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DNAJC15 is a mitochondrial TIMM23-related co-chaperonin known for its role in regulating oxidative phosphorylation efficiency, oxidative stress response and lipid metabolism. Recently, it has been proposed that the loss of DNAJC15 correlates with cisplatin (CDDP)-resistance onset in ovarian cancer (OC), suggesting this protein as a potential prognostic factor during OC progression. However, the molecular mechanisms through which DNAJC15 contributes to CDDP response remains poorly investigated. Here, we show that high levels of DNAJC15 are associated with accumulation of lipid droplets, decreased tumorigenic features and increased sensitivity to CDDP in OC cells. When overexpressed, DNAJC15 induced a phenotype displaying increased lipid peroxidation and subsequent ferroptosis induction. To prove a role for DNAJC15-induced ferroptosis in promoting sensitivity to CDDP, we reduced lipid peroxidation upon Ferrostatin 1 treatment, which decreased cells' vulnerability to ferroptosis ultimately recovering their CDDP-resistant phenotype. In conclusion, our study uncovers the role of DNAJC15 in modulating ferroptosis activation and in the onset of CDDP resistance in OC cells.

Colorectal cancer (CRC) is a major public health concern and identifying prognostic molecular biomarkers can help stratify patients based on risk profiles, thus enabling personalized medicine. Epitranscriptomic modifications play a relevant role in controlling gene expression, N6-methyladenosine (m6A) regulators play crucial roles in cancer progression, but their clinical significance in CRC cancer has thus far not been elucidated. Thus, we aimed to examine by immunohistochemical techniques and RT-qPCR, protein levels and RNAs expression of m6A writers (METTL3, WTAP) and eraser (FTO) in a cohort of 10 patients affected by CRC. The patients were followed for 5 years and values of METTL3, WTAP and FTO RNAs in alive vs dead patients were compared. Proteins expression and RNAs expression had a different trend, METTL3, WTAP and FTO proteins' expression showed an increasing trend from non-cancerous adjacent (N) tissue vs carcinoma (CA) tissue G1 stage, and then a decreasing trend from G1 to G2 and G3 stages. The most marked increase was observed in WTAP that, from a 40% of protein expression positivity in N tissue raised to the 81% of positivity in G1 stage K tissue. RNAs expression of METTL3, WTAP and FTO genes in N tissue vs G1 stage CA tissue was significantly different, the analysis and comparison of RNAs values in patient alive after 5 years (0.58±0.04) vs patients dead after 5 years (1.69±0.29) showed that only WTAP values resulted significantly high in dead patients. The fact that WTAP protein expression levels lower while WTAP RNA expression remains high, lets us hypothesize a sort of inhibition of protein expression, but further studies are needed to clarify the mechanism. Although the results suggest a relationship between biological meaning and prognostic utility of WTAP, this prognostic utility must be confirmed by further studies on a larger sample.

Mucopolysaccharidosis type I Hurler (MPSIH) is characterized by severe and progressive skeletal dysplasia that is not fully addressed by allogeneic hematopoietic stem cell transplantation (HSCT). Autologous hematopoietic stem progenitor cell-gene therapy (HSPC-GT) provides superior metabolic correction in patients with MPSIH compared with HSCT; however, its ability to affect skeletal manifestations is unknown. Eight patients with MPSIH (mean age at treatment: 1.9 years) received lentiviral-based HSPC-GT in a phase 1/2 clinical trial (NCT03488394). Clinical (growth, measures of kyphosis and genu velgum), functional (motor function, joint range of motion), and radiological [acetabular index (AI), migration percentage (MP) in hip x-rays and MRIs and spine MRI score] parameters of skeletal dysplasia were evaluated at baseline and multiple time points up to 4 years after treatment. Specific skeletal measures were retrospectively compared with an external cohort of HSCT-treated patients. At a median follow-up of 3.78 years after HSPC-GT, all patients treated with HSPC-GT exhibited longitudinal growth within WHO reference ranges and a median height gain greater than that observed in patients treated with HSCT after 3-year follow-up. Patients receiving HSPC-GT experienced complete and earlier normalization of joint mobility compared with patients treated with HSCT. Mean AI and MP showed progressive decreases after HSPC-GT, suggesting a reduction in acetabular dysplasia. Typical spine alterations measured through a spine MRI score stabilized after HSPC-GT. Clinical, functional, and radiological measures suggested an early beneficial effect of HSPC-GT on MPSIH-typical skeletal features. Longer follow-up is needed to draw definitive conclusions on HSPC-GT's impact on MPSIH skeletal dysplasia.

