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Lamellar ichthyosis (LI) is a chronic disease, mostly caused by mutations in the TGM1 gene, marked by impaired skin barrier formation. No definitive therapies are available, and current treatments aim at symptomatic relief. LI mouse models often fail to faithfully replicate the clinical and histopathological features of human skin conditions. To develop advanced therapeutic approaches, such as combined ex vivo cell and gene therapy, we established a human cellular model of LI by efficient CRISPR-Cas9-mediated gene ablation of the TGM1 gene in human primary clonogenic keratinocytes. Gene-edited cells showed complete absence of transglutaminase 1 (TG1) expression and recapitulated a hyperkeratotic phenotype with most of the molecular hallmarks of LI in vitro. Using a self-inactivating γ-retroviral (SINγ-RV) vector expressing transgenic TGM1 under the control of its own promoter, we tested an ex vivo gene therapy approach and validate the model of LI as a platform for pre-clinical evaluation studies. Gene-corrected TGM1-null keratinocytes displayed proper TG1 expression, enzymatic activity, and cornified envelope formation and, hence, restored proper epidermal architecture. Single-cell multiomics analysis demonstrated proviral integrations in holoclone-forming epidermal stem cells, which are crucial for epidermal regeneration. This study serves as a proof of concept for assessing the potential of this therapeutic approach in treating TGM1-dependent LI.

miRNAs circulating in whole serum and HBsAg-particles are differentially expressed in chronic hepatitis B (CHB) and HBeAg-negative-HBV infection (ENI); their profiles are unknown in chronic hepatitis D (CHD). Serum- and HBsAg-associated miRNAs were analyzed in 75 subjects of 3 well-characterized groups (CHB 25, CHD 25, ENI 25) using next-generation sequencing (NGS). Overall miRNA profiles were consonant in serum and HBsAg-particles but significantly different according to the presence of hepatitis independently of Hepatitis D Virus (HDV)-co-infection. Stringent (Bonferroni Correction < 0.001) differential expression analysis showed 39 miRNAs upregulated in CHB vs. ENI and 31 of them also in CHD vs. ENI. miRNA profiles were coincident in CHB and CHD with only miR-200a-3p upregulated in CHB. Three miRNAs (miR-625-3p, miR-142-5p, and miR-223-3p) involved in immune response were upregulated in ENI. All 3 hepatocellular miRNAs of MiR-B-Index (miR-122-5p, miR-99a-5p, miR-192-5p) were overexpressed in both CHB and CHD patients. In conclusion, CHD and CHB patients showed highly similar serum miRNA profiling that was significantly different from that of individuals with HBeAg-negative infection and without liver disease.

Background and Aim: Ultrasound (US) imaging is increasingly preferred over other more invasive modalities in preclinical studies using animal models. However, this technique has some limitations, mainly related to operator dependence. To overcome some of the current drawbacks, sophisticated data processing models are proposed, in particular artificial intelligence models based on deep learning (DL) networks. This systematic review aims to overview the application of DL algorithms in assisting US analysis of images acquired in in vivo preclinical studies on animal models. Methods: A literature search was conducted using the Scopus and PubMed databases. Studies published from January 2012 to November 2022 that developed DL models on US images acquired in preclinical/animal experimental scenarios were eligible for inclusion. This review was conducted according to PRISMA guidelines. Results: Fifty-six studies were enrolled and classified into five groups based on the anatomical district in which the DL models were used. Sixteen studies focused on the cardiovascular system and fourteen on the abdominal organs. Five studies applied DL networks to images of the musculoskeletal system and eight investigations involved the brain. Thirteen papers, grouped under a miscellaneous category, proposed heterogeneous applications adopting DL systems. Our analysis also highlighted that murine models were the most common animals used in in vivo studies applying DL to US imaging. Conclusion: DL techniques show great potential in terms of US images acquired in preclinical studies using animal models. However, in this scenario, these techniques are still in their early stages, and there is room for improvement, such as sample sizes, data preprocessing, and model interpretability.

