Sagittal kinematics and imbalance of the spine and whole body during walking in late-onset Pompe disease.

Late-onset Pompe disease (LOPD) is characterized by postural abnormalities mainly due to involvement of paraspinal lumbar and abdominal-pelvic muscles. Previous studies quantitatively analyzed static upright posture, spatial-temporal parameters, and kinematics of the lower limbs and trunk, considered as single bone segment. Sagittal plane analysis of the spine and whole body during walking has never been investigated in patients with LOPD. The aim of the study was to evaluate sagittal kinematics and imbalance of the spine and whole body in patients with LOPD by three-dimensional (3-D)-motion analysis using an appropriate marker set protocol and introducing innovative kinematic parameters. Seven siblings with LOPD were assessed by 3-D-stereophotogrammetry using the DB-total protocol, which allows to analyze sagittal alignment of whole body. Fourteen age- and sex-matched healthy subjects were used as controls. LOPD group showed a flattening of the spinal curvatures, with a head and neck posteriorization with respect to sacrum, a significant increase of concavity in Heel-S2-Nasion/C7 angles, a rear-position of upper limbs with respect to pelvis, a shorter pendular activity, and a trend of elbow extension during ambulation. Moreover, a significant increase of excursion range in most of sagittal parameters was found. The present study highlighted a specific pathological postural pattern, resembling "man falling backwards," which reveals a biomechanical compensation strategy of patients with LOPD to maintain the balance against the instability of the spinopelvic region, kinematically verified by increase of the excursion ranges. DB-total kinematic parameters might be useful for functional evaluation and for monitoring response to enzyme replacement therapy, rehabilitation project, and disease progression.NEW & NOTEWORTHY This study is the first to quantitatively characterize the sagittal spine and whole body posture of patients with late-onset Pompe disease during walking, showing a pathological kinematic pattern defined "man falling backwards." 3-D-motion analysis, with a specific marker set (DB-total protocol) introducing new whole body kinematic parameters, may be useful for accurate functional evaluation and monitoring this rare disease.

POLR3A variants in hereditary spastic paraparesis and ataxia: clinical, genetic, and neuroradiological findings in a cohort of Italian patients.

Mutations in POLR3A are characterized by high phenotypic heterogeneity, with manifestations ranging from severe childhood-onset hypomyelinating leukodystrophic syndromes to milder and later-onset gait disorders with central hypomyelination, with or without additional non-neurological signs. Recently, a milder phenotype consisting of late-onset spastic ataxia without hypomyelinating leukodystrophy has been suggested to be specific to the intronic c.1909 + 22G > A mutation in POLR3A. Here, we present 10 patients from 8 unrelated families with POLR3A-related late-onset spastic ataxia, all harboring the c.1909 + 22G > A variant. Most of them showed an ataxic-spastic picture, two a "pure" cerebellar phenotype, and one a "pure" spastic presentation. The non-neurological findings typically associated with POLR3A mutations were absent in all the patients. The main findings on brain MRI were bilateral hyperintensity along the superior cerebellar peduncles on FLAIR sequences, observed in most of the patients, and cerebellar and/or spinal cord atrophy, found in half of the patients. Only one patient exhibited central hypomyelination. The POLR3A mutations present in this cohort were the c.1909 + 22G > A splice site variant found in compound heterozygosity with six additional variants (three missense, two nonsense, one splice) and, in one patient, with a novel large deletion involving exons 14-18. Interestingly, this patient had the most "complex" presentation among those observed in our cohort; it included some neurological and non-neurological features, such as seizures, neurosensory deafness, and lipomas, that have not previously been reported in association with late-onset POLR3A-related disorders, and therefore further expand the phenotype.

Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington's disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer.

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies.

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.

Novel autophagic vacuolar myopathies: Phenotype and genotype features.