The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03878-4., Competing Interests: Conflict of interestOn behalf of all authors, the corresponding author states that there is no conflict of interest., (© The Author(s) 2024.)

It is known that exposure to heavy metal such as lead (Pb) and cadmium (Cd) has several adverse effects, particularly on the human reproductive system. Pb and Cd have been associated with infertility in both men and women. In pregnant women, they have been associated with spontaneous abortion, preterm birth, and impairment of the development of the fetus. Since these heavy metals come from both natural and anthropogenic activities and their harmful effects have been observed even at low levels of exposure, exposure to them remains a public health issue, especially for the reproductive system. Given this, the present study aimed to investigate the potential reproductive effects of Pb and Cd levels in the follicular fluid (FF) of infertile women and non-smokers exposed to heavy metals for professional reasons or as a result of living in rural areas near landfills and waste disposal areas in order to correlate the intrafollicular presence of these metals with possible alterations in the ultrastructure of human cumulus-oocyte complexes (COCs), which are probably responsible for infertility. Blood and FF metals were measured using atomic absorption spectrometry. COCs corresponding to each FF analyzed were subjected to ultrastructural analyses using transmission electron microscopy. We demonstrated for the first time that intrafollicular levels of Pb (0.66 µg/dL-0.85 µg/dL) and Cd (0.26 µg/L-0.41 µg/L) could be associated with morphological alterations of both the oocyte and cumulus cells' (CCs) ultrastructure. Since blood Cd levels (0.54 µg/L-1.87 µg/L) were above the current reference values established by the guidelines of the Agency for Toxic Substances and Disease Registry (ATSDR) and the Environmental Protection Agency (EPA) (0.4 µg/L), whereas blood Pb levels (1.28 µg/dL-3.98 µg/dL) were below the ATSDR reference values (≤5 µg/dL), we believe that these alterations could be due especially to Cd, even if we cannot exclude a possible additional effect of Pb. Our results highlighted that oocytes were affected in maturation and quality, whereas CCs showed scarcely active steroidogenic elements. Regressing CCs, with cytoplasmic alterations, were also numerous. According to Cd's endocrine-disrupting activity, the poor steroidogenic activity of CCs might correlate with delayed oocyte cytoplasmic maturation. So, we conclude that levels of heavy metals in the blood and the FF might negatively affect fertilization, embryo development, and pregnancy, compromising oocyte competence in fertilization both directly and indirectly, impairing CC steroidogenic activity, and inducing CC apoptosis.

A systematic and narrative literature review was performed, focusing attention on the anatomy of the area located at the junction of the sphenoid and the basal portion of the temporal bone (petrous bone, petrous apex, upper petro-clival region) encircled by the free edge of the tentorium, the insertion of the tentorium itself to the petrous apex and the anterior and posterior clinoid processes that give rise to three distinct dural folds or ligaments: the anterior petroclinoid ligament, the posterior petroclinoid ligament and the interclinoid ligament. These dural folds constitute the posterior portion of the roof of the cavernous sinus denominated "the oculomotor triangle". The main purpose of this review study was to describe this anatomical region, particularly in the light of the relationships between the anterior margin of the free edge of the tentorium and the above-mentioned components of the sphenoid and petrous bone.

Background: Acute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A)., Methods: We treated FLT3-ITD + AML cells with low doses of the aforementioned drugs, used alone or in combinations and we investigated the induction of ER and oxidative stress. We then performed the same experiments in an in vitro co-culture system of FLT3-ITD + AML cells and bone marrow stromal cells (BMSCs) to assess the protective role of the niche on AML blasts. Eventually, we tested the combination of drugs in an orthotopic murine model of human AML., Results: The combination RBA exerts strong cytotoxic activity on FLT3-ITD + AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with BMSCs. Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD + AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML and BMSCs upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSCs., Conclusions: Our findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD + AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML and BMSCs, involving mechanotransduction and YAP signaling., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