Endosomal trafficking is essential for cellular homeostasis. At the crossroads of distinct intracellular pathways, the endolysosomal system is crucial to maintain critical functions and adapt to the environment. Alterations of endosomal compartments were observed in cells from adult individuals with Down syndrome (DS), suggesting that the dysfunction of the endosomal pathway may contribute to the pathogenesis of DS. However, the nature and the degree of impairment, as well as the timing of onset, remain elusive. Here, by applying imaging and biochemical approaches, we demonstrate that the structure and dynamics of early endosomes are altered in DS cells. Furthermore, we found that recycling trafficking is markedly compromised in these cells. Remarkably, our results in 18–20 week-old human fetal fibroblasts indicate that alterations in the endolysosomal pathway are already present early in development. In addition, we show that overexpression of the polyphosphoinositide phosphatase synaptojanin 1 (Synj1) recapitulates the alterations observed in DS cells, suggesting a role for this lipid phosphatase in the pathogenesis of DS, likely already early in disease development. Overall, these data strengthen the link between the endolysosomal pathway and DS, highlighting a dangerous liaison among Synj1, endosomal trafficking and DS.

PurposeThe aims of this study were to evaluate the reproducibility of a new multi-parametric steatoscore (new SteatoScore) in oncologic patients with non-alcoholic fatty liver disease (NAFLD) and to compare it with computed tomography (CT).Materials and MethodsFifty-one (31 men, 20 women) oncologic patients, with a mean age and weight of 63.9 years and 78.33 kg, respectively, were retrospectively enrolled in the study. Patients underwent ultrasound (US) and computed tomography (CT) examinations as part of their oncologic follow-up protocol. US examinations were performed by using a 3.5-MHz convex probe. During the US examination, three standardized clips were obtained in each patient. Two operators performed all measurements, one of whom repeated the processing twice in 1 year. Hepatic/renal ratio (HR), attenuation rate (AR), diaphragm visualization (DV), hepatic/portal vein ratio (HPV), and portal vein wall visualization (PVW) were acquired and calculated by using Matlab and inserted in a multi-parametric algorithm called new SteatoScore. On unenhanced CT scan, hepatic attenuation (HA), liver-spleen difference (L-S), and liver/spleen ratio (L/S) were measured by placement of a region of interest (ROI) within liver and spleen parenchyma, avoiding areas with vessels and biliary ducts.ResultsThe intra-observer variability was greater than the inter-observer one, with intraclass correlation coefficient (ICC) values of 0.94 and 0.97, respectively. Correlation between single US and CT parameters provided an agreement in no case exceeding 50%. New SteatoScore showed high reproducibility, and high coefficient of correlation with L-S (R = −0.64; p < 0.0001) and L/S (R = −0.62; p < 0.0001) at CT.ConclusionNew SteatoScore has a high reproducibility and shows a good correlation with unenhanced CT in evaluation of oncologic patients with NAFLD.

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.

Epidermolysis bullosa (EB) is a group of devastating genetic diseases characterized by skin and mucosal fragility and formation of blisters, which develop either spontaneously or in response to minor mechanical trauma. There is no definitive therapy for any form of EB. Intermediate junctional EB (JEB) caused by mutations in the gene LAMB3 has been the first genetic skin disease successfully tackled by ex vivo gene therapy. Here, we present a multicenter, open-label, uncontrolled phase II/III study that aims at confirming the efficacy of Hologene 5, a graft consisting of cultured transgenic keratinocytes and epidermal stem cells and meant to combine cell and gene therapy for the treatment of LAMB3-related JEB. Autologous clonogenic keratinocytes will be isolated from patients’ skin biopsies, genetically corrected with a gamma-retroviral vector (γRV) carrying the full-length human LAMB3 cDNA and plated onto a fibrin support (144cm2). The transgenic epidermis will be transplanted onto surgically prepared selected skin areas of at least six JEB patients (four pediatric and two adults). Evaluation of clinical efficacy will include, as primary endpoint, a combination of clinical parameters, such as percentage of re-epithelialization, cellular, molecular, and functional parameters, mechanical stress tests, and patient-reported outcome (PRO), up to 12months after transplantation. Safety and further efficacy endpoints will also be assessed during the clinical trial and for additional 15years in an interventional non-pharmacological follow-up study. If successful, this clinical trial would provide a therapeutic option for skin lesions of JEB patients with LAMB3 mutations and pave the way to a combined cell and gene therapy platform tackling other forms of EB and different genodermatoses.Clinical Trial Registration: EudraCT Number: 2018-000261-36.

