on potential anti-aging factors.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of cellular senescence by transcriptionally and post-transcriptionally modulating the expression of many important genes involved in senescence-associated pathways and processes. Among the different lncRNAs associated to senescence, Senescence Associated Long Non-coding RNA (SALNR) was found to be down-regulated in different cellular models of senescence. Since its release in 2015, SALNR has not been annotated in any database or public repository, and no other experimental data have been published. The SALNR sequence is located on the long arm of chromosome 10, at band 10q23.33, and it overlaps the 3' end of the HELLS gene. This investigation helped to unravel the mystery of the existence of SALNR by analyzing publicly available short- and long-read RNA sequencing data sets and RT-PCR analysis in human tissues and cell lines. Additionally, the expression of HELLS has been studied in cellular models of replicative senescence, both in silico and in vitro. Our findings, while not supporting the actual existence of SALNR as an independent transcript in the analyzed experimental models, demonstrate the expression of a predicted HELLS isoform entirely covering the SALNR genomic region. Furthermore, we observed a strong down-regulation of HELLS in senescent cells versus proliferating cells, supporting its role in the senescence and aging process., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Consiglio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Bladder cancer assessment with non-invasive gene expression signatures facilitates the detection of patients at risk and surveillance of their status, bypassing the discomforts given by cystoscopy. To achieve accurate cancer estimation, analysis pipelines for gene expression data (GED) may integrate a sequence of several machine learning and bio-statistical techniques to model complex characteristics of pathological patterns., Methods: Numerical experiments tested the combination of GED preprocessing by discretization with tree ensemble embeddings and nonlinear dimensionality reductions to categorize oncological patients comprehensively. Modeling aimed to identify tumor stage and distinguish survival outcomes in two situations: complete and partial data embedding. This latter experimental condition simulates the addition of new patients to an existing model for rapid monitoring of disease progression. Machine learning procedures were employed to identify the most relevant genes involved in patient prognosis and test the performance of preprocessed GED compared to untransformed data in predicting patient conditions., Results: Data embedding paired with dimensionality reduction produced prognostic maps with well-defined clusters of patients, suitable for medical decision support. A second experiment simulated the addition of new patients to an existing model (partial data embedding): Uniform Manifold Approximation and Projection (UMAP) methodology with uniform data discretization led to better outcomes than other analyzed pipelines. Further exploration of parameter space for UMAP and t-distributed stochastic neighbor embedding (t-SNE) underlined the importance of tuning a higher number of parameters for UMAP rather than t-SNE. Moreover, two different machine learning experiments identified a group of genes valuable for partitioning patients (gene relevance analysis) and showed the higher precision obtained by preprocessed data in predicting tumor outcomes for cancer stage and survival rate (six classes prediction)., Conclusions: The present investigation proposed new analysis pipelines for disease outcome modeling from bladder cancer-related biomarkers. Complete and partial data embedding experiments suggested that pipelines employing UMAP had a more accurate predictive ability, supporting the recent literature trends on this methodology. However, it was also found that several UMAP parameters influence experimental results, therefore deriving a recommendation for researchers to pay attention to this aspect of the UMAP technique. Machine learning procedures further demonstrated the effectiveness of the proposed preprocessing in predicting patients' conditions and determined a sub-group of biomarkers significant for forecasting bladder cancer prognosis., (© 2022. The Author(s).)

Psychiatric disorders represent a heterogeneous class of multifactorial mental diseases whose origin entails a pathogenic integration of genetic and environmental influences. Incidence of these pathologies is dangerously high, as more than 20% of the Western population is affected. Despite the diverse origins of specific molecular dysfunctions, these pathologies entail disruption of fine synaptic regulation, which is fundamental to behavioral adaptation to the environment. The synapses, as functional units of cognition, represent major evolutionary targets. Consistently, fine synaptic tuning occurs at several levels, involving a novel class of molecular regulators known as long non-coding RNAs (lncRNAs). Non-coding RNAs operate mainly in mammals as epigenetic modifiers and enhancers of proteome diversity. The prominent evolutionary expansion of the gene number of lncRNAs in mammals, particularly in primates and humans, and their preferential neuronal expression does represent a driving force that enhanced the layering of synaptic control mechanisms. In the last few years, remarkable alterations of the expression of lncRNAs have been reported in psychiatric conditions such as schizophrenia, autism, and depression, suggesting unprecedented mechanistic insights into disruption of fine synaptic tuning underlying severe behavioral manifestations of psychosis. In this review, we integrate literature data from rodent pathological models and human evidence that proposes the biology of lncRNAs as a promising field of neuropsychiatric investigation.