Heart failure is a major side effect of doxorubicin (DOX) treatment in patients with cancer. However, the mechanisms underlying the development of DOX-induced heart failure need to be addressed. This study aims to test whether the serine/threonine kinase MST1, a major Hippo pathway component, contributes to the development of DOX-induced myocardial injury. C57BL/6J WT mice and mice with cardiomyocyte-specific dominant-negative MST1 (kinase-dead) overexpression received three weekly injections of DOX, reaching a final cumulative dose of 18 mg/kg. Echocardiographic, histological and biochemical analyses were performed six weeks after the first DOX administration. The effects of MST1 inhibition on DOX-induced cardiomyocyte injury were also tested in vitro. MST1 signaling was significantly activated in cardiomyocytes in response to DOX treatment in vitro and in vivo. Wild-type (WT) mice treated with DOX developed cardiac dysfunction and mitochondrial abnormalities. However, these detrimental effects were abolished in mice with cardiomyocyte-specific overexpression of dominant-negative MST1 (DN-MST1) or treated with XMU-MP-1, a specific MST1 inhibitor, indicating that MST1 inhibition attenuates DOX-induced cardiac dysfunction. DOX treatment led to a significant downregulation of cardiac levels of SIRT3, a deacetylase involved in mitochondrial protection, in WT mice, which was rescued by MST1 inhibition. Pharmacological inhibition of SIRT3 blunted the protective effects of MST1 inhibition, indicating that SIRT3 downregulation mediates the cytotoxic effects of MST1 activation in response to DOX treatment. Finally, we found a significant upregulation of MST1 and downregulation of SIRT3 levels in human myocardial tissue of cancer patients treated with DOX. In summary, MST1 contributes to DOX-induced cardiomyopathy through SIRT3 downregulation., (© 2023. The Author(s).)

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies., (© 2023. The Author(s).)

The Gravity Force to which living beings are subjected on Earth rules the functionality of most biological processes in many tissues. It has been reported that a situation of Microgravity (such as that occurring in space) causes negative effects on living beings. Astronauts returning from space shuttle missions or from the International Space Station have been diagnosed with various health problems, such as bone demineralization, muscle atrophy, cardiovascular deconditioning, and vestibular and sensory imbalance, including impaired visual acuity, altered metabolic and nutritional status, and immune system dysregulation. Microgravity has profound effects also on reproductive functions. Female astronauts, in fact, suppress their cycles during space travels, and effects at the cellular level in the early embryo development and on female gamete maturation have also been observed. The opportunities to use space flights to study the effects of gravity variations are limited because of the high costs and lack of repeatability of the experiments. For these reasons, the use of microgravity simulators for studying, at the cellular level, the effects, such as those, obtained during/after a spatial trip, are developed to confirm that these models can be used in the study of body responses under conditions different from those found in a unitary Gravity environment (1 g). In view of this, this study aimed to investigate in vitro the effects of simulated microgravity on the ultrastructural features of human metaphase II oocytes using a Random Positioning Machine (RPM). We demonstrated for the first time, by Transmission Electron Microscopy analysis, that microgravity might compromise oocyte quality by affecting not only the localization of mitochondria and cortical granules due to a possible alteration of the cytoskeleton but also the function of mitochondria and endoplasmic reticulum since in RPM oocytes we observed a switch in the morphology of smooth endoplasmic reticulum (SER) and associated mitochondria from mitochondria-SER aggregates to mitochondria-vesicle complexes. We concluded that microgravity might negatively affect oocyte quality by interfering in vitro with the normal sequence of morphodynamic events essential for acquiring and maintaining a proper competence to fertilization in human oocytes.

