Studio morfologico di un modello sperimentale di atrofia muscolare spinale (SMA) in condizioni basali e dopo trattamento con litio

L’Atrofia Muscolare Spinale (SMA) è la più comune malattia neurodegenerativa infantile. La SMA è una malattia autosomica recessiva caratterizzata dalla degenerazione dei motoneuroni delle corna anteriori del midollo spinale ed è associata ad atrofia e a paralisi muscolare. Esistono quattro forme di SMA (I-IV), classificate sulla base dell’età di esordio della malattia e della sua gravità. I meccanismi molecolari che causano la malattia non sono ancora completamente conosciuti e, attualmente, non esistono cure efficaci per il trattamento dei pazienti SMA. Tutte le forme di SMA sono caratterizzate dalla riduzione dei livelli di espressione della proteina SMN (Survival Motor Neuron), codificata nell’Uomo da due geni omologhi (SMN1 e SMN2) localizzati sul cromosoma 5. I geni SMN1 e SMN2 differiscono solo per 5 nucleotidi di cui uno è localizzato nell’esone 7, responsabile dello splicing alternativo del gene. Il gene SMN1 codifica per una proteina full lenght contenente anche il trascritto dell’esone 7, mentre il gene SMN2 codifica la proteina full lenght solo nel 10% dei casi e nel restante 90% una proteina mancante dell’esone 7, tronca ed instabile. Il gene SMN1 presenta mutazioni nel 98% dei casi di malattia, mentre in tutti i pazienti il gene SMN2 risulta inalterato. Tuttavia, la quantità di proteina full lenght prodotta dal gene SMN2 non riesce a compensare i bassi livelli di proteina SMN. Pertanto varie strategie terapeutiche hanno come target la proteina SMN ed in particolare hanno lo scopo di aumentarne i livelli. Ad esempio, studi in vitro hanno dimostrato che l’acido valproico, un farmaco stabilizzante dell’umore, che inibisce la proteina glicogeno sintasi chinasi 3beta (GSK-3beta), aumenta i livelli della proteina SMN nei fibroblasti di pazienti SMA. In vivo l’acido valproico aumenta i livelli di SMN nel midollo spinale di modelli murini di SMA e rallenta la progressione della malattia. Nel presente studio abbiamo indagato su modelli murini di SMA-III gli effetti dei sali di litio, un altro stabilizzante dell’umore che, come l’acido valproico, inibisce la proteina GSK-3beta. Per valutare gli effetti protettivi del litio è stato utilizzato un modello murino di SMA a lenta progressione (SMA-III) che possiede il genotipo SMN1A2G+/- SMN2+/+ SMN-/- (knock out doppio transgenico). Gli animali SMA-III sono stati trattati con sali di litio (1 mEq/kg) per circa 15 mesi a giorni alterni. Durante il periodo di trattamento gli animali sono stati sottoposti a test motori per valutare gli effetti del farmaco sulla forza muscolare. Quindi gli animali sono stati sacrificati ed il midollo spinale è stato valutato con un approccio morfologico in microscopia ottica ed elettronica e mediante analisi di Western Blot. Dall’indagine è emerso che il litio ha rallentato la progressione della malattia. Inoltre, il litio riduce la perdita dei motoneuroni e ne preserva la morfologia. Le indagini di immunocitochimica e di Western Blot confermano un aumento dei livelli di espressione di SMN dopo trattamento con litio. Questi dati dimostrano che il litio potrebbe avere un ruolo nel contrastare il progredire della malattia. Inoltre, poiché la proteina SMN è espressa in maniera ubiquitaria in vari tipi cellulari, è stata valutata la modulazione della sua espressione da parte dei sali di litio anche nelle cellule PC12, che rappresentano un modello di neurone. Anche negli esperimenti in vitro il litio ha aumentato i livelli della proteina SMN, come osservato in microscopia elettronica e confermato dall’analisi di Western Blot. Tali dati indicano che il litio induce neuroprotezione nei topi SMA producendo un aumento della proteina SMN il cui deficit è responsabile della malattia. L’aumento della proteina SMN indotto da litio, dimostrato anche nella coltura di cellule PC12, fa supporre che tra le molteplici azioni di questa molecola vi sia anche la capacità di modulare l’espressione della proteina. Questa proprietà potrebbe essere dovuta inibizione della proteina GSK-3beta che da altri Autori è stato dimostrata aumentare i livelli della proteina SMN

The type of DNA glycosylase determines the base excision repair pathway in mammalian cells.

