Deficiency of Glucocerebrosidase Activity beyond Gaucher Disease: PSAP and LIMP-2 Dysfunctions.

Glucocerebrosidase (GCase) is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide in the presence of its activator saposin C (SapC). SapC arises from the proteolytical cleavage of prosaposin (encoded by PSAP gene), which gives rise to four saposins. GCase is targeted to the lysosomes by LIMP-2, encoded by SCARB2 gene. GCase deficiency causes Gaucher Disease (GD), which is mainly due to biallelic pathogenetic variants in the GCase-encoding gene, GBA1. However, impairment of GCase activity can be rarely caused by SapC or LIMP-2 deficiencies. We report a new case of LIMP-2 deficiency and a new case of SapC deficiency (missing all four saposins, PSAP deficiency), and measured common biomarkers of GD and GCase activity. Glucosylsphingosine and chitotriosidase activity in plasma were increased in GCase deficiencies caused by PSAP and GBA1 mutations, whereas SCARB2-linked deficiency showed only Glucosylsphingosine elevation. GCase activity was reduced in fibroblasts and leukocytes: the decrease was sharper in GBA1- and SCARB2-mutant fibroblasts than PSAP-mutant ones; LIMP-2-deficient leukocytes displayed higher residual GCase activity than GBA1-mutant ones. Finally, we demonstrated that GCase mainly undergoes proteasomal degradation in LIMP-2-deficient fibroblasts and lysosomal degradation in PSAP-deficient fibroblasts. Thus, we analyzed the differential biochemical profile of GCase deficiencies due to the ultra-rare PSAP and SCARB2 biallelic pathogenic variants in comparison with the profile observed in GBA1-linked GCase deficiency.

Steps dominate gas evasion from a mountain headwater stream.

Steps are dominant morphologic traits of high-energy streams, where climatically- and biogeochemically-relevant gases are processed, transported to downstream ecosystems or released into the atmosphere. Yet, capturing the imprint of the small-scale morphological complexity of channel forms on large-scale river outgassing represents a fundamental unresolved challenge. Here, we combine theoretical and experimental approaches to assess the contribution of localized steps to the gas evasion from river networks. The framework was applied to a representative, 1 km-long mountain reach in Italy, where carbon dioxide concentration drops across several steps and a reference segment without steps were measured under different hydrologic conditions. Our results indicate that local steps lead the reach-scale outgassing, especially for high and low discharges. These findings suggest that steps are key missing components of existing scaling laws used for the assessment of gas fluxes across water-air interfaces. Therefore, global evasion from rivers may differ substantially from previously reported estimates.

Beating heart implantation of transventricular artificial cordae: How can access site selection and leaflet insertion improve mitral regurgitation correction?

NeoChord-DS1000-System (NC) and The Harpoon-Mitral-Repair-System (H-MRS) are two trans-apical chordal implantation devices developed for the treatment of degenerative mitral valve (MV) regurgitation (DMR) either if as Fibroelastic-Deficiency (FED), Forma-Frusta (FF), or Barlow (B) presentation. The aim of this study is to evaluate some of the advantages and disadvantages of these two different devices by performing numerical simulation analyses focused on different transventricular access sites in all subsets of DMR presentations. By applying a novel approach for the development of patient-specific MV domains we worked out a set of numerical simulations of the artificial chordae implantation. Different leaflet insertions and ventricle access sites were investigated, and resulting contact-area (CA), tensioning-forces (F) and leaflet's stress (LS) were calculated. The analyses showed that: i) NC-approach maintains low LS when performed with a posterior access site and optimizes the overlap between the leaflets at the systolic peak; ii) H-MRS-system presents better results in case of a more anterior ventricular entry site; however, for FED prolapse large variation of F and LS with respect to NC-approach are found; iii) an accidental contact between artificial sutures and the anterior leaflet may occur when valve function is restored through an excessive anterior access site. Present findings set light on specific technical aspects of transapical off-pump chords implantation, either performed with NC and H-MRS systems and highlight the advantages and disadvantages proper to the two devices. Our study also paves the basis for a systematic application of computational methodology, in order to plan a patient-specific mini-invasive approach thus maximizing the outcomes.