The incidence of cancer and Alzheimer's disease (AD) increases exponentially with age. A growing body of epidemiological evidence and molecular investigations inspired the hypothesis of an inverse relationship between these two pathologies. It has been proposed that the two diseases might utilize the same proteins and pathways that are, however, modulated differently and sometimes in opposite directions. Investigation of the common processes underlying these diseases may enhance the understanding of their pathogenesis and may also guide novel therapeutic strategies. Starting from a text-mining approach, our in silico study integrated the dispersed biological evidence by combining data mining, gene set enrichment, and protein-protein interaction (PPI) analyses while searching for common biological hallmarks linked to AD and cancer. We retrieved 138 genes (ALZCAN gene set), computed a significant number of enriched gene ontology clusters, and identified four PPI modules. The investigation confirmed the relevance of autophagy, ubiquitin proteasome system, and cell death as common biological hallmarks shared by cancer and AD. Then, from a closer investigation of the PPI modules and of the miRNAs enrichment data, several genes ( SQSTM1 , UCHL1 , STUB1 , BECN1 , CDKN2A , TP53 , EGFR , GSK3B , and HSPA9 ) and miRNAs (miR-146a-5p, MiR-34a-5p, miR-21-5p, miR-9-5p, and miR-16-5p) emerged as promising candidates. The integrative approach uncovered novel miRNA-gene networks (e.g., miR-146 and miR-34 regulating p62 and Beclin1 in autophagy) that might give new insights into the complex regulatory mechanisms of gene expression in AD and cancer., (Copyright © 2019 Battaglia, Venturin, Sojic, Jesuthasan, Orro, Spinelli, Musicco, De Bellis and Adorni.)

The abnormal deposition of proteins in brain tissue is a common feature of neurodegenerative diseases (NDs) often accompanied by the spread of mutated proteins, causing neuronal toxicity. Exosomes play a fundamental role on their releasing in extracellular space after endosomal pathway activation, allowing to remove protein aggregates by lysosomal degradation or their inclusion into multivesicular bodies (MVBs), besides promoting cellular cross-talk. The emerging evidence of pathogenic mutations associated to ND susceptibility, leading to impairment of exosome production and secretion, opens a new perspective on the mechanisms involved in neurodegeneration. Recent findings suggest to investigate the genetic mechanisms regulating the different exosome functions in central nervous system (CNS), to understand their role in the pathogenesis of NDs, addressing the identification of diagnostic and pharmacological targets. This review aims to summarize the mechanisms underlying exosome biogenesis, their molecular composition and functions in CNS, with a specific focus on the recent findings invoking a defective exosome biogenesis as a common biological feature of the major NDs, caused by genetic alterations. Further definition of the consequences of specific genetic mutations on exosome biogenesis and release will improve diagnostic and pharmacological studies in NDs.