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.

Fowler-Stephens Laparoscopic Orchiopexy (FSLO) permits the mobilization of Intra-Abdominal Testis (IAT) to the scrotal position after spermatic vessel ligation. We reported our experience of FSLO for IAT. The charts of all boys who underwent a FSLO were retrospectively reviewed. Data were analysed for demographic data, procedure, complications and follow-up results. From January 2008 to June 2016, 160 laparoscopies for Non Palpable Testis (NPT) were performed at a mean age of 3,2 years. 61% of patients had a right NPT, while 6% were bilateral. In 64 cases, an IAT was found: 20 were managed by FSLO with a two-stage procedure in 11 patients. There were no differences in hospitalisations; one patient had a prolonged ileus. Follow-up ranged from 1 to 8 years. Of the 20 patients who underwent FSLO, testicular atrophy developed in three; the remaining testes were in the scrotal position, with normal consistency. FSLO was applied in 31% of IAT. The overall success rate of the technique was 85 %. The percentage of atrophy associated after spermatic vessels interruption appears to provide a good chance of testicular survival.

Non-alcoholic fatty liver disease (NAFLD) is becoming a global public health issue and the identification of the steatosis severity is very important for the patients’ health. Ultrasound (US) images of 214 patients were acquired in two different scan views (subcostal and intercostal). A classification of the level of steatosis was made by a qualitative evaluation of the liver ultrasound images. Furthermore, an US image processing algorithm provided quantitative parameters (hepatic–renal ratio (HR) and Steato-score) designed to quantifying the fatty liver content. The aim of the study is to evaluate the differences in the assessment of hepatic steatosis acquiring and processing different US scan views. No significant differences were obtained calculating the HR and the Steato-score parameters, not even with the classification of patients on the basis of body mass index (BMI) and of different classes of steatosis severity. Significant differences between the two parameters were found only for patients with absence or mild level of steatosis. These results show that the two different scan projections do not greatly affect HR and the Steato-score assessment. Accordingly, the US-based steatosis assessment is independent from the view of the acquisitions, thus making the subcostal and intercostal scans interchangeable, especially for patients with moderate and severe steatosis.

Summary: Laminin 332-deficient junctional epidermolysis bullosa (JEB) is a severe genetic skin disease. JEB is marked by epidermal stem cell depletion, the origin of which is unknown. We show that dysregulation of the YAP and TAZ pathway underpins such stem cell depletion. Laminin 332-mediated YAP activity sustains human epidermal stem cells, detected as holoclones. Ablation of YAP selectively depletes holoclones, while enforced YAP blocks conversion of stem cells into progenitors and indefinitely extends the keratinocyte lifespan. YAP is dramatically decreased in JEB keratinocytes, which contain only phosphorylated, inactive YAP. In normal keratinocytes, laminin 332 and α6β4 ablation abolish YAP activity and recapitulate the JEB phenotype. In JEB keratinocytes, laminin 332-gene therapy rescues YAP activity and epidermal stem cells in vitro and in vivo. In JEB cells, enforced YAP recapitulates laminin 332-gene therapy, thus uncoupling adhesion from proliferation in epidermal stem cells. This work has important clinical implication for ex vivo gene therapy of JEB. : Gene therapy of junctional epidermolysis bullosa is hampered by the epidermal stem cell loss marking this disease. De Rosa et al. find that YAP dysregulation underpins such loss, which is recapitulated by laminin 332 and α6β4 integrin ablation. Combined cell and gene therapy rescues adhesion, YAP function, and stem cells. Keywords: epidermal stem cells, epidermolysis bullosa, YAP, cell and gene therapy