Distensibility of Deformable Aortic Replicas Assessed by an Integrated In-Vitro and In-Silico Approach.

The correct estimation of the distensibility of deformable aorta replicas is a challenging issue, in particular when its local characterization is necessary. We propose a combined in-vitro and in-silico approach to face this problem. First, we tested an aortic silicone arch in a pulse-duplicator analyzing its dynamics under physiological working conditions. The aortic flow rate and pressure were measured by a flow meter at the inlet and two probes placed along the arch, respectively. Video imaging analysis allowed us to estimate the outer diameter of the aorta in some sections in time. Second, we replicated the in-vitro experiment through a Fluid-Structure Interaction simulation. Observed and computed values of pressures and variations in aorta diameters, during the cardiac cycle, were compared. Results were considered satisfactory enough to suggest that the estimation of local distensibility from in-silico tests is reliable, thus overcoming intrinsic experimental limitations. The aortic distensibility (AD) is found to vary significantly along the phantom by ranging from 3.0 × 10-3 mmHg-1 in the ascending and descending tracts to 4.2 × 10-3 mmHg-1 in the middle of the aortic arch. Interestingly, the above values underestimate the AD obtained in preliminary tests carried out on straight cylindrical samples made with the same material of the present phantom. Hence, the current results suggest that AD should be directly evaluated on the replica rather than on the samples of the adopted material. Moreover, tests should be suitably designed to estimate the local rather than only the global distensibility.

Numerical Models Can Assist Choice of an Aortic Phantom for In Vitro Testing.

(1) Background: The realization of appropriate aortic replicas for in vitro experiments requires a suitable choice of both the material and geometry. The matching between the grade of details of the geometry and the mechanical response of the materials is an open issue that deserves attention. (2) Methods: To explore this issue, we performed a series of Fluid-Structure Interaction simulations, which compared the dynamics of three aortic models. Specifically, we reproduced a patient-specific geometry with a wall of biological tissue or silicone, and a parametric geometry based on in vivo data made in silicone. The biological tissue and the silicone were modeled with a fiber-oriented anisotropic and isotropic hyperelastic model, respectively. (3) Results: Clearly, both the aorta's geometry and its constitutive material contribute to the determination of the aortic arch deformation; specifically, the parametric aorta exhibits a strain field similar to the patient-specific model with biological tissue. On the contrary, the local geometry affects the flow velocity distribution quite a lot, although it plays a minor role in the helicity along the arch. (4) Conclusions: The use of a patient-specific prototype in silicone does not a priori ensure a satisfactory reproducibility of the real aorta dynamics. Furthermore, the present simulations suggest that the realization of a simplified replica with the same compliance of the real aorta is able to mimic the overall behavior of the vessel.

Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test.

Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.

Understanding and recognition of the right ventricular function and dysfunction via a numerical study.

The role played by the right ventricular (RV) dysfunction has long been underestimated in clinical practice. Recent findings are progressively confirming that when the RV efficiency deteriorates both the right and the left circulation is (significantly) affected, but studies dedicated to a detailed description of RV hemodynamic role still lack. In response to such a gap in knowledge, this work proposes a numerical model that for the first time evaluates the effect of isolated RV dysfunction on the whole circulation. Lumped parameter modelling was applied to represent the physio-pathological hemodynamics. Different grades of impairment were simulated for three dysfunctions i.e., systolic, diastolic, and combined systolic and diastolic. Hemodynamic alterations (i.e., of blood pressure, flow, global hemodynamic parameters), arising from the dysfunctions, are calculated and analysed. Results well accord with clinical observations, showing that RV dysfunction significantly affects both the pulmonary and systemic hemodynamics. Successful verification against in vivo data proved the clinical potentiality of the model i.e., the capability of identifying the degree of RV impairment for given hemodynamic conditions. This study aims at contributing to the improvement of RV dysfunction recognition and treatment, and to the development of tools for the clinical management of pathologies involving the right heart.