Cyclin-dependent kinase 5 regulatory subunit 1 ( CDK5R1 ) gene encodes for p35, the main activator of Cyclin-dependent kinase 5 (CDK5). The active p35/CDK5 complex is involved in numerous aspects of brain development and function, and its deregulation is closely associated to Alzheimer's disease (AD) onset and progression. We recently showed that miR-15/107 family can negatively regulate CDK5R1 expression modifying mRNA stability. Interestingly, miRNAs belonging to miR-15/107 family are downregulated in AD brain while CDK5R1 is upregulated. Long non-coding RNAs (lncRNAs) are emerging as master regulators of gene expression, including miRNAs, and their dysregulation has been implicated in the pathogenesis of AD. Here, we evaluated the existence of an additional layer of CDK5R1 expression regulation provided by lncRNAs. In particular, we focused on three lncRNAs potentially regulating CDK5R1 expression levels, based on existing data: NEAT1, HOTAIR, and MALAT1. We demonstrated that NEAT1 and HOTAIR negatively regulate CDK5R1 mRNA levels, while MALAT1 has a positive effect. We also showed that all three lncRNAs positively control miR-15/107 family of miRNAs. Moreover, we evaluated the expression of NEAT1, HOTAIR, and MALAT1 in AD and control brain tissues. Interestingly, NEAT1 displayed increased expression levels in temporal cortex and hippocampus of AD patients. Interestingly, we observed a strong positive correlation between CDK5R1 and NEAT1 expression levels in brain tissues, suggesting a possible neuroprotective role of NEAT1 in AD to compensate for increased CDK5R1 levels. Overall, our work provides evidence of another level of CDK5R1 expression regulation mediated by lncRNAs and points to NEAT1 as a biomarker, as well as a potential pharmacological target for AD therapy.

Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) encodes p35, the main activatory subunit of cyclin-dependent kinase 5 (CDK5). The p35/CDK5 active complex plays a fundamental role in brain development and functioning, but its deregulated activity has also been implicated in various neurodegenerative disorders, including Alzheimer's disease (AD). CDK5R1 displays a large and highly evolutionarily conserved 3'-untranslated region (3'-UTR), a fact that has suggested a role for this region in the post-transcriptional control of CDK5R1 expression. Our group has recently demonstrated that two miRNAs, miR-103 and miR-107, regulate CDK5R1 expression and affect the levels of p35. MiR-103 and miR-107 belong to the miR-15/107 family, a group of evolutionarily conserved miRNAs highly expressed in human cerebral cortex. In this work, we tested the hypothesis that other members of this group of miRNAs, in addition to miR-103 and miR-107, were able to modulate CDK5R1 expression. We provide evidence that several miRNAs belonging to the miR-15/107 family regulate p35 levels. BACE1 expression levels were also found to be modulated by different members of this family. Furthermore, overexpression of these miRNAs led to reduced APP phosphorylation levels at the CDK5-specific Thr668 residue. We also show that miR-15/107 miRNAs display reduced expression levels in hippocampus and temporal cortex, but not in cerebellum, of AD brains. Moreover, increased CDK5R1 mRNA levels were observed in AD hippocampus tissues. Our results suggest that the downregulation of the miR-15/107 family might have a role in the pathogenesis of AD by increasing the levels of CDK5R1/p35 and consequently enhancing CDK5 activity.

Cyclin-dependent kinase 5 (CDK5) and cyclin-dependent kinase 5, regulatory subunit 1 (CDK5R1), encoding CDK5 activator p35, have a fundamental role in central nervous system (CNS) development and function, and are involved in the pathogenesis of several neurodegenerative disorders, thus constituting strong candidate genes for the onset of intellectual disability (ID). We carried out a mutation screening of CDK5 and CDK5R1 coding regions and CDK5R1 3'-UTR on a cohort of 360 patients with non-syndromic ID (NS-ID) using denaturing high performance liquid chromatography (DHPLC) and direct sequencing. We found one novel silent mutation in CDK5 and one novel silent mutation in CDK5R1 coding regions, three novel intronic variations in CDK5, not causing any splicing defect, and four novel heterozygous variations in CDK5R1 3'-UTR. None of these variations was present in 450 healthy controls and single-nucleotide polymorphism (SNP) databases. The functional study of CDK5R1 p.A108V mutation evidenced an impaired p35 cleavage by the calcium-dependent protease calpain. Moreover, luciferase constructs containing the CDK5R1 3'-UTR mutations showed altered gene expression levels. Eight known polymorphisms were also identified displaying different frequencies in NS-ID patients compared with the controls. In particular, the minor allele of CDK5R1 3'-UTR rs735555 polymorphism was associated with increased risk for NS-ID. In conclusion, our data suggest that mutations and polymorphisms in CDK5 and CDK5R1 genes may contribute to the onset of the NS-ID phenotype.

