Artificial Intelligence-Powered Molecular Docking and Steered Molecular Dynamics for Accurate scFv Selection of Anti-CD30 Chimeric Antigen Receptors.

Chimeric antigen receptor (CAR) T cells represent a revolutionary immunotherapy that allows specific tumor recognition by a unique single-chain fragment variable (scFv) derived from monoclonal antibodies (mAbs). scFv selection is consequently a fundamental step for CAR construction, to ensure accurate and effective CAR signaling toward tumor antigen binding. However, conventional in vitro and in vivo biological approaches to compare different scFv-derived CARs are expensive and labor-intensive. With the aim to predict the finest scFv binding before CAR-T cell engineering, we performed artificial intelligence (AI)-guided molecular docking and steered molecular dynamics analysis of different anti-CD30 mAb clones. Virtual computational scFv screening showed comparable results to surface plasmon resonance (SPR) and functional CAR-T cell in vitro and in vivo assays, respectively, in terms of binding capacity and anti-tumor efficacy. The proposed fast and low-cost in silico analysis has the potential to advance the development of novel CAR constructs, with a substantial impact on reducing time, costs, and the need for laboratory animal use.

Lentiviral haematopoietic stem-cell gene therapy for early-onset metachromatic leukodystrophy: long-term results from a non-randomised, open-label, phase 1/2 trial and expanded access.

BACKGROUND: Effective treatment for metachromatic leukodystrophy (MLD) remains a substantial unmet medical need. In this study we investigated the safety and efficacy of atidarsagene autotemcel (arsa-cel) in patients with MLD. METHODS: This study is an integrated analysis of results from a prospective, non-randomised, phase 1/2 clinical study and expanded-access frameworks. 29 paediatric patients with pre-symptomatic or early-symptomatic early-onset MLD with biochemical and molecular confirmation of diagnosis were treated with arsa-cel, a gene therapy containing an autologous haematopoietic stem and progenitor cell (HSPC) population transduced ex vivo with a lentiviral vector encoding human arylsulfatase A (ARSA) cDNA, and compared with an untreated natural history (NHx) cohort of 31 patients with early-onset MLD, matched by age and disease subtype. Patients were treated and followed up at Ospedale San Raffaele, Milan, Italy. The coprimary efficacy endpoints were an improvement of more than 10% in total gross motor function measure score at 2 years after treatment in treated patients compared with controls, and change from baseline of total peripheral blood mononuclear cell (PBMC) ARSA activity at 2 years after treatment compared with values before treatment. This phase 1/2 study is registered with ClinicalTrials.gov, NCT01560182. FINDINGS: At the time of analyses, 26 patients treated with arsa-cel were alive with median follow-up of 3·16 years (range 0·64-7·51). Two patients died due to disease progression and one due to a sudden event deemed unlikely to be related to treatment. After busulfan conditioning, all arsa-cel treated patients showed sustained multilineage engraftment of genetically modified HSPCs. ARSA activity in PBMCs was significantly increased above baseline 2 years after treatment by a mean 18·7-fold (95% CI 8·3-42·2; p<0·0001) in patients with the late-infantile variant and 5·7-fold (2·6-12·4; p<0·0001) in patients with the early-juvenile variant. Mean differences in total scores for gross motor function measure between treated patients and age-matched and disease subtype-matched NHx patients 2 years after treatment were significant for both patients with late-infantile MLD (66% [95% CI 48·9-82·3]) and early-juvenile MLD (42% [12·3-71·8]). Most treated patients progressively acquired motor skills within the predicted range of healthy children or had stabilised motor performance (maintaining the ability to walk). Further, most displayed normal cognitive development and prevention or delay of central and peripheral demyelination and brain atrophy throughout follow-up; treatment benefits were particularly apparent in patients treated before symptom onset. The infusion was well tolerated and there was no evidence of abnormal clonal proliferation or replication-competent lentivirus. All patients had at least one grade 3 or higher adverse event; most were related to conditioning or to background disease. The only adverse event related to arsa-cel was the transient development of anti-ARSA antibodies in four patients, which did not affect clinical outcomes. INTERPRETATION: Treatment with arsa-cel resulted in sustained, clinically relevant benefits in children with early-onset MLD by preserving cognitive function and motor development in most patients, and slowing demyelination and brain atrophy. FUNDING: Orchard Therapeutics, Fondazione Telethon, and GlaxoSmithKline.