BACKGROUND: Autophagic vacuolar myopathies (AVMs) are an emerging group of heterogeneous myopathies sharing histopathological features on muscle pathology, in which autophagic vacuoles are the pathognomonic morphologic hallmarks. Glycogen storage disease type II (GSDII) caused by lysosomal acid α-glucosidase (GAA) deficiency is the best-characterised AVM. AIMS: This study aimed to investigate the mutational profiling of seven neuromuscular outpatients sharing clinical, myopathological and biochemical findings with AVMs. METHODS: We applied a diagnostic protocol, recently published by our research group for suspected late-onset GSDII (LO-GSDII), including counting PAS-positive lymphocytes on blood smears, dried blood spot (DBS)-GAA, muscle biopsy histological and immunofluorescence studies, GAA activity assay and expression studies on muscle homogenate, GAA sequencing, GAA multiplex ligation-dependent probe amplification (MLPA) and whole exome sequencing (WES). RESULTS: The patients had a limb girdle-like muscular pattern with persistent hyperCKaemia; vacuolated PAS-positive lymphocytes, glycogen accumulation and impaired autophagy at muscle biopsy. Decreased GAA activity was also measured. While GAA sequencing identified no pathogenic mutations, WES approach allowed us to identify for each patient an unexpected mutational pattern in genes cooperating in lysosomal-autophagic machinery, some of which have never been linked to human diseases. CONCLUSIONS: Our data suggest that reduced GAA activity may occur in any condition of impaired autophagy and that WES approach is advisable in all genetically undefined cases of autophagic myopathy. Therefore, deficiency of GAA activity and PAS-positive lymphocytes should be considered as AVM markers together with LC3/p62-positive autophagic vacuoles.

Rare Variants in Autophagy and Non-Autophagy Genes in Late-Onset Pompe Disease: Suggestions of Their Disease-Modifying Role in Two Italian Families.

Pompe disease is an autosomal recessive disorder caused by a deficiency in the enzyme acid alpha-glucosidase. The late-onset form of Pompe disease (LOPD) is characterized by a slowly progressing proximal muscle weakness, often involving respiratory muscles. In LOPD, the levels of GAA enzyme activity and the severity of the clinical pictures may be highly variable among individuals, even in those who harbour the same combination of GAA mutations. The result is an unpredictable genotype-phenotype correlation. The purpose of this study was to identify the genetic factors responsible for the progression, severity and drug response in LOPD. We report here on a detailed clinical, morphological and genetic study, including a whole exome sequencing (WES) analysis of 11 adult LOPD siblings belonging to two Italian families carrying compound heterozygous GAA mutations. We disclosed a heterogeneous pattern of myopathic impairment, associated, among others, with cardiac defects, intracranial vessels abnormality, osteoporosis, vitamin D deficiency, obesity and adverse response to enzyme replacement therapy (ERT). We identified deleterious variants in the genes involved in autophagy, immunity and bone metabolism, which contributed to the severity of the clinical symptoms observed in the LOPD patients. This study emphasizes the multisystem nature of LOPD and highlights the polygenic nature of the complex phenotype disclosed in these patients.

The Discovery of Highly Potent THP Derivatives as OCTN2 Inhibitors: From Structure-Based Virtual Screening to In Vivo Biological Activity.

A mismatch between ß-oxidation and the tricarboxylic acid cycle (TCA) cycle flux in mitochondria produces an accumulation of lipid metabolic intermediates, resulting in both blunted metabolic flexibility and decreased glucose utilization in the affected cells. The ability of the cell to switch to glucose as an energy substrate can be restored by reducing the reliance of the cell on fatty acid oxidation. The inhibition of the carnitine system, limiting the carnitine shuttle to the oxidation of lipids in the mitochondria, allows cells to develop a high plasticity to metabolic rewiring with a decrease in fatty acid oxidation and a parallel increase in glucose oxidation. We found that 3-(2,2,2-trimethylhydrazine)propionate (THP), which is able to reduce cellular carnitine levels by blocking both carnitine biosynthesis and the cell membrane carnitine/organic cation transporter (OCTN2), was reported to improve mitochondrial dysfunction in several diseases, such as Huntington's disease (HD). Here, new THP-derived carnitine-lowering agents (TCL), characterized by a high affinity for the OCTN2 with a minimal effect on carnitine synthesis, were developed, and their biological activities were evaluated in both in vitro and in vivo HD models. Certain compounds showed promising biological activities: reducing protein aggregates in HD cells, ameliorating motility defects, and increasing the lifespan of HD Drosophila melanogaster.

Metabolic syndrome, Mediterranean diet, and polyphenols: Evidence and perspectives.