Background: Long-non-coding RNAs (lncRNAs), RNA molecules longer than 200 nucleotides, have been involved in several biological processes and in a growing number of diseases, controlling gene transcription, pre-mRNA processing, the transport of mature mRNAs to specific cellular compartments, the regulation of mRNA stability, protein translation and turnover. The fundamental role of lncRNAs in central nervous system (CNS) is becoming increasingly evident. LncRNAs are abundantly expressed in mammalian CNS in a specific spatio-temporal manner allowing a quick response to environmental/molecular changes., Methods: This article reviews the biology and mechanisms of action of lncRNAs underlying their potential role in CNS and in some neurodegenerative diseases., Results: an increasing number of studies report on lncRNAs involvement in different molecular mechanisms of gene expression modulation in CNS, from neural stem cell differentiation mainly by chromatin remodeling, to control of neuronal activities. More recently, lncRNAs have been implicated in neurodegenerative diseases, including Alzheimer's Disease, where the role of BACE1-AS lncRNA has been widely defined. BACE1-AS levels are up-regulated in AD brains where BACE1-AS acts by stabilizing BACE1 mRNA thereby increasing BACE1 protein content and Aβ42 formation. In Frontotemporal dementia and Amyotrophic lateral sclerosis the lncRNAs NEAT1&#95;2 and MALAT1 co-localize at nuclear paraspeckles with TDP-43 and FUS proteins and their binding to TDP-43 is markedly increased in affected brains. In Parkinson's Disease the lncRNA UCHL1-AS1 acts by directly promoting translation of UCHL1 protein leading to perturbation of the ubiquitin-proteasome system. Different lncRNAs, such as HTT-AS, BDNF-AS and HAR1, were found to be dysregulated in their expression also in Huntington's Disease. In Fragile X syndrome (FXS) and Fragile X tremor/ataxia syndrome (FXTAS) patients, the presence of CGG repeats expansion alters the expression of the lncRNAs FMR1-AS1 and FMR6. Interestingly, they are expressed in peripheral blood leukocytes, suggesting these lncRNAs may represent biomarkers for FXS/FXTAS early detection and therapy. Finally, the identification of the antisense RNAs SCAANT1-AS and ATXN8OS in spinocerebellar ataxia 7 and 8, respectively, suggests that very different mechanisms of action driven by lncRNAs may trigger neurodegeneration in these disorders., Conclusion: The emerging role of lncRNAs in neurodegenerative diseases suggests that their dysregulation could trigger neuronal death via still unexplored RNA-based regulatory mechanisms which deserve further investigation. The evaluation of their diagnostic significance and therapeutic potential could also address the setting up of novel treatments in diseases where no cure is available to date.