Piezo1 controls cell volume and migration by modulating swelling-activated chloride current through Ca2+ influx.

Regulatory volume decrease (RVD), a homeostatic process responsible for the re-establishment of the original cell volume upon swelling, is critical in controlling several functions, including migration. RVD is mainly sustained by the swelling-activated Cl- current (ICl,swell ), which can be modulated by cytoplasmic Ca2+ . Cell swelling also activates mechanosensitive channels, including the ubiquitously expressed Ca2+ -permeable channel Piezo1. We hypothesized that, by controlling cytoplasmic Ca2+ and in turn ICl,swell , Piezo1 is involved in the fine regulation of RVD and cell migration. We compared RVD and ICl,swell in wild-type (WT) HEK293T cells, which express endogenous levels of Piezo1, and in cells overexpressing (OVER) or knockout (KO) for Piezo1. Compared to WT, RVD was markedly increased in OVER, while virtually absent in KO cells. Consistently, ICl,swell amplitude was highest in OVER and lowest in KO cells, with WT cells displaying an intermediate level, suggesting a Ca2+ -dependent modulation of the current by Piezo1 channels. Indeed, in the absence of external Ca2+ , ICl,swell in both WT and OVER cells, as well as the RVD probed in OVER cells, were significantly lower than in the presence of Ca2+ and no longer different compared to KO cells. However, the Piezo-mediated Ca2+ influx was ineffective in enhancing ICl,swell in the absence of releasable Ca2+ from intracellular stores. The different expression levels of Piezo1 affected also cell migration which was strongly enhanced in OVER, while reduced in KO cells, as compared to WT. Taken together, our data indicate that Piezo1 controls RVD and migration in HEK293T cells by modulating ICl,swell through Ca2+ influx.

The role of physical cues in the development of stem cell-derived organoids.

Organoids are a novel three-dimensional stem cells' culture system that allows the in vitro recapitulation of organs/tissues structure complexity. Pluripotent and adult stem cells are included in a peculiar microenvironment consisting of a supporting structure (an extracellular matrix (ECM)-like component) and a cocktail of soluble bioactive molecules that, together, mimic the stem cell niche organization. It is noteworthy that the balance of all microenvironmental components is the most critical step for obtaining the successful development of an accurate organoid instead of an organoid with heterogeneous morphology, size, and cellular composition. Within this system, mechanical forces exerted on stem cells are collected by cellular proteins and transduced via mechanosensing-mechanotransduction mechanisms in biochemical signaling that dictate the stem cell specification process toward the formation of organoids. This review discusses the role of the environment in organoids formation and focuses on the effect of physical components on the developmental system. The work starts with a biological description of organoids and continues with the relevance of physical forces in the organoid environment formation. In this context, the methods used to generate organoids and some relevant published reports are discussed as examples showing the key role of mechanosensing-mechanotransduction mechanisms in stem cell-derived organoids.

De novo ssRNA Aptamers against the SARS-CoV-2 Main Protease: In Silico Design and Molecular Dynamics Simulation.