Mitochondria act as key organelles in cellular bioenergetics and biosynthetic processes producing signals that regulate different molecular networks for proliferation and cell death. This ability is also preserved in pathologic contexts such as tumorigenesis, during which bioenergetic changes and metabolic reprogramming confer flexibility favoring cancer cell survival in a hostile microenvironment. Although different studies epitomize mitochondrial dysfunction as a protumorigenic hit, genetic ablation or pharmacological inhibition of respiratory complex I causing a severe impairment is associated with a low-proliferative phenotype. In this scenario, it must be considered that despite the initial delay in growth, cancer cells may become able to resume proliferation exploiting molecular mechanisms to overcome growth arrest. Here, we highlight the current knowledge on molecular responses activated by complex I-defective cancer cells to bypass physiological control systems and to re-adapt their fitness during microenvironment changes. Such adaptive mechanisms could reveal possible novel molecular players in synthetic lethality with complex I impairment, thus providing new synergistic strategies for mitochondrial-based anticancer therapy., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Inhibition of respiratory complex I (CI) is becoming a promising anti-cancer strategy, encouraging the design and the use of inhibitors, whose mechanism of action, efficacy and specificity remain elusive. As CI is a central player of cellular bioenergetics, a finely tuned dosing of targeting drugs is required to avoid side effects. We compared the specificity and mode of action of CI inhibitors metformin, BAY 87-2243 and EVP 4593 using cancer cell models devoid of CI. Here we show that both BAY 87-2243 and EVP 4593 were selective, while the antiproliferative effects of metformin were considerably independent from CI inhibition. Molecular docking predictions indicated that the high efficiency of BAY 87-2243 and EVP 4593 may derive from the tight network of bonds in the quinone binding pocket, although in different sites. Most of the amino acids involved in such interactions are conserved across species and only rarely found mutated in human. Our data make a case for caution when referring to metformin as a CI-targeting compound, and highlight the need for dosage optimization and careful evaluation of molecular interactions between inhibitors and the holoenzyme.

Objectives: Extracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here, we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL., Methods: We tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL-derived EVs., Results: A subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in human umbilical vein endothelial cells (HUVECs) and increasing the angiogenic tubularly-like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched with small RNAs and a high amount of miR-126, the most abundant angiogenic miRNA in platelets. The transfer of miR-126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as a downstream target in this system., Conclusion: PL is a biological source of available EVs with angiogenic effects involving a miRNAs-based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs., (© 2022 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Up-to-date in vitro and in vivo preclinical models expressing the patient-specific cancer lineage responsible for CRC and its metastatic behavior and responsiveness to therapy are needed. Exosomes' role in tumorigenesis and the metastatic process was demonstrated, and the material content and size of the exosomes are associated with a poor prognosis of CRC. Exosomes are generally imagined after their recovery from blood serum as isolated entities, and our work aims to investigate them "in situ" in their native environment by scanning and transmission electron microscopy to understand their secretion modalities. We studied CRC stem cells in patient-derived multicellular tumor spheroids (MTSs) and in their mouse xenograft to find possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models. We observed that MTSs' exosome secretion patterns depend on their structural complexity: few-layer MTSs show a lesser exosome secretion, limited to the apical domain of cancer cells, secretion increases in multilayered MTSs, and it develops from apical and basolateral cancer cells domains. In xenograft models, exosome secretion occurs from all cancer cell domains, and it is quantitatively greater than that observed in MTSs. This difference in exosome secretion pattern between MTSs and xenografts may be due to the influence of surrounding non-tumor cells.

Small cell neuroendocrine carcinoma is most frequently found in the lung (SCLC), but it has been also reported, albeit with a very low incidence, in the ovary. Here, we analyze a case of primary small cell carcinoma of the ovary of pulmonary type (SCCOPT), a rare and aggressive tumor with poor prognosis, whose biology and molecular features have not yet been thoroughly investigated. The patient affected by SCCOPT had a residual tumor following chemotherapy which displayed pronounced similarity with neuroendocrine tumors and lung cancer in terms of its microRNA expression profile and mTOR-downstream activation. By analyzing the metabolic markers of the neoplastic lesion, we established a likely glycolytic signature. In conclusion, this in-depth characterization of SCCOPT could be useful for future diagnoses, possibly aided by microRNA profiling, allowing clinicians to adopt the most appropriate therapeutic strategy.