Background: Cardiovascular malformations have a higher incidence in patients with NF1 microdeletion syndrome compared to NF1 patients with intragenic mutation, presumably owing to haploinsufficiency of one or more genes included in the deletion interval and involved in heart development. In order to identify which genes could be responsible for cardiovascular malformations in the deleted patients, we carried out expression studies in mouse embryos and functional studies in zebrafish., Methods and Results: The expression analysis of three candidate genes included in the NF1 deletion interval, ADAP2, SUZ12 and UTP6, performed by in situ hybridisation, showed the expression of ADAP2 murine ortholog in heart during fundamental phases of cardiac morphogenesis. In order to investigate the role of ADAP2 in cardiac development, we performed loss-of-function experiments of zebrafish ADAP2 ortholog, adap2, by injecting two different morpholino oligos (adap2-MO and UTR-adap2-MO). adap2-MOs-injected embryos (morphants) displayed in vivo circulatory and heart shape defects. The molecular characterisation of morphants with cardiac specific markers showed that the injection of adap2-MOs causes defects in heart jogging and looping. Additionally, morphological and molecular analysis of adap2 morphants demonstrated that the loss of adap2 function leads to defective valvulogenesis, suggesting a correlation between ADAP2 haploinsufficiency and the occurrence of valve defects in NF1-microdeleted patients., Conclusions: Overall, our findings indicate that ADAP2 has a role in heart development, and might be a reliable candidate gene for the occurrence of cardiovascular malformations in patients with NF1 microdeletion and, more generally, for the occurrence of a subset of congenital heart defects., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) encodes p35, a specific activator of cyclin-dependent kinase 5 (CDK5). CDK5 and p35 have a fundamental role in neuronal migration and differentiation during CNS development. Both the CDK5R1 3'-UTR's remarkable size and its conservation during evolution strongly indicate an important role in post-transcriptional regulation. We previously validated different regulatory elements in the 3'-UTR of CDK5R1, which affect transcript stability, p35 levels and cellular migration through the binding with nELAV proteins and miR-103/7 miRNAs. Interestingly, a 138 bp-long region, named C2.1, was identified as the most mRNA destabilizing portion within CDK5R1 3'-UTR. This feature was maintained by a shorter region of 73 bp, characterized by two poly-U stretches. UV-CL experiments showed that this region interacts with protein factors. UV-CLIP assays and pull-down experiments followed by mass spectrometry analysis demonstrated that nELAV and hnRNPA2/B1 proteins bind to the same U-rich element. These RNA-binding proteins (RBPs) were shown to oppositely control CDK5R1 mRNA stability and p35 protein content at post-trascriptional level. While nELAV proteins have a positive regulatory effect, hnRNPA2/B1 has a negative action that is responsible for the mRNA destabilizing activity both of the C2.1 region and of the full-length 3'-UTR. In co-expression experiments of hnRNPA2/B1 and nELAV RBPs we observed an overall decrease of p35 content. We also demonstrated that hnRNPA2/B1 can downregulate nELAV protein content but not vice versa. This study, by providing new insights on the combined action of different regulatory factors, contributes to clarify the complex post-transcriptional control of CDK5R1 gene expression., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this report, we describe two adult brothers affected by moderate non-specific intellectual disability (ID). They showed minor facial anomalies, not clearly ascribable to any specific syndromic patterns, microcephaly, brachydactyly and broad toes. Both brothers presented seizures. Karyotype, subtelomeric and FMR1 analysis were normal in both cases. We performed array-CGH analysis that revealed no copy-number variations potentially associated with ID. Subsequent exome sequence analysis allowed the identification of the ATRX c.109C>T (p.R37X) mutation in both the affected brothers. Sanger sequencing confirmed the presence of the mutation in the brothers and showed that the mother is a healthy carrier. Mutations in the ATRX gene cause the X-linked alpha thalassemia/mental retardation (ATR-X) syndrome (MIM #301040), a severe clinical condition usually associated with profound ID, facial dysmorphism and alpha thalassemia. However, the syndrome is clinically heterogeneous and some mutations, including the c.109C>T, are associated with a broad phenotypic spectrum, with patients displaying a less severe phenotype with only mild-moderate ID. In the case presented here, exome sequencing provided an effective strategy to achieve the molecular diagnosis of ATR-X syndrome, which otherwise would have been difficult to consider due to the mild non-specific phenotype and the absence of a family history with typical severe cases.

Centaurin-α₂ is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-α₂ can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-α₂ interacting protein, β-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of β-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-α₂ overexpression in HeLa cells and extraction of soluble (αβ dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-α₂ mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-α₂ remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-α₂ and tubulin confirmed that Centaurin-α₂ promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-α₂ overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-α₂ role on MT stabilization. Centaurin-α₂ interacts with β-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-α₂ as a new microtubule-associated protein (MAP) increasing MT stability.