Deferoxamine mesylate improves splicing and GAA activity of the common c.-32-13T>G allele in late-onset PD patient fibroblasts.

Pompe disease (PD) is an autosomal recessive lysosomal storage disorder due to deficient activity of the acid alpha glucosidase enzyme (GAA). As a consequence of the enzymatic defect, undigested glycogen accumulates within lysosomes. Most patients affected by the late-onset (LO) phenotype carry in at least one allele the c.-32-13T>G variant, which leads to exon 2 exclusion from the pre-mRNA. These patients display a variable and suboptimal response to enzyme replacement therapy. To identify novel therapeutic approaches, we developed a fluorescent GAA exon 2 splicing assay and screened a library of US Food and Drug Administration (FDA)-approved compounds. This led to the identification of several drugs able to restore normal splicing. Among these, we further validated the effects of the iron chelator deferoxamine (Defe) in c.-32-13T>G fibroblasts. Defe treatment resulted in a 2-fold increase of GAA exon 2 inclusion and a 40% increase in enzymatic activity. Preliminary results suggest that this effect is mediated by the regulation of iron availability, at least partially. RNA-seq experiments also showed that Defe might shift the balance of splicing factor levels toward a profile promoting GAA exon 2 inclusion. This work provides the basis for drug repurposing and development of new chemically modified molecules aimed at improving the clinical outcome in LO-PD patients.

Heterogeneity Matters: Aggregation Bias of Gas Transfer Velocity Versus Energy Dissipation Rate Relations in Streams.

The gas transfer velocity, k , modulates gas fluxes across air-water interfaces in rivers. While the theory postulates a local scaling law between k and the turbulent kinetic energy dissipation rate ε , empirical studies usually interpret this relation at the reach-scale. Here, we investigate how local k ( ε ) laws can be integrated along heterogeneous reaches exploiting a simple hydrodynamic model, which links stage and velocity to the local slope. The model is used to quantify the relative difference between the gas transfer velocity of a heterogeneous stream and that of an equivalent homogeneous system. We show that this aggregation bias depends on the exponent of the local scaling law, b , and internal slope variations. In high-energy streams, where b > 1 , spatial heterogeneity of ε significantly enhances reach-scale values of k as compared to homogeneous settings. We conclude that small-scale hydro-morphological traits bear a profound impact on gas evasion from inland waters.

CRISPR/Cas9 Editing for Gaucher Disease Modelling.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid ß-glucosidase gene (GBA1). Besides causing GD, GBA1 mutations constitute the main genetic risk factor for developing Parkinson's disease. The molecular basis of neurological manifestations in GD remain elusive. However, neuroinflammation has been proposed as a key player in this process. We exploited CRISPR/Cas9 technology to edit GBA1 in the human monocytic THP-1 cell line to develop an isogenic GD model of monocytes and in glioblastoma U87 cell lines to generate an isogenic GD model of glial cells. Both edited (GBA1 mutant) cell lines presented low levels of mutant acid ß-glucosidase expression, less than 1% of residual activity and massive accumulation of substrate. Moreover, U87 GBA1 mutant cells showed that the mutant enzyme was retained in the ER and subjected to proteasomal degradation, triggering unfolded protein response (UPR). U87 GBA1 mutant cells displayed an increased production of interleukin-1ß, both with and without inflammosome activation, α-syn accumulation and a higher rate of cell death in comparison with wild-type cells. In conclusion, we developed reliable, isogenic, and easy-to-handle cellular models of GD obtained from commercially accessible cells to be employed in GD pathophysiology studies and high-throughput drug screenings.