Before entering human clinical studies to evaluate their safety and effectiveness, new drugs and novel medical treatments are subject to extensive animal testing that are expensive and time-consuming. By contrast, advanced technologies enable the development of animal-free models that allow the efficacy of innovative therapies to be studied without sacrificing animals, while providing helpful information and details. We report on the powerful combination of 3D bioprinting (3DB) and photo-thermal therapy (PTT) applications. To this end, we realize a 3DB construct consisting of glioblastoma U87-MG cells in a 3D geometry, incorporating biomimetic keratin-coated gold nanoparticles (Ker-AuNPs) as a photo-thermal agent. The resulting plasmonic 3DB structures exhibit a homogeneous cell distribution throughout the entire volume while promoting the localization of Ker-AuNPs within the cells. A 3D immunofluorescence assay and transmission electron microscopy (TEM) confirm the uniform distribution of fluorescent-labeled Ker-AuNPs in the volume and their capability to enter the cells. Laser-assisted (λ = 532 nm) PTT experiments demonstrate the extraordinary ability of Ker-AuNPs to generate heating, producing the highest temperature rise of about 16 °C in less than 2 min.

Large amounts of spent coffee grounds (SCGs) are produced annually worldwide. SCGs contain high levels of phenolics and other bioactive compounds that make them a potential source of reducing and stabilizing agents for the synthesis of metal nanoparticles. This study investigates the use of SCG extracts as a green strategy to produce silver nanoparticles (AgNPs). SCG extracts were obtained using aqueous ethanol as the solvent and then contacted with a silver nitrate solution under the selected conditions. A central composite design coupled with response surface methodology was used to evaluate the effects of solvent composition (C = 30-70% v / v ), silver-to-phenolic ratio (R = 3-7 mol/mol), temperature (T = 25-55 °C) and pH (10-12) on the production of AgNPs. Characterization of AgNPs by DLS, TEM and XRD techniques showed that they were highly crystalline with a narrow size distribution. Under optimal reaction conditions, AgNPs with an average size of about 10 nm and a zeta potential of -30.5 to -20.7 mV were obtained. Overall, the results of this study indicate that SCGs are a promising material for the green synthesis of small-sized and stable AgNPs.

Anticancer strategies aimed at inhibiting Complex I of the mitochondrial respiratory chain are increasingly being attempted in solid tumors, as functional oxidative phosphorylation is vital for cancer cells. Using ovarian cancer as a model, we show that a compensatory response to an energy crisis induced by Complex I genetic ablation or pharmacological inhibition is an increase in the mitochondrial biogenesis master regulator PGC1α, a pleiotropic coactivator of transcription regulating diverse biological processes within the cell. We associate this compensatory response to the increase in PGC1α target gene expression, setting the basis for the comprehension of the molecular pathways triggered by Complex I inhibition that may need attention as drawbacks before these approaches are implemented in ovarian cancer care., (© 2022. The Author(s).)

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., Competing Interests: Declaration of interests FFu, VC, MGNS, AA, CB, FB, FFe, MM, FT, VG, SR, ESF, MPC, and MEB are investigators of gene therapy clinical trials for MLD sponsored by Orchard Therapeutics, the licence holder of investigational medicinal product arsa-cel. FFu and VC have acted as ad-hoc consultants for an Orchard Therapeutics advisory board. The MLD gene therapy was licensed to GlaxoSmithKline in 2014, and then to Orchard Therapeutics in 2018. Telethon and Ospedale San Raffaele are entitled to receive milestone payments and royalties for such a therapy. MSe and AB left San Raffaele Hospital and their role as principal investigators of the pivotal study on November, 2014, and September, 2015, respectively. AA subsequently became study principal investigator and responsible physician for treatment under expanded access frameworks. AB is currently a member of the scientific advisory board of Orchard Therapeutics and holds stock in Orchard Therapeutics. PMVR and CDS have a contract with Orchard Therapeutics to perform statistical analyses of gene therapy clinical trials for MLD. SMa and FM have a service contract with Ospedale San Raffaele. SMa has a contract with Orchard Therapeutics to perform ARSA activity on CSF in the clinical trial NCT03392987. JG-S, LCS, and GFD are former employees and hold stock in Orchard Therapeutics, which sponsored the clinical trial. All other authors declare that they have no financial interest related to the work described in the manuscript., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The replication method is a widely used technique to produce bioactive glass (BG) scaffolds mimicking trabecular bone. However, these scaffolds usually exhibit poor mechanical reliability and fast degradation, which can be improved by coating them with a polymer. In this work, we proposed the use of custom-made poly(urethane)s (PURs) as coating materials for 45S5 Bioglass ® -based scaffolds. In detail, BG scaffolds were dip-coated with two PURs differing in their soft segment (poly(ε-caprolactone) or poly(ε-caprolactone)/poly(ethylene glycol) 70/30 w / w ) (PCL-PUR and PCL/PEG-PUR) or PCL (control). PUR-coated scaffolds exhibited biocompatibility, high porosity (ca. 91%), and improved mechanical properties compared to BG scaffolds (2-3 fold higher compressive strength). Interestingly, in the case of PCL-PUR, compressive strength significantly increased by coating BG scaffolds with an amount of polymer approx. 40% lower compared to PCL/PEG-PUR- and PCL-coated scaffolds. On the other hand, PEG presence within PCL/PEG-PUR resulted in a fast decrease in mechanical reliability in an aqueous environment. PURs represent promising coating materials for BG scaffolds, with the additional pros of being ad-hoc customized in their physico-chemical properties. Moreover, PUR-based coatings exhibited high adherence to the BG surface, probably because of the formation of hydrogen bonds between PUR N-H groups and BG surface functionalities, which were not formed when PCL was used.