Herein, we have generated ssRNA aptamers to inhibit SARS-CoV-2 Mpro, a protease necessary for the SARS-CoV-2 coronavirus replication. Because there is no aptamer 3D structure currently available in the databanks for this protein, first, we modeled an ssRNA aptamer using an entropic fragment-based strategy. We refined the initial sequence and 3D structure by using two sequential approaches, consisting of an elitist genetic algorithm and an RNA inverse process. We identified three specific aptamers against SARS-CoV-2 Mpro, called MAptapro, MAptapro-IR1, and MAptapro-IR2, with similar 3D conformations and that fall in the dimerization region of the SARS-CoV-2 Mpro necessary for the enzymatic activity. Through the molecular dynamic simulation and binding free energy calculation, the interaction between the MAptapro-IR1 aptamer and the SARS-CoV-2 Mpro enzyme resulted in the strongest and the highest stable complex; therefore, the ssRNA MAptapro-IR1 aptamer was selected as the best potential candidate for the inhibition of SARS-CoV-2 Mpro and a perspective therapeutic drug for the COVID-19 disease.

Interfacial Compatibilization into PLA/Mg Composites for Improved In Vitro Bioactivity and Stem Cell Adhesion.

The present work highlights the crucial role of the interfacial compatibilization on the design of polylactic acid (PLA)/Magnesium (Mg) composites for bone regeneration applications. In this regard, an amphiphilic poly(ethylene oxide-b-L,L-lactide) diblock copolymer with predefined composition was synthesised and used as a new interface to provide physical interactions between the metallic filler and the biopolymer matrix. This strategy allowed (i) overcoming the PLA/Mg interfacial adhesion weakness and (ii) modulating the composite hydrophilicity, bioactivity and biological behaviour. First, a full study of the influence of the copolymer incorporation on the morphological, wettability, thermal, thermo-mechanical and mechanical properties of PLA/Mg was investigated. Subsequently, the bioactivity was assessed during an in vitro degradation in simulated body fluid (SBF). Finally, biological studies with stem cells were carried out. The results showed an increase of the interfacial adhesion by the formation of a new interphase between the hydrophobic PLA matrix and the hydrophilic Mg filler. This interface stabilization was confirmed by a decrease in the damping factor (tanδ) following the copolymer addition. The latter also proves the beneficial effect of the composite hydrophilicity by selective surface localization of the hydrophilic PEO leading to a significant increase in the protein adsorption. Furthermore, hydroxyapatite was formed in bulk after 8 weeks of immersion in the SBF, suggesting that the bioactivity will be noticeably improved by the addition of the diblock copolymer. This ceramic could react as a natural bonding junction between the designed implant and the fractured bone during osteoregeneration. On the other hand, a slight decrease of the composite mechanical performances was noted.

Novel bicistronic lentiviral vectors correct ß-Hexosaminidase deficiency in neural and hematopoietic stem cells and progeny: implications for in vivo and ex vivo gene therapy of GM2 gangliosidosis.

The favorable outcome of in vivo and ex vivo gene therapy approaches in several Lysosomal Storage Diseases suggests that these treatment strategies might equally benefit GM2 gangliosidosis. Tay-Sachs and Sandhoff disease (the main forms of GM2 gangliosidosis) result from mutations in either the HEXA or HEXB genes encoding, respectively, the α- or ß-subunits of the lysosomal ß-Hexosaminidase enzyme. In physiological conditions, α- and ß-subunits combine to generate ß-Hexosaminidase A (HexA, αß) and ß-Hexosaminidase B (HexB, ßß). A major impairment to establishing in vivo or ex vivo gene therapy for GM2 gangliosidosis is the need to synthesize the α- and ß-subunits at high levels and with the correct stoichiometric ratio, and to safely deliver the therapeutic products to all affected tissues/organs. Here, we report the generation and in vitro validation of novel bicistronic lentiviral vectors (LVs) encoding for both the murine and human codon optimized Hexa and Hexb genes. We show that these LVs drive the safe and coordinate expression of the α- and ß-subunits, leading to supranormal levels of ß-Hexosaminidase activity with prevalent formation of a functional HexA in SD murine neurons and glia, murine bone marrow-derived hematopoietic stem/progenitor cells (HSPCs), and human SD fibroblasts. The restoration/overexpression of ß-Hexosaminidase leads to the reduction of intracellular GM2 ganglioside storage in transduced and in cross-corrected SD murine neural progeny, indicating that the transgenic enzyme is secreted and functional. Importantly, bicistronic LVs safely and efficiently transduce human neurons/glia and CD34+ HSPCs, which are target and effector cells, respectively, in prospective in vivo and ex vivo GT approaches. We anticipate that these bicistronic LVs may overcome the current requirement of two vectors co-delivering the α- or ß-subunits genes. Careful assessment of the safety and therapeutic potential of these bicistronic LVs in the SD murine model will pave the way to the clinical development of LV-based gene therapy for GM2 gangliosidosis.