CDK5R1 encodes p35, a specific activator of the serine/threonine kinase CDK5, which plays crucial roles in CNS development and maintenance. CDK5 activity strongly depends on p35 levels and p35/CDK5 misregulation is deleterious for correct CNS function, suggesting that a tightly controlled regulation of CDK5R1 expression is needed for proper CDK5 activity. Accordingly, CDK5R1 expression was demonstrated to be controlled at both transcriptional and post-transcriptional levels, but a possible regulation through microRNAs (miRNAs) has never been investigated. We predicted, within the large CDK5R1 3'UTR several miRNA target sites. Among them, we selected for functional studies miR-103 and miR-107, whose expression has shown a strong inverse correlation with p35 levels in different cell lines. A significant reduction of CDK5R1 mRNA and p35 levels was observed after transfection of SK-N-BE neuroblastoma cells with the miR-103 or miR-107 precursor (pre-miR-103 or pre-miR-107). Conversely, p35 levels significantly increased following transfection of the corresponding antagonists (anti-miR-103 or anti-miR-107). Moreover, the level of CDK5R1 transcript shifts from the polysomal to the subpolysomal mRNA fraction after transfection with pre-miR-107 and, conversely, from the subpolysomal to the polysolmal mRNA fraction after transfection with anti-miR-107, suggesting a direct action on translation efficiency. We demonstrate, by means of luciferase assays, that miR-103 and miR-107 are able to directly interact with the CDK5R1 3'-UTR, in correspondence of a specific target site. Finally, miR-103 and miR-107 overexpression, as well as CDK5R1 silencing, caused a reduction in SK-N-BE migration ability, indicating that these miRNAs affect neuronal migration by modulating CDK5R1 expression. These findings indicate that miR-103 and miR-107 regulate CDK5R1 expression, allowing us to hypothesize that a miRNA-mediated mechanism may influence CDK5 activity and the associated molecular pathways.

Noonan syndrome is a genetic condition characterized by congenital heart defects, short stature, and characteristic facial features. Familial or de novo mutations in PTPN11, RAF1, SOS1, KRAS, and NRAS are responsible for 60-75% of the cases, thus, additional genes are expected to be involved in the pathogenesis. In addition, the genotype-phenotype correlation has been hindered by the highly variable expressivity of the disease. For all these reasons, expanding the genotyped and clinically evaluated case numbers will benefit the clinical community. A mutation analysis has been performed on RAF1, SOS1, and GRB2, in 24 patients previously found to be negative for PTPN11 and KRAS mutations. We identified four mutations in SOS1 and one in RAF1, while no GRB2 variants have been found. Interestingly, the RAF1 mutation was present in a patient also carrying a newly identified p.R497Q familial SOS1 mutation, segregating with a typical Noonan Syndrome SOS1 cutaneous phenotype. Functional analysis demonstrated that the R497Q SOS1 mutation leads to Jnk activation, but has no effect on the Ras effector Erk1. We propose that this variant might contribute to the onset of the peculiar ectodermal traits displayed by the propositus amidst the more classical Noonan syndrome presentation. To our knowledge, this is the first reported case of a patient harboring mutations in two genes, with an involvement of both Ras and Rac1 pathways, indicating that SOS1 may have a role of modifier gene that might contribute the variable expressivity of the disease, evidencing a genotype-phenotype correlation in the family., (Copyright 2010 Wiley-Liss, Inc.)

Urokinase-type plasminogen activator (uPA) and c-met play a major role in cancer invasion and metastasis. Evidence has suggested that uPA and c-met overexpression may be coordinated in human hepatocellular carcinoma (HCC). In the present study, to understand whether the expression of these genes might be coregulated by specific microRNAs (miRs) in human cells, we predicted that Homo sapiens microRNA-23b could recognize two sites in the 3'-UTR of uPA and four sites in the c-met 3'-UTR by the algorithm pictar. The miR-23b expression analysis in human tumor and normal cells revealed an inverse trend with uPA and c-met expression, indicating that uPA and c-met negative regulation might depend on miR-23b expression. Transfection of miR-23b molecules in HCC cells (SKHep1C3) led to inhibition of protein expression of the target genes and caused a decrease in cell migration and proliferation capabilities. Furthermore, anti-miR-23b transfection in human normal AB2 dermal fibroblasts upregulated the expression of endogenous uPA and c-met. Cotransfection experiments in HCC cells of the miR-23b with pGL4.71 Renilla luciferase reporter gene constructs, containing the putative uPA and c-met 3'-UTR target sites, and with the pGL3 firefly luciferase-expressing vector showed a decrease in the relative luciferase activity. This would indicate that miR-23b can recognize target sites in the 3'-UTR of uPA and of c-met mRNAs and translationally repress the expression of uPA and c-met in HCC cells. The evidence obtained shows that overexpression of miR-23b leads to uPA and c-met downregulation and to decreased migration and proliferation abilities of HCC cells.