Metabolic syndrome (MetS) is defined as the co-occurrence of metabolic risk factors that includes insulin resistance, hyperinsulinemia, impaired glucose tolerance, type 2 diabetes mellitus, dyslipidemia, and visceral obesity. The clinical significance of MetS consists of identifying a subgroup of patients sharing a common physiopathological state predisposing to chronic diseases. Clinical and scientific studies pinpoint lifestyle modification as an effective strategy aiming to reduce several features accountable for the risk of MetS onset. Among the healthy dietary patterns, the Mediterranean diet (MedDiet) emerges in terms of beneficial properties associated with longevity. Current evidence highlights the protective effect exerted by MedDiet on the different components of MetS. Interestingly, the effect exerted by polyphenols contained within the representative MedDiet components (i.e., olive oil, red wine, and nuts) seems to be accountable for the beneficial properties associated to this dietary pattern. In this review, we aim to summarize the principal evidence regarding the effectiveness of MedDiet-polyphenols in preventing or delaying the physiopathological components accountable for MetS onset. These findings may provide useful insights concerning the health properties of MedDiet-polyphenols as well as the novel targets destined to a tailored approach to MetS.

Meldonium improves Huntington's disease mitochondrial dysfunction by restoring peroxisome proliferator-activated receptor γ coactivator 1α expression.

Mitochondrial dysfunction seems to play a fundamental role in the pathogenesis of neurodegeneration in Huntington's disease (HD). We assessed possible neuroprotective actions of meldonium, a small molecule affecting mitochondrial fuel metabolism, in in vitro and in vivo HD models. We found that meldonium was able to prevent cytotoxicity induced by serum deprivation, to reduce the accumulation of mutated huntingtin (mHtt) aggregates, and to upregulate the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in mHTT-expressing cells. The PGC-1α increase was accompanied by the increment of mitochondrial mass and by the rebalancing of mitochondrial dynamics with a promotion of the mitochondrial fusion. Meldonium-induced PGC-1α significantly alleviated motor dysfunction and prolonged the survival of a transgenic HD Drosophila model in which mHtt expression in the nervous system led to progressive motor performance deficits. Our study strongly suggests that PGC-1α, as a master coregulator of mitochondrial biogenesis, energy homeostasis, and antioxidant defense, is a potential therapeutic target in HD.

Senescence Phenomena and Metabolic Alteration in Mesenchymal Stromal Cells from a Mouse Model of Rett Syndrome.

Chromatin modifiers play a crucial role in maintaining cell identity through modulation of gene expression patterns. Their deregulation can have profound effects on cell fate and functions. Among epigenetic regulators, the MECP2 protein is particularly attractive. Mutations in the Mecp2 gene are responsible for more than 90% of cases of Rett syndrome (RTT), a progressive neurodevelopmental disorder. As a chromatin modulator, MECP2 can have a key role in the government of stem cell biology. Previously, we showed that deregulated MECP2 expression triggers senescence in mesenchymal stromal cells (MSCs) from (RTT) patients. Over the last few decades, it has emerged that senescent cells show alterations in the metabolic state. Metabolic changes related to stem cell senescence are particularly detrimental, since they contribute to the exhaustion of stem cell compartments, which in turn determine the falling in tissue renewal and functionality. Herein, we dissect the role of impaired MECP2 function in triggering senescence along with other senescence-related aspects, such as metabolism, in MSCs from a mouse model of RTT. We found that MECP2 deficiencies lead to senescence and impaired mitochondrial energy production. Our results support the idea that an alteration in mitochondria metabolic functions could play an important role in the pathogenesis of RTT.

Verapamil Inhibits Ser202/Thr205 Phosphorylation of Tau by Blocking TXNIP/ROS/p38 MAPK Pathway.

PURPOSE: Oxidative stress is a hallmark of Alzheimer's Disease (AD) and promotes tau phosphorylation. Since Thioredoxin Interacting protein (TXNIP), the inhibitor of the anti-oxidant system of Thioredoxin, is up regulated in the hippocampus of AD patients, we investigated whether TXNIP plays a role in promoting tau phosphorylation and whether Verapamil, an inhibitor of TXNIP expression, prevents TXNIP downstream effects. METHODS: We analyzed TXNIP expression and tau phosphorylation in the hippocampus of the 5xFAD mice in the absence and presence of a pharmacological treatment with Verapamil. Using SH-SY5Y cells, we verified the causative role of TXNIP in promoting tau phosphorylation at Ser202/Thr205, by inducing TXNIP silencing. RESULTS: The amyloid beta peptide (Aß1-42) leads to TXNIP over-expression in SH-SY5Y cells, which in turns induces oxidative stress and the activation of p38 MAPK, promoting tau phosphorylation at Ser202/Thr205. Silencing of TXNIP abolishes Aß1-42-induced tau phosphorylation, p38 MAPK phosphorylation and subsequent tau phosphorylation. Verapamil prevents TXNIP expression as well as p38 MAPK and tau phosphorylation at Ser202/Thr205 in the hippocampus of the 5xFAD mice. CONCLUSIONS: Our study unveil a novel pathway involved in AD progression that is inhibited by Verapamil, shedding new light on the understanding of the therapeutic potential of Verapamil in AD.