Background: Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications., Methods: We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an α-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years., Results: We now report interim results. The children's mean (±SD) age at the time of HSPC gene therapy was 1.9±0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts., Conclusions: The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, NCT03488394; EudraCT number, 2017-002430-23.)., (Copyright © 2021 Massachusetts Medical Society.)

Background and Aim: Diarrhea, abdominal pain, and bloating are frequent in irritable bowel syndrome (IBS)-like disorders, although little is known about their intestinal ultrastructural alterations. The aim of the present study was to study duodenal biopsies from IBS-like patients to find ultrastructural alterations., Materials and Methods: Study design: descriptive comparative pilot study. Thirty outpatients (9 male and 21 female; median age 37.7 years; range, 20 to 65 years) complaining of IBS-like symptoms were enrolled between January 2015 to May 2019 and were divided into 6 groups, each equally consisting of 5 patients: (A) untreated celiac disease (uCD); (B) treated celiac disease (tCD); (C) wheat allergy (WA); (D) Non-celiac gluten sensitivity (NCGS); (E) Nickel allergic contact mucositis (Ni ACM); (F) controls affected by GERD. Transmission electron microscopy (TEM) morphological characteristics were: microvilli length, intermicrovillar distance, junctional complexes (JC) gap width, autophagic bodies, apoptosis, altered mitochondria, lipid/chylomicron droplets, and mast cells. Regarding JC, we focused on tight junctions (TJ), adherens junctions (AJ), and desmosomes., Results: Major alterations in microvilli length and intermicrovillar distance have been observed in the subjects affected by uCD. Microvilli of tCD patients showed marked recovery after adequate GFD, although not comparable to controls. Intermediate microvillar alterations were instead observed in NCGS and Ni ACM, while characteristics of WA subjects appeared more similar to tCD. Regarding JC, TJ did not show significant differences between all groups studied, including controls. The AJ were significantly more dilated in all groups compared to controls, while no significant differences were found between the pathological groups. The distance between desmosomes was greater in uCD, NCGS, and Ni ACM than in tCD, WA, and controls. Finally, intracellular alterations have been detected in most of the groups studied although they seemed more unspecific., Conclusions: TEM analysis confirmed damages to the intestinal barrier and defense mechanisms by enterocytes in IBS-like patients, probably linked to low-grade inflammation or adverse reactions triggered by food allergens, heavy metals, or other unknown. On the other hand, our study needs confirmation and further investigations with larger populations to facilitate diagnosis, therapy, and prevention of IBS-like disorders in the future.

Spheroids from primary colorectal cancer cells and their mice xenografts have emerged as useful preclinical models for cancer research as they replicate tumor features more faithfully as compared to cell lines. While 3D models provide a reliable system for drug discovery and testing, their structural complexity represents a challenge and their structure-function relationships are only partly understood. Here, we present a comparative ultrastructural and flow citometric analysis of patient colorectal cancer-derived spheroids and their mice xenografts. Ultrastructural observations highlighted that multicellular spheroids and their xenografts contain the same cancer cell types but with different ratios, specifically multicellular spheroids were enriched in cells with a stem-like phenotype, while xenografts had an increased amount of lipid droplets-containing cells. The flow cytometric analysis for stem cell marker and activity showed enrichment of stem-like cells presence and activity in spheroids while xenografts had the inverse response. Our results evidence the effects on cancer cells of different in vitro and in vivo microenvironments. Those differences have to be paid into account in designing innovative experimental models for personalized drug testing.