Unpatterned Bioactive Poly(Butylene 1,4-Cyclohexanedicarboxylate)-Based Film Fast Induced Neuronal-Like Differentiation of Human Bone Marrow-Mesenchymal Stem Cells.

Herein, we present poly(butylene 1,4-cyclohexanedicarboxylate) (PBCE) films characterized by an unpatterned microstructure and a specific hydrophobicity, capable of boosting a drastic cytoskeleton architecture remodeling, culminating with the neuronal-like differentiation of human bone marrow-mesenchymal stem cells (hBM-MSCs). We have used two different filming procedures to prepare the films, solvent casting (PBCE) and compression-moulding (PBCE*). PBCE film had a rough and porous surface with spherulite-like aggregations (Ø = 10-20 µm) and was characterized by a water contact angle = 100°. PBCE* showed a smooth and continuous surface without voids and visible spherulite-like aggregations and was more hydrophobic (WCA = 110°). Both surface characteristics were modulated through the copolymerization of different amounts of ether-oxygen-containing co-units into PBCE chemical structure. We showed that only the surface characteristics of PBCE-solvent-casted films steered hBM-MSCs toward a neuronal-like differentiation. hBM-MSCs lost their canonical mesenchymal morphology, acquired a neuronal polarized shape with a long cell protrusion (≥150 µm), expressed neuron-specific class III ß-tubulin and microtubule-associated protein 2 neuronal markers, while nestin, a marker of uncommitted stem cells, was drastically silenced. These events were observed as early as 2-days after cell seeding. Of note, the phenomenon was totally absent on PBCE* film, as hBM-MSCs maintained the mesenchymal shape and behavior and did not express neuronal/glial markers.

Insight into Mechanobiology: How Stem Cells Feel Mechanical Forces and Orchestrate Biological Functions.

The cross-talk between stem cells and their microenvironment has been shown to have a direct impact on stem cells' decisions about proliferation, growth, migration, and differentiation. It is well known that stem cells, tissues, organs, and whole organisms change their internal architecture and composition in response to external physical stimuli, thanks to cells' ability to sense mechanical signals and elicit selected biological functions. Likewise, stem cells play an active role in governing the composition and the architecture of their microenvironment. Is now being documented that, thanks to this dynamic relationship, stemness identity and stem cell functions are maintained. In this work, we review the current knowledge in mechanobiology on stem cells. We start with the description of theoretical basis of mechanobiology, continue with the effects of mechanical cues on stem cells, development, pathology, and regenerative medicine, and emphasize the contribution in the field of the development of ex-vivo mechanobiology modelling and computational tools, which allow for evaluating the role of forces on stem cell biology.

Integrated Computational Analysis Highlights unique miRNA Signatures in the Subventricular Zone and Striatum of GM2 Gangliosidosis Animal Models.

This work explores for the first time the potential contribution of microRNAs (miRNAs) to the pathophysiology of the GM2 gangliosidosis, a group of Lysosomal Storage Diseases. In spite of the genetic origin of GM2 gangliosidosis, the cascade of events leading from the gene/protein defects to the cell dysfunction and death is not fully elucidated. At present, there is no cure for patients. Taking advantage of the animal models of two forms of GM2 gangliosidosis, Tay-Sachs (TSD) and Sandhoff (SD) diseases, we performed a microRNA screening in the brain subventricular zone (SVZ) and striatum (STR), which feature the neurogenesis and neurodegeneration states, respectively, in adult mutant mice. We found abnormal expression of a panel of miRNAs involved in lipid metabolism, CNS development and homeostasis, and neuropathological processes, highlighting region- and disease-specific profiles of miRNA expression. Moreover, by using a computational analysis approach, we identified a unique disease- (SD or TSD) and brain region-specific (SVZ vs. STR) miRNAs signatures of predicted networks potentially related to the pathogenesis of the diseases. These results may contribute to the understanding of GM2 gangliosidosis pathophysiology, with the aim of developing effective treatments.