Neuro-Behçet's disease presenting as an isolated progressive cognitive and behavioral syndrome.

Behçet's disease is a chronic inflammatory disorder manifesting as a vasculitis that affects arteries and veins of any size. Up to 44% of cases may also present with neurological symptoms, thus defining Neuro-Behçet's disease. We describe a case of Neuro-Behçet's disease characterized by progressive behavioral and cognitive deterioration prevailing over other neurological symptoms, without evident systemic involvement.

Identification of the first dominant mutation of LAMA5 gene causing a complex multisystem syndrome due to dysfunction of the extracellular matrix.

BACKGROUND: The laminin alpha 5 gene (LAMA5) plays a master role in the maintenance and function of the extracellular matrix (ECM) in mammalian tissues, which is critical in developmental patterning, stem cell niches, cancer and genetic diseases. Its mutations have never been reported in human disease so far. The aim of this study was to associate the first mutation in LAMA5 gene to a novel multisystem syndrome. METHODS: A detailed characterisation of a three-generation family, including clinical, biochemical, instrumental and morphological analysis, together with genetics and expression (WES and RNAseq) studies, was performed. RESULTS: The heterozygous LAMA5 mutation c.9418G>A (p.V3140M) was associated with skin anomalies, impaired scarring, night blindness, muscle weakness, osteoarthritis, joint and internal organs ligaments laxity, malabsorption syndrome and hypothyroidism. We demonstrated that the mutation alters the amount of LAMA5 peptides likely derived from protein cleavage and perturbs the activation of the epithelial-mesenchymal signalling, producing an unbalanced expression of Sonic hedgehog and GLI1, which are upregulated in cells from affected individuals, and of ECM proteins (COL1A1, MMP1 and MMP3), which are strongly inhibited. Studies carried out using human skin biopsies showed alteration of dermal papilla with a reduction of the germinative layer and an early arrest of hair follicle downgrowth. The knock-in mouse model, generated in our laboratory, shows similar changes in the tissues studied so far. CONCLUSIONS: This is the first report of a disease phenotype associated with LAMA5 mutation in humans.

Neurofibromatous neuropathy: An ultrastructural study.

Plexiform neurofibroma is pathognomonic of neurofibromatosis 1 (NF1). An NF1-associated peripheral neuropathy has been described in a small minority of NF1 patients but its histopathological features are poorly characterized. We report the case of a 46-year-old woman presenting with bilateral supraclavicular painful masses without other stigmata of NF1. MRI showed bilateral plexiform lesions extending from cervical roots to the elbows. Nerve conduction studies documented a sensory motor polyneuropathy. Morphometric analysis of sural nerve biopsy showed a preferential loss of large-caliber myelinated fibers with a g ratio of 0.515, and the presence of regeneration clusters. By electron microscopy, marked and diffuse endoneurial fibrosis with an altered relationship between Schwann cells (SC) and collagen fibrils was observed. Moreover both myelinating and non-myelinating SC were characterized by the presence of various cell degradation products. These changes suggest that, in neurofibromatous neuropathy, a widespread axonal atrophy and degeneration take place independently on the presence of tumoral infiltration, possibly due to an impairment in SC-axon cross talk. In this case, the coexistence of plexiform neurofibromas with a peripheral neuropathy strongly suggests a diagnosis of NF1 even without fulfillment of clinical criteria. We propose that in the presence of plexiform neurofibromas, electrophysiological studies should be performed also in asymptomatic patients, in order to detect the existence of a subclinical neuropathy.

Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes.

Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity.

Human blood lipid profiles after dietary supplementation of different omega 3 ethyl esters formulations.