Molecular Genetics of Niemann-Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder caused by mutations in NPC1 or NPC2 genes. In 2009, the molecular characterization of 44 NPC Italian patients has been published. Here, we present an update of the genetic findings in 105 Italian NPC patients belonging to 83 unrelated families (77 NPC1 and 6 NPC2). NPC1 and NPC2 genes were studied following an algorithm recently published. Eighty-four different NPC1 and five NPC2 alleles were identified. Only two NPC1 alleles remained non detected. Sixty-two percent of NPC1 alleles were due to missense variants. The most frequent NPC1 mutation was the p.F284Lfs*26 (5.8% of the alleles). All NPC2 mutations were found in the homozygous state, and all but one was severe. Among newly diagnosed patients, 18 novel NPC1 mutations were identified. The pathogenic nature of 7/9 missense alleles and 3/4 intronic variants was confirmed by filipin staining and NPC1 protein analysis or mRNA expression in patient's fibroblasts. Taken together, our previous published data and new results provide an overall picture of the molecular characteristics of NPC patients diagnosed so far in Italy.

Changes in global gene expression indicate disordered autophagy, apoptosis and inflammatory processes and downregulation of cytoskeletal signalling and neuronal development in patients with Niemann-Pick C disease.

Changes in gene expression profiles were investigated in 23 patients with Niemann-Pick C1 disease (NPC). cDNA expression microarrays with subsequent validation by qRT-PCR were used. Comparison of NPC to control samples revealed upregulation of genes involved in inflammation (MMP3, THBS4), cytokine signalling (MMP3), extracellular matrix degradation (MMP3, CTSK), autophagy and apoptosis (CTSK, GPNMB, PTGIS), immune response (AKR1C3, RCAN2, PTGIS) and processes of neuronal development (RCAN2). Downregulated genes were associated with cytoskeletal signalling (ACTG2, CNN1); inflammation and oxidative stress (CNN1); inhibition of cell proliferation, migration and differentiation; ERK-MAPK pathway (COL4A1, COL4A2, CPA4); cell adhesion (IGFBP7); autophagy and apoptosis (CDH2, IGFBP7, COL4A2); neuronal function and development (CSRP1); and extracellular matrix stability (PLOD2). When comparing NPC and Gaucher patients together versus controls, upregulation of SERPINB2 and IL13RA2 and downregulation of CSRP1 and CNN1 were characteristic. Notably, in NPC patients, the expression of PTGIS is upregulated while the expression of PLOD2 is downregulated when compared to Gaucher patients or controls and potentially could serve to differentiate these patients. Interestingly, in NPC patients with (i) jaundice, splenomegaly and cognitive impairment/psychomotor delay-the expression of ACTG2 was especially downregulated; (ii) ataxia-the expression of ACTG2 and IGFBP5 was especially downregulated; and (iii) VSGP, dysarthria, dysphagia and epilepsy-the expression of AKR1C3 was especially upregulated while the expression of ACTG2 was downregulated. These results indicate disordered apoptosis, autophagy and cytoskeleton remodelling as well as upregulation of immune response and inflammation to play an important role in the pathogenesis of NPC in humans.

The neochord mitral valve repair procedure: Numerical simulation of different neochords tensioning protocols.

Transapical off-pump mitral valve repair with neochord implantation is an established technique for minimally-invasive intervention on mitral valve prolapse/flail. The procedure involves the positioning of artificial chords, whose length/tension is adjusted intraoperatively, adopting different methods based on the experience of the surgeon. This unsystematic approach occasionally leads to complications such as leaflet rupture and excessive/insufficient load on the neochords. In this study, finite element models of a generalized prolapsing mitral valve are used to verify the effect of two alternative tensioning approaches (AT - All together and 1-by-1 - one by one sequences) on the coaptation area and valve biomechanics, comparing results with a corresponding healthy configuration. The total force of about 1 N is exerted by the chords in both strategies, but the maximum stress and coaptation area are closer to those of the healthy configuration in the 1-by-1 sequence. However, the analysis also provides an explanation for the chords unloading in the 1-by-1 strategy observed in the clinical practice, and suggests an optimum tensioning methodology for NeoChord procedures. The study also reveals the potential power of the implemented numerical approach to serve as a tool for procedural planning, supporting the identification of the most suitable ventricular access site and the most effective stitching points for the artificial chords.