Neurofibromatosis type 1 (NF1) is a common genetic disease caused by haploinsufficiency of the NF1 tumor-suppressor gene. Different pathogenetic mechanisms have been identified, with the majority (95%) causing intragenic lesions. Single or multiexon NF1 copy number changes occur in about 2% of patients, but little is known about the molecular mechanisms behind these intragenic deletions. We report here on the molecular characterization of a novel NF1 multiexonic deletion. The application of a multidisciplinary approach including multiplex ligation-dependent probe amplification, allelic segregation analysis, and fluorescent in situ hybridization allowed us to map the breakpoints in IVS27b and IVS48. Furthermore, the breakpoint junction was characterized by sequencing. Using bioinformatic analysis, we identified some recombinogenic motifs in close proximity to the centromeric and telomeric breakpoints and predicted the presence of a mutated messenger ribonucleic acid, which was deleted between exons 28 and 48 and encodes a neurofibromin that lacks some domains essential for its function. Through reverse transcriptase-polymerase chain reaction, the expression of the mutated allele was verified, showing the junction between exons 27b and 49 and, as expected, was not subjected to nonsense-mediated decay. Multiexonic deletions represent 2% of NF1 mutations, and until now, the breakpoint has been identified in only a few cases. The fine characterization of multiexonic deletions broadens the mutational repertoire of the NF1 gene, allowing for the identification of different pathogenetic mechanisms causing NF1.

Background: CDK5R1 plays a central role in neuronal migration and differentiation during central nervous system development. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3'-untranslated region (3'-UTR) suggests a role in post-transcriptional regulation of CDK5R1 expression., Results: The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs). The insertion of CDK5R1 3'-UTR into luciferase 3'-UTR causes a decreased luciferase activity in four transfected cell lines. We identified 3'-UTR subregions which tend to reduce the reporter gene expression, sometimes in a cell line-dependent manner. In most cases the quantitative analysis of luciferase mRNA suggests that CDK5R1 3'-UTR affects mRNA stability. A region, leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. The 3' end of the transcript, containing a class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. We also observed the interaction of the stabilizing neuronal RNA-binding proteins ELAV with the CDK5R1 transcript in SH-SY5Y cells and identified three 3'-UTR sub-regions showing affinity for ELAV proteins., Conclusion: Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3'-UTR and support the hypothesis that CDK5R1 gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3'-UTR in the modulation of CDK5R1 expression. The fine tuning of CDK5R1 expression by 3'-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders.

Mental retardation (MR) is displayed by 57% of NF1 patients with microdeletion syndrome as a result of 17q11.2 region haploinsufficiency. We considered the cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) and oligodendrocyte-myelin glycoprotein (OMG) genes, mapping in the NF1 microdeleted region, as candidate genes for MR susceptibility. CDK5R1 encodes for a neurone-specific activator of cyclin-dependent kinase 5 (CDK5) involved in neuronal migration during central nervous system development. OMG encodes for an inhibitor of neurite outgrowth by the binding to the Nogo-66 receptor (RTN4R). CDK5R1 and OMG genes are characterized by large 3' and 5' untranslated regions (UTRs), where we predict the presence of several transcription/translation regulatory elements. We screened 100 unrelated Italian patients affected by unspecific MR for mutations in CDK5R1 and OMG coding regions and in their 3' or 5' UTRs. Four novel mutations and two novel polymorphisms for CDK5R1 and three novel mutations for OMG were detected, including two missense changes (c.323C>T; A108V in CDK5R1 and c.1222A>G; T408A in OMG), one synonymous codon variant (c.532C>T; L178L in CDK5R1), four variants in CDK5R1 3'UTR and two changes in OMG 5'UTR. All the mutations were absent in 370 chromosomes from normal subjects. The allelic frequencies of the two novel polymorphisms in CDK5R1 3'UTR were established in both 185 normal and 100 mentally retarded subjects. Prediction of mRNA and protein secondary structures revealed that two changes lead to putative structural alterations in the mutated c.2254C>G CDK5R1 3'UTR and in OMG T408A gene product.