Adipose Stem Cell Translational Applications: From Bench-to-Bedside.

During the last five years, there has been a significantly increasing interest in adult adipose stem cells (ASCs) as a suitable tool for translational medicine applications. The abundant and renewable source of ASCs and the relatively simple procedure for cell isolation are only some of the reasons for this success. Here, we document the advances in the biology and in the innovative biotechnological applications of ASCs. We discuss how the multipotential property boosts ASCs toward mesenchymal and non-mesenchymal differentiation cell lineages and how their character is maintained even if they are combined with gene delivery systems and/or biomaterials, both in vitro and in vivo.

Combined gene/cell therapies provide long-term and pervasive rescue of multiple pathological symptoms in a murine model of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) is a lysosomal storage disease caused by deficient activity of ß-galactocerebrosidase (GALC). The infantile forms manifest with rapid and progressive central and peripheral demyelination, which represent a major hurdle for any treatment approach. We demonstrate here that neonatal lentiviral vector-mediated intracerebral gene therapy (IC GT) or transplantation of GALC-overexpressing neural stem cells (NSC) synergize with bone marrow transplant (BMT) providing dramatic extension of lifespan and global clinical-pathological rescue in a relevant GLD murine model. We show that timely and long-lasting delivery of functional GALC in affected tissues ensured by the exclusive complementary mode of action of the treatments underlies the outstanding benefit. In particular, the contribution of neural stem cell transplantation and IC GT during the early asymptomatic stage of the disease is instrumental to enhance long-term advantage upon BMT. We clarify the input of central nervous system, peripheral nervous system and periphery to the disease, and the relative contribution of treatments to the final therapeutic outcome, with important implications for treatment strategies to be tried in human patients. This study gives proof-of-concept of efficacy, tolerability and clinical relevance of the combined gene/cell therapies proposed here, which may constitute a feasible and effective therapeutic opportunity for children affected by GLD.

Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial.