The validity of omega 3 fatty acids (ω3 FAs), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as dietary supplements has been widely proved. It's well known in fact, that they protect against cardiovascular diseases, reduce the levels of triacylglycerides (TAGs) and cholesteryl esters (CEs) in blood, and have anti-inflammatory activity. For these reasons, in the last few years the production of dietary supplement containing ω3 has increased significantly. In this context, the possibility to obtain ω3 and other high value molecules from alternative sources as fish waste, in accordance with the principles of circular economy, becomes an enormous attractive. In addition, the opportunity of creating new products, with greater health benefits, represents an interesting challenge. The current study was focused on the extraction of ω3 fatty acids and peptides from tuna waste industry, to realize a new dietary supplement. To this purpose, a supercritical fluid extraction (SFE) method was developed to separate, isolate, and enrich the different fractions subsequently used to produce an innovative formulate. The obtained supplement was characterized in terms of fatty acids esterified ester (FAEE) composition by gas chromatography (GC) coupled to both flame ionization detection (FID) and mass spectrometry (MS), and content of heavy metals by inductively coupled plasma-mass spectrometry (ICP-MS). The effects of ω3 supplementation on metabolism and circulating lipid profiles was tested on 12 volunteers and assessed by GC-FID analysis of whole blood collected on paper support (Dried Blood Spot, DBS) at the beginning of the study and after thirty days. The results of plasma fatty acids levels after 30 days showed a significant decrease in the ω6/ω3 ratio, as well as the saturated/polyunsaturated fatty acids (SFA/PUFA) ratio, compared to subjects who took the ω3 ethyl esters unformulated. The novel formulated supplements proved to be extremely interesting and promising products, due to a significant increase in bioavailability, that makes it highly competitive in the current panorama of the nutraceutical industry.

A Translational Approach to Spinal Neurofibromatosis: Clinical and Molecular Insights from a Wide Italian Cohort.

Spinal neurofibromatosis (SNF), a phenotypic subclass of neurofibromatosis 1 (NF1), is characterized by bilateral neurofibromas involving all spinal roots. In order to deepen the understanding of SNF's clinical and genetic features, we identified 81 patients with SNF, 55 from unrelated families, and 26 belonging to 19 families with at least 1 member affected by SNF, and 106 NF1 patients aged >30 years without spinal tumors. A comprehensive NF1 mutation screening was performed using NGS panels, including NF1 and several RAS pathway genes. The main features of the SNF subjects were a higher number of internal neurofibromas (p < 0.001), nerve root swelling (p < 0.001), and subcutaneous neurofibromas (p = 0.03), while hyperpigmentation signs were significantly less frequent compared with the classical NF1-affected cohorts (p = 0.012). Fifteen patients underwent neurosurgical intervention. The histological findings revealed neurofibromas in 13 patients and ganglioneuromas in 2 patients. Phenotypic variability within SNF families was observed. The proportion of missense mutations was higher in the SNF cases than in the classical NF1 group (21.40% vs. 7.5%, p = 0.007), conferring an odds ratio (OR) of 3.34 (CI = 1.33−10.78). Two unrelated familial SNF cases harbored in trans double NF1 mutations that seemed to have a subclinical worsening effect on the clinical phenotype. Our study, with the largest series of SNF patients reported to date, better defines the clinical and genetic features of SNF, which could improve the management and genetic counseling of NF1.

Genotype-Phenotype Correlations in Neurofibromatosis Type 1: Identification of Novel and Recurrent NF1 Gene Variants and Correlations with Neurocognitive Phenotype.

Neurofibromatosis type 1 (NF1) is one of the most common genetic tumor predisposition syndrome, caused by mutations in the NF1. To date, few genotype-phenotype correlations have been discerned in NF1, due to a highly variable clinical presentation. We aimed to study the molecular spectrum of NF1 and genotype-phenotype correlations in a monocentric study cohort of 85 NF1 patients (20 relatives, 65 sporadic cases). Clinical data were collected at the time of the mutation analysis and reviewed for accuracy in this investigation. An internal phenotypic categorization was applied. The 94% of the patients enrolled showed a severe phenotype with at least one systemic complication and a wide range of associated malignancies. Spine deformities were the most common complications in this cohort. We also reported 66 different NF1 mutations, of which 7 are novel mutations. Correlation analysis identified a slight significant inverse correlation between age at diagnosis and delayed acquisition of psychomotor skills with residual multi-domain cognitive impairment. Odds ratio with 95% confidence interval showed a higher prevalence of learning disabilities in patients carrying frameshift mutations. Overall, our results aim to offer an interesting contribution to studies on the genotype-phenotype of NF1 and in genetic management and counselling.