We report our experience with a sutureless technique for laparoscopic inguinal hernia repair in children. Twenty-eight children, 12 girls and 16 boys, aged 3 months to 7 years, underwent sutureless laparoscopic inguinal hernia repair. In girls, we utilized simple cauterization of the internal inguinal ring with a single trocar technique. In boys denudation of the peritoneum was obtained utilizing a three-trocar technique. The peritoneum around the internal inguinal ring was peeled off and detached from the vas and the vessels, and then wrapped around the grasper, resulting in a large area of denudation. No perioperative complications were observed. No recurrences nor testicular damage were noted after a mean follow-up of 18 months (range 6-35 months). The mean operative time was 12 min (range 7-20 min) for girls and 19 min (range 15-30 min) for boys. A 6-year-old girl exhibited a lymphocele. The sutureless technique appears to be easy, safe, and effective, provided that the patients are carefully selected. In boys, special attention needs to be paid to preservation of the vas and the testicular vessels.

We retrospectively reviewed the results of transscrotal orchidopexy in the surgical management of palpable testis. From January 2014 to June 2017, 130 male children with a total of 140 palpable undescended testes (UDT) underwent transscrotal orchidopexy. The charts were retrospectively reviewed for demographic data, preoperative position and mobility of the testis, patency of the peritoneal vaginal duct (PVD), and post-operative complications. The resting position of the testis and its traction towards the scrotum were assessed before surgery and under anaesthesia. The mean age of the patients was 4.6 years. The position of the testis assessed at surgery was in most cases at the external inguinal ring (62.8%), at the neck of the scrotum (15.7%), in the inguinal canal (12.8%), or in an ectopic position (8,5%). A PVD was found in 66 testes (47.1%). Two surgical cases required an inguinal incision. In each patient, the postoperative course was unremarkable. The testicle at 1-year follow-up was in a scrotal position in 134 cases, but 6 patients required a second surgical intervention for re-ascent of the testis. No testicular atrophy or inguinal hernias were observed. Transscrotal orchidopexy is a simple and effective procedure for the treatment of palpable UDT. The incidence of complications is low and manageable, with rapid postoperative recovery and early resumption of normal activities.

We report a long-term follow-up (6.5 years) of a phase I/II clinical trial envisaging the use of autologous genetically modified cultured epidermal stem cells for gene therapy of junctional epidermolysis bullosa, a devastating genetic skin disease. The critical goals of the trial were to evaluate the safety and long-term persistence of genetically modified epidermis. A normal epidermal-dermal junction was restored and the regenerated transgenic epidermis was found to be fully functional and virtually indistinguishable from a normal control. The epidermis was sustained by a discrete number of long-lasting, self-renewing transgenic epidermal stem cells that maintained the memory of the donor site, whereas the vast majority of transduced transit-amplifying progenitors were lost within the first few months after grafting. These data pave the way for the safe use of epidermal stem cells in combined cell and gene therapy for genetic skin diseases.

The usefulness of polyunsaturated fatty acids on inflammatory, cardiovascular, and the nervous system was studied in the last decades, but the mechanisms underlying their benefic properties are still partially unknown. These agents seem to express their action on the membrane phospholipid composition and permeability and modulation of second messenger cascades. In psychiatry, the efficacy and tolerability of omega-3 fatty acids were investigated in several psychiatric disorders, including major depression, bipolar disorder, personality disorders, high-risk conditions to develop psychosis, attention-deficit hyperactivity disorder, and autism spectrum disorders. Initial findings in this field are promising, and some relevant questions need to be addressed. In particular, the effects of these agents on the main symptom dimensions have to be investigated in a trans-diagnostic perspective. The present systematic review is aimed to examine the available data on the efficacy of omega-3 fatty acids on domains of psychotic symptoms, affective symptoms, impulsivity, and aggressiveness, and harmful behaviors, and suicide risk.