Intramedullary spinal cord metastases (ISCMs) are infrequent lesions. Their incidence is estimated to range from 0.9 to 2.1%, found in autopsies of cancer patients. However, as the life expectancy of malignant tumour patients constantly increases, the reported incidences of ISCMs are consequently rising. This report presents a case of the misdiagnosis of an anaplastic astrocytoma type of tumour due to its similarities to small-cell neuroendocrine carcinoma. Therefore, we would like to underline the importance of further investigation that could assist and support the surgeon in the making of the differential diagnosis. We present the clinical case of a 73-year-old woman with a solitary intramedullary spinal cord metastasis as the initial manifestation of a carcinoid type of tumour. The patient was admitted to our department while presenting a rapid onset of paraparesis. Magnetic resonance imaging was performed, which showed an intramedullary mass at the C2-C6 vertebral level with a heterogeneous contrast enhancement. In light of these findings, the patient underwent surgery for a partial tumour resection. The lesion resulted in being a small-cell neuroendocrine type of carcinoma. This peculiar type of tumour presents similar radiological characteristics to the anaplastic astrocytoma type, which is why our diagnostical mismatch occurred. This is the report of a rare case of solitary intramedullary spinal cord metastasis, which is the result of an initial presentation of a lung small-cell neuroendocrine type of carcinoma. We conclude that ISCMs should be regularly considered as a part of the differential diagnosis of intramedullary lesions, especially in the case of a rapid onset and deterioration of neurological symptoms.

Background: Galectin-3 ( LGALS3 ) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm, and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common single-nucleotide polymorphism (SNP) (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the rs4644 SNP as risk factor for differentiated thyroid cancer (DTC) and to determine the effect on the transcriptome in thyrocytes. Methods: A case/control association study in 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. We used the nonmalignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed were validated by quantitative reverse transcription PCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent on galectin-3 levels. Results: rs4644 AA genotype was associated with a reduced risk of DTC (compared with CC, OR adj  = 0.66; 95% confidence interval = 0.46-0.93; P ass  = 0.02). We found that rs4644 affects galectin-3 as a transcriptional coregulator. Among 34 genes affected by rs4644, HES1 , HSPA6 , SPC24 , and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others), also in 574 thyroid tissues of Genotype-Tissue Expression (GTEx) biobank. Moreover, the expression of these genes was regulated by LGALS3 -silencing. Using the proximity ligation assay in Nthy-Ori cells, we found that the TTF-1 interaction was genotype dependent. Conclusions: Our data show that in thyroid, rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1.