In Vitro and Ex Vivo Hemodynamic Testing of an Innovative Occluder for Paravalvular Leak After Transcather Aortic Valve Implantation.

This study aims at achieving a proof-of-concept for a novel device designed to occlude the orifices that may form between transcatheter valves and host tissues after TAVI. The device effect on the performance of a SAPIEN XT with a paravalvular gap was assessed into an in vitro and ex vivo pulse duplicator. The in vitro tests were performed complying with the standard international regulations, measuring the trasvalvular pressure and regurgitant volumes with and without the paravalvular gap, and with the occluder correctly positioned into the gap. In the second series of tests, the leakage reduction due to the presence of the occluder was assessed for the same setup, into a beating swine heart. The occluder implantation decreased the regurgitant fraction of about 50% for the in vitro assessment and 75% for the ex vivo test, under rest operating conditions. These results suggest that suitably designed occluders can lead to important benefit in the PVL treatment.

Molecular genetics of Pompe disease: a comprehensive overview.

Pompe disease (PD) is an autosomal recessive lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme due to mutations in the GAA gene. The enzymatic deficiency leads to the accumulation of glycogen within the lysosomes. Clinically, the disease has been classically classified in infantile and childhood/adult forms. The GAA gene has been localized to chromosome 17q25.2-q25.3 and to date, 582 mutations distributed throughout the whole gene have been reported (HGMD: http://www.hgmd.cf.ac.uk/ac/). All types of mutations have been described; missense variants are the most frequent type followed by small deletions. Most GAA mutations are private or found in a small number of families. However, an exception is represented by the c.-32-13T>G splice mutation that is very common in patients of Caucasian origin affected by the childhood/adult form of the disease, with an allelic frequency ranging from 40% to 70%. In this article, we review the spectrum of GAA mutations, their distribution in different populations, and their classification according to their impact on GAA splicing process, protein expression and activity. In addition, whenever possible, we discuss the phenotype/genotype correlation. The information collected in this review provides an overview of the molecular genetics of PD and can be used to facilitate diagnosis and genetic counseling of families affected by this disorder.

Form follows function: estimation of CSF flow in the third ventricle-aqueduct-fourth ventricle complex modeled as a diffuser/nozzle pump.

OBJECTIVE: In the last 20 years, researchers have debated cerebrospinal fluid (CSF) dynamics theories, commonly based on the classic bulk flow perspective. New hypotheses do not consider a possible hydraulic impact of the ventricular morphology. The present study investigates, by means of a mathematical model, the eventual role played by the geometric shape of the "third ventricle-aqueduct-fourth ventricle" complex in CSF circulation under the assumption that the complex behaves like a diffuser/nozzle (DN) pump. METHODS: DN pumps are quite recent devices introduced as valveless micropumps in various industrial applications given their property of driving net flow when subjected to rhythmic pulsations. A novel peculiar DN pump configuration was adopted in this study to mimic the ventricular complex, with two reservoirs (the ventricles) and one tube provided with a conical reach (the aqueduct-proximal fourth ventricle). The flow was modeled according to the classic equations of laminar flow, and the external rhythmic pulsations forcing the system were reproduced as a pulsatile pressure gradient between the chambers. Several physiological scenarios were implemented with the integration of data acquired by MRI in 10 patients with no known pathology of CSF dynamics, and a quantitative analysis of the effect of geometric and hydraulic parameters (diverging angle, sizes, frequency of pulsations) on the CSF net flow was performed. RESULTS: The results showed a craniocaudal net flow in all the given values, consistent with the findings of cine MRI studies. Moreover, the net flow estimated for the analyzed cohort of patients ranged from 0.221 to 0.505 ml/min, remarkably close to the values found on phase contrast cine MRI in healthy subjects. Sensitivity analysis underlines the pivotal role of the DN configuration, as well as of the frequency of forcing pressure, which promotes a relevant net flow considering both the heart and respiration rate. CONCLUSIONS: This work suggests that the geometry of the third ventricle-aqueduct-fourth ventricle complex, which resembles a diverter, appears to be functional in the generation of a net craniocaudal flow and potentially has an impact on CSF dynamics. These conclusions can be drawn by observing the analogies between the shape of the ventricles and the geometry of DN pumps and by recognizing the basis of the mathematical model of the simplified third ventricle-aqueduct-fourth ventricle complex proposed.