The base excision repair (BER) of modified nucleotides is initiated by damage-specific DNA glycosylases. The repair of the resulting apurinic/apyrimidinic site involves the replacement of either a single nucleotide (short patch BER) or of several nucleotides (long patch BER). The mechanism that controls the selection of either BER pathway is unknown. We tested the hypothesis that the type of base damage present on DNA, by determining the specific DNA glycosylase in charge of its excision, drives the repair of the resulting abasic site intermediate to either BER branch. In mammalian cells hypoxanthine (HX) and 1,N6-ethenoadenine (epsilonA) are both substrates for the monofunctional 3-methyladenine DNA glycosylase, the ANPG protein, whereas 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by the bifunctional DNA glycosylase/beta-lyase 8-oxoG-DNA gly- cosylase (OGG1). Circular plasmid molecules containing a single HX, epsilonA, or 8-oxoG were constructed. In vitro repair assays with HeLa cell extracts revealed that HX and epsilonA are repaired via both short and long patch BER, whereas 8-oxoG is repaired mainly via the short patch pathway. The preferential repair of 8-oxoG by short patch BER was confirmed by the low efficiency of repair of this lesion by DNA polymerase beta-deficient mouse cells as compared with their wild-type counterpart. These data fit into a model where the intrinsic properties of the DNA glycosylase that recognizes the lesion selects the branch of BER that will restore the intact DNA template

Piezoelectric nanocomposite bioink and ultrasound stimulation modulate early skeletal myogenesis

Despite the significant progress in bioprinting for skeletal muscle tissue engineering, new stimuli-responsive bioinks to boost the myogenesis process are highly desirable. In this work, we developed a printable alginate/Pluronic-based bioink including piezoelectric barium titanate nanoparticles (nominal diameter: ∼60 nm) for the 3D bioprinting of muscle cell-laden hydrogels. The aim was to investigate the effects of the combination of piezoelectric nanoparticles with ultrasound stimulation on early myogenic differentiation of the printed structures. After the characterization of nanoparticles and bioinks, viability tests were carried out to investigate three nanoparticle concentrations (100, 250, and 500 μg mL−1) within the printed structures. An excellent cytocompatibility was confirmed for nanoparticle concentrations up to 250 μg mL−1. TEM imaging demonstrated the internalization of BTNPs in intracellular vesicles. The combination of piezoelectric nanoparticles and ultrasound stimulation upregulated the expression of MYOD1, MYOG, and MYH2 and enhanced cell aggregation, which is a crucial step for myoblast fusion, and the presence of MYOG in the nuclei. These results suggest that the direct piezoelectric effect induced by ultrasound on the internalized piezoelectric nanoparticles boosts myogenesis in its early phases

Sub-cellular motor neuron analysis in a model of spinal muscle atrophy

Spinal muscular atrophy (SMA) is a neurogenetic autosomal recessive disorder characterized by degeneration of lower motor neurons associated with muscle atrophy and paralysis. The disease course including onset and severity depends by reduced amounts of the survival motor neuron (SMN) protein. Such a protein is increased when the enzyme glycogen synthase kinase-3beta (GSK3beta) is inhibited. In the present study we used a knockout double transgenic mouse (Smn−/−; SMN1A2G; SMN2) modelling SMAIII to dissect the spinal cord pathology at ultrastructural analysis at prolonged survival time (18 months). We analysed the subcellular structure of spinal cord motor neurons both in baseline conditions and following the administration of a GSK3beta inhibitor. We found that motor neurons increased their diameter confirming our previous light microscopy data. The amount of immunogold labelled SMN particles was dramatically reduced in the whole cell body incuding nucleus and cytoplasm. Remarkably, at nuclear level we could detect marked reduction of the SMN protein with Cajal-like bodies thus mimicking the human disease. In mice receiving long-term lithium administration the level of the SMN protein were massively increase way more than other SMAIII mice and significantly exceeding the levels counted in controls. When compared with control mice administered long-term lithium SMN levels in SMA III mice were overlapping with healthy animals, at large. The effects of lithium on ultrastructural morphology of motor neurons extended to the preservation of mitochondrial compartment which was slightly affected in motor neurons from SMA III mice. These data confirm the essential role of GSK3beta inhibition in increasing the amount of the SMN protein and provide a novel action for an old drug which increases SMN level exceeding any other compound tested so far in this motor neuron pathology. At the same time the beneficial effects of lithium on mitochondrial morphology are confirmed. As an appendix to the present study we wish to mention the ubiquitous nature of these effects which were replicated in non-motor neuron cell lines. Apart from the significance in cell biology this latter observation provide the basis to analyze the effects of a lithium treatment on