BACKGROUND: Metachromatic leukodystrophy (a deficiency of arylsulfatase A [ARSA]) is a fatal demyelinating lysosomal disease with no approved treatment. We aimed to assess the long-term outcomes in a cohort of patients with early-onset metachromatic leukodystrophy who underwent haemopoietic stem-cell gene therapy (HSC-GT). METHODS: This is an ad-hoc analysis of data from an ongoing, non-randomised, open-label, single-arm phase 1/2 trial, in which we enrolled patients with a molecular and biochemical diagnosis of metachromatic leukodystrophy (presymptomatic late-infantile or early-juvenile disease or early-symptomatic early-juvenile disease) at the Paediatric Clinical Research Unit, Ospedale San Raffaele, in Milan. Trial participants received HSC-GT, which consisted of the infusion of autologous HSCs transduced with a lentiviral vector encoding ARSA cDNA, after exposure-targeted busulfan conditioning. The primary endpoints of the trial are safety (toxicity, absence of engraftment failure or delayed haematological reconstitution, and safety of lentiviral vector-tranduced cell infusion) and efficacy (improvement in Gross Motor Function Measure [GMFM] score relative to untreated historical controls, and ARSA activity, 24 months post-treatment) of HSC-GT. For this ad-hoc analysis, we assessed safety and efficacy outcomes in all patients who had received treatment and been followed up for at least 18 months post-treatment on June 1, 2015. This trial is registered with ClinicalTrials.gov, number NCT01560182. FINDINGS: Between April, 2010, and February, 2013, we had enrolled nine children with a diagnosis of early-onset disease (six had late-infantile disease, two had early-juvenile disease, and one had early-onset disease that could not be definitively classified). At the time of analysis all children had survived, with a median follow-up of 36 months (range 18-54). The most commonly reported adverse events were cytopenia (reported in all patients) and mucositis of different grades of severity (in five of nine patients [grade 3 in four of five patients]). No serious adverse events related to the medicinal product were reported. Stable, sustained engraftment of gene-corrected HSCs was observed (a median of 60·4% [range 14·0-95·6] lentiviral vector-positive colony-forming cells across follow-up) and the engraftment level was stable during follow-up; engraftment determinants included the duration of absolute neutropenia and the vector copy number of the medicinal product. A progressive reconstitution of ARSA activity in circulating haemopoietic cells and in the cerebrospinal fluid was documented in all patients in association with a reduction of the storage material in peripheral nerve samples in six of seven patients. Eight patients, seven of whom received treatment when presymptomatic, had prevention of disease onset or halted disease progression as per clinical and instrumental assessment, compared with historical untreated control patients with early-onset disease. GMFM scores for six patients up to the last follow-up showed that gross motor performance was similar to that of normally developing children. The extent of benefit appeared to be influenced by the interval between HSC-GT and the expected time of disease onset. Treatment resulted in protection from CNS demyelination in eight patients and, in at least three patients, amelioration of peripheral nervous system abnormalities, with signs of remyelination at both sites. INTERPRETATION: Our ad-hoc findings provide preliminary evidence of safety and therapeutic benefit of HSC-GT in patients with early-onset metachromatic leukodystrophy who received treatment in the presymptomatic or very early-symptomatic stage. The results of this trial will be reported when all 20 patients have achieved 3 years of follow-up. FUNDING: Italian Telethon Foundation and GlaxoSmithKline.

A Comparison of Lysosomal Enzymes Expression Levels in Peripheral Blood of Mild- and Severe-Alzheimer's Disease and MCI Patients: Implications for Regenerative Medicine Approaches.

The association of lysosomal dysfunction and neurodegeneration has been documented in several neurodegenerative diseases, including Alzheimer's Disease (AD). Herein, we investigate the association of lysosomal enzymes with AD at different stages of progression of the disease (mild and severe) or with mild cognitive impairment (MCI). We conducted a screening of two classes of lysosomal enzymes: glycohydrolases (ß-Hexosaminidase, ß-Galctosidase, ß-Galactosylcerebrosidase, ß-Glucuronidase) and proteases (Cathepsins S, D, B, L) in peripheral blood samples (blood plasma and PBMCs) from mild AD, severe AD, MCI and healthy control subjects. We confirmed the lysosomal dysfunction in severe AD patients and added new findings enhancing the association of abnormal levels of specific lysosomal enzymes with the mild AD or severe AD, and highlighting the difference of AD from MCI. Herein, we showed for the first time the specific alteration of ß-Galctosidase (Gal), ß-Galactosylcerebrosidase (GALC) in MCI patients. It is notable that in above peripheral biological samples the lysosomes are more sensitive to AD cellular metabolic alteration when compared to levels of Aß-peptide or Tau proteins, similar in both AD groups analyzed. Collectively, our findings support the role of lysosomal enzymes as potential peripheral molecules that vary with the progression of AD, and make them useful for monitoring regenerative medicine approaches for AD.