In vitro assessment of pacing as therapy for aortic regurgitation.

Background and objective: Clinical evaluation of pacing therapy in mitigating the aortic insufficiency after transchateter aortic valve implantation often gives contradictory outcomes. This study presents an in vitro investigation aimed at clarifying the effect of pacing on paravalvular leakage. Methods: A series of in vitro tests reproducing the heart operating changes clinically obtained by pacing was carried out in a 26 mm Edwards Sapien XT prosthesis with mild paravalvular leakage. The effect of pacing on the regurgitant volumes per cycle and per minute was quantified, and the energy and power consumed by the left ventricle were calculated. Results: Results indicate that though pacing results in some reduction in the total regurgitation per cycle, the volume of fluid regurgitating per minute increases substantially, causing overload of left ventricle. Conclusions: Our tests indicate no effective haemodynamic benefit from pacing, suggesting a prudential clinical use of this therapy for the treatment of postoperative aortic regurgitation.

Experimental Setup and Measuring System to Study Solitary Wave Interaction with Rigid Emergent Vegetation.

The aim of this study is to present a peculiar experimental setup, designed to investigate the interaction between solitary waves and rigid emergent vegetation. Flow rate changes due to the opening and closing of a software-controlled electro-valve generate a solitary wave. The complexity of the problem required the combined use of different measurement systems of water level and velocity. Preliminary results of the experimental investigation, which allow us to point out the effect of the vegetation on the propagation of a solitary wave and the effectiveness of the measuring system, are also presented. In particular, water level and velocity field changes due to the interaction of the wave with rigid vegetation are investigated in detail.

The heat shock protein amplifier arimoclomol improves refolding, maturation and lysosomal activity of glucocerebrosidase.

BACKGROUND: Gaucher Disease is caused by mutations of the GBA gene which encodes the lysosomal enzyme acid beta-glucosidase (GCase). GBA mutations commonly affect GCase function by perturbing its protein homeostasis rather than its catalytic activity. Heat shock proteins are well known cytoprotective molecules with functions in protein homeostasis and lysosomal function and their manipulation has been suggested as a potential therapeutic strategy for GD. The investigational drug arimoclomol, which is in phase II/III clinical trials, is a well-characterized HSP amplifier and has been extensively clinically tested. Importantly, arimoclomol efficiently crosses the blood-brain-barrier presenting an opportunity to target the neurological manifestations of GD, which remains without a disease-modifying therapy. METHODS: We used a range of biological and biochemical in vitro assays to assess the effect of arimoclomol on GCase activity in ex vivo systems of primary fibroblasts and neuronal-like cells from GD patients. FINDINGS: We found that arimoclomol induced relevant HSPs such as ER-resident HSP70 (BiP) and enhanced the folding, maturation, activity, and correct cellular localization of mutated GCase across several genotypes including the common L444P and N370S mutations in primary cells from GD patients. These effects where recapitulated in a human neuronal model of GD obtained by differentiation of multipotent adult stem cells. INTERPRETATION: These data demonstrate the potential of HSP-targeting therapies in GCase-deficiencies and strongly support the clinical development of arimoclomol as a potential therapeutic option for the neuronopathic forms of GD. FUNDING: The research was funded by Orphazyme A/S, Copenhagen, Denmark.