Characterizing animal dispersal patterns and the rational behind individuals' transfer choices is a long-standing question of interest in evolutionary biology. In wild western gorillas ( Gorilla gorilla ), a one-male polygynous species, previous genetic findings suggested that, when dispersing, females might favor groups with female kin to promote cooperation, resulting in higher-than-expected within-group female relatedness. The extent of male dispersal remains unclear with studies showing conflicting results. To investigate male and female dispersal patterns and extragroup paternity, we analyzed long-term field observations, including female spatial proximity data, together with genetic data (10 autosomal microsatellites) on individuals from a unique set of four habituated western gorilla groups, and four additional extragroup males (49 individuals in total). The majority of offspring (25 of 27) were sired by the group male. For two offspring, evidence for extragroup paternity was found. Contrarily to previous findings, adult females were not significantly more related within groups than across groups. Consistently, adult female relatedness within groups did not correlate with their spatial proximity inferred from behavioral data. Adult females were similarly related to adult males from their group than from other groups. Using R ST statistics, we found significant genetic structure and a pattern of isolation by distance, indicating limited dispersal in this species. Comparing relatedness among females and among males revealed that males disperse farer than females, as expected in a polygamous species. Our study on habituated western gorillas shed light on the dispersal dynamics and reproductive behavior of this polygynous species and challenge some of the previous results based on unhabituated groups., Competing Interests: The authors declare to have no conflicts of interest., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Electrochemotherapy (ECT) is an emerging treatment for solid tumors and an attractive research field due to its clinical results. This therapy represents an alternative local treatment to the standard ones and is based on the tumor-directed delivery of non-ablative electrical pulses to maximize the action of specific cytotoxic drugs such as cisplatin (CSP) and bleomycin (BLM) and to promote cancer cell death. Nowadays, ECT is mainly recommended as palliative treatment. However, it can be applied to a wide range of superficial cancers, having an impact in preventing or delaying tumor progression and therefore in improving quality of life. In addition, during the natural history of the tumor, early ECT may improve patient outcomes. Our group has extensive clinical and research experience on ECT in vulvar tumors in the palliative setting, with 70% overall response rate. So far, in most studies, ECT was based on BLM. However, the potential of CSP in this setting seems interesting due to some theoretical advantages. The purpose of this report is to: (i) compare the efficacy of CSP and BLM-based ECT through a systematic literature review; (ii) report the results of our studies on CSP-resistant squamous cell tumors cell lines and the possibility to overcome chemoresistance using ECT; (iii) discuss the future ECT role in gynecological tumors and in particular in vulvar carcinoma.

Application of nonspecific phosphodiesterases inhibitors, such as pentoxifylline (PTX), is a strategy utilised to aid sperm selection from immotile sperm samples prior to ICSI. No extensive studies have yet been performed to verify the safety of the clinical outcomes of ICSI after PTX administration. In this article, we summarise the data reported in the literature that assess the implication of in vitro usage of PTX on sperm parameters, as well as clinical outcomes during assisted male reproduction programme., (© 2020 Blackwell Verlag GmbH.)

Aim: To realize and characterize a new generation of keratin-coated gold nanoparticles (Ker-AuNPs) as highly efficient photosensitive nanosized therapeutics for plasmonic photothermal (PPT) therapy. Materials & methods: The chemical, physical, morphological and photothermal properties of Ker-AuNPs are investigated using dynamic light scattering, ζ-potential, UV-Visible, Fourier transform infrared spectroscopy, x-ray photoelectron spectroscopy, transmission electron microscopy and high-resolution thermography. In vitro experiments are performed on a human glioblastoma cell line (i.e., U87-MG), using viability assays, transmission electron microscopy, fluorescence microscopy, cytometric analyses and PPT experiments. Results: Experiments confirm the excellent biocompatibility of Ker-AuNPs, their efficient cellular uptake and localized photothermal heating capabilities. Conclusion: The reported structural and functional properties pointed out these Ker-AuNPs as a promising new tool in the field of biocompatible photothermal agents for PPT treatments against cancer-related diseases.

Long non-coding RNAs are emerging as new molecular players involved in many biological processes, such as proliferation, apoptosis, cell cycle, migration, and differentiation. Their aberrant expression has been reported in variety of diseases. The aim of this study is the identification and functional characterization of clinically relevant lncRNAs responsible for the inhibition of miR-145-5p, a key tumor suppressor in thymic epithelial tumors (TETs). Starting from gene expression analysis by microarray in a cohort of fresh frozen thymic tumors and normal tissues, we identified LINC00174 as upregulated in TET. Interestingly, LINC00174 expression is positively correlated with a 5-genes signature in TETs. Survival analyses, performed on the TCGA dataset, showed that LINC00174 and its associated 5-genes signature are prognostic in TETs. Specifically, we show that LINC00174 favors the expression of SYBU, FEM1B, and SCD5 genes by sponging miR-145-5p, a well-known tumor suppressor microRNA downregulated in a variety of tumors, included TETs. Functionally, LINC00174 impacts on cell migration and lipid metabolism. Specifically, SCD5, one of the LINC00174-associated genes, is implicated in the control of lipid metabolism and promotes thymic cancer cells migration. Our study highlights that LINC00174 and its associated gene signature are relevant prognostic indicators in TETs. Of note, we here show that a key controller of lipid metabolism, SCD5, augments the migration ability of TET cells, creating a link between lipids and motility, and highlighting these pathways as relevant targets for the development of novel therapeutic approaches for TET.