Therapeutic benefit of lentiviral-mediated neonatal intracerebral gene therapy in a mouse model of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) is an inherited lysosomal storage disease caused by ß-galactocerebrosidase (GALC) deficiency. Gene therapy (GT) should provide rapid, extensive and lifetime GALC supply in central nervous system (CNS) tissues to prevent or halt irreversible neurologic progression. Here we used a lentiviral vector (LV) to transfer a functional GALC gene in the brain of Twitcher mice, a severe GLD model. A single injection of LV.GALC in the external capsule of Twitcher neonates resulted in robust transduction of neural cells with minimal and transient activation of inflammatory and immune response. Importantly, we documented a proficient transduction of proliferating and post-mitotic oligodendroglia, a relevant target cell type in GLD. GALC activity (30-50% of physiological levels) was restored in the whole CNS of treated mice as early as 8 days post-injection. The early and stable enzymatic supply ensured partial clearance of storage and reduction of psychosine levels, translating in amelioration of histopathology and enhanced lifespan. At 6 months post-injection in non-affected mice, LV genome persisted exclusively in the injected region, where transduced cells overexpressed GALC. Integration site analysis in transduced brain tissues showed no aberrant clonal expansion and preferential targeting of neural-specific genes. This study establishes neonatal LV-mediated intracerebral GT as a rapid, effective and safe therapeutic intervention to correct CNS pathology in GLD and provides a strong rationale for its application in this and similar leukodystrophies, alone or in combination with therapies targeting the somatic pathology, with the final aim of providing an effective and timely treatment of these global disorders.

A new analytical bench assay for the determination of arylsulfatase a activity toward galactosyl-3-sulfate ceramide: implication for metachromatic leukodystrophy diagnosis.

Here, we present the design and validation of a new assay for the diagnosis of metachromatic leukodystrophy. The method is highly specific, simple, reproducible, and straightforward. In our spectrophotometric method, the determination of arylsulfatase A (ARSA) activity toward the natural substrate, galactosyl-3-sulfate ceramide (or sulfatide), is performed using neat sulfatide without chemical modification. This confers to the assay high analytical specificity. The hydrolyzed sulfatide is monitored upon inclusion of the colorimetric reagent Azure A. The nonhydrolyzed sulfatide-Azure A is recovered and measured at a wavelength of λ = 650 nm. Thus, ARSA activity toward the sulfatide is obtained by subtracting the nonhydrolyzed sulfatide from the total sulfatide used in the enzyme reaction (sulfatide-Azure A present in a parallel assay performed in the absence of ARSA). Within a clinical context, our method definitely discriminated between healthy subject samples and metachromatic leukodystrophy patient samples, and, therefore, it is suitable for diagnostic applications and for monitoring the efficacy of therapeutic treatments in patients or animal models.

Piezo1 - Serine/threonine-protein phosphatase 2A - Cofilin1 biochemical mechanotransduction axis controls F-actin dynamics and cell migration.

This study sheds light on a ground-breaking biochemical mechanotransduction pathway and reveals how Piezo1 channels orchestrate cell migration. We observed an increased cell migration rate in HEK293T (HEK) cells treated with Yoda1, a Piezo1 agonist, or in HEK cells overexpressing Piezo1 (HEK + P). Conversely, a significant reduction in cell motility was observed in HEK cells treated with GsMTx4 (a channel inhibitor) or upon silencing Piezo1 (HEK-P). Our findings establish a direct correlation between alterations in cell motility, Piezo1 expression, abnormal F-actin microfilament dynamics, and the regulation of Cofilin1, a protein involved in severing F-actin microfilaments. Here, the conversion of inactive pCofilin1 to active Cofilin1, mediated by the serine/threonine-protein phosphatase 2A catalytic subunit C (PP2AC), resulted in increased severing of F-actin microfilaments and enhanced cell migration in HEK + P cells compared to HEK controls. However, this effect was negligible in HEK-P and HEK cells transfected with hsa-miR-133b, which post-transcriptionally inhibited PP2AC mRNA expression. In summary, our study suggests that Piezo1 regulates cell migration through a biochemical mechanotransduction pathway involving PP2AC-mediated Cofilin1 dephosphorylation, leading to changes in F-actin microfilament dynamics.

Impact of Soil Fertilization with Pig Slurry on Antibiotic Residues and Resistance Genes: A Longitudinal Study.

The impact of soil fertilization with animal manure on the spread and persistence of antibiotic resistance in the environment is far from being fully understood. To add knowledge about persistence and correlations between antibiotic residues and antibiotic resistance genes (ARGs) in fertilized soil, a longitudinal soil mesocosm study was conducted. Soil samples were collected from the mesocosms immediately before spreading and then afterward at fifteen time points during a 320-day observation period. Eight ARGs (ermB, sul1, tetA, tetG, tetM, cfr, fexA, and optrA) and the class 1 integron-integrase gene, intI1, were determined in both pig slurry and soil, as well as residues of 36 antibiotics. Soil chemical and biochemical parameters were also measured. Twelve antibiotics were detected in the slurry in the range of 3 µg kg-1-3605 µg kg-1, with doxycycline, lincomycin, and tiamulin being the most abundant, whereas ermB, sul1, and tetM were the predominant ARGs. Before spreading, neither antibiotic residues nor ARGs were detectable in the soil; afterwards, their concentrations mirrored those in the slurry, with a gradual decline over the duration of the experiment. After about three months, the effect of the amendment was almost over, and no further evolution was observed.

Tight Regulation of Mechanotransducer Proteins Distinguishes the Response of Adult Multipotent Mesenchymal Cells on PBCE-Derivative Polymer Films with Different Hydrophilicity and Stiffness.

Mechanotransduction is a molecular process by which cells translate physical stimuli exerted by the external environment into biochemical pathways to orchestrate the cellular shape and function. Even with the advancements in the field, the molecular events leading to the signal cascade are still unclear. The current biotechnology of tissue engineering offers the opportunity to study in vitro the effect of the physical stimuli exerted by biomaterial on stem cells and the mechanotransduction pathway involved in the process. Here, we cultured multipotent human mesenchymal/stromal cells (hMSCs) isolated from bone marrow (hBM-MSCs) and adipose tissue (hASCs) on films of poly(butylene 1,4-cyclohexane dicarboxylate) (PBCE) and a PBCE-based copolymer containing 50 mol% of butylene diglycolate co-units (BDG50), to intentionally tune the surface hydrophilicity and the stiffness (PBCE = 560 Mpa; BDG50 = 94 MPa). We demonstrated the activated distinctive mechanotransduction pathways, resulting in the acquisition of an elongated shape in hBM-MSCs on the BDG50 film and in maintaining the canonical morphology on the PBCE film. Notably, hASCs acquired a new, elongated morphology on both the PBCE and BDG50 films. We found that these events were mainly due to the differences in the expression of Cofilin1, Vimentin, Filamin A, and Talin, which established highly sensitive machinery by which, rather than hASCs, hBM-MSCs distinguished PBCE from BDG50 films.

Novel Nanostructured Scaffolds of Poly(butylene trans-1,4-cyclohexanedicarboxylate)-Based Copolymers with Tailored Hydrophilicity and Stiffness: Implication for Tissue Engineering Modeling.

Here, we present novel biocompatible poly(butylene trans-1,4-cyclohexanedicarboxylate) (PBCE)-based random copolymer nanostructured scaffolds with tailored stiffness and hydrophilicity. The introduction of a butylene diglycolate (BDG) co-unit, containing ether oxygen atoms, along the PBCE chain remarkably improved the hydrophilicity and chain flexibility. The copolymer containing 50 mol% BDG co-units (BDG50) and the parent homopolymer (PBCE) were synthesized and processed as electrospun scaffolds and compression-molded films, added for the sake of comparison. We performed thermal, wettability, and stress-strain measures on the PBCE-derived scaffolds and films. We also conducted biocompatibility studies by evaluating the adhesion and proliferation of multipotent mesenchymal/stromal cells (hBM-MSCs) on each polymeric film and scaffold. We demonstrated that solid-state properties can be tailored by altering sample morphology besides chemical structure. Thus, scaffolds were characterized by a higher hydrophobicity and a lower elastic modulus than the corresponding films. The three-dimensional nanostructure conferred a higher adsorption protein capability to the scaffolds compared to their film counterparts. Finally, the PBCE and BDG50 scaffolds were suitable for the long-term culture of hBM-MSCs. Collectively, the PBCE homopolymer and copolymer are good candidates for tissue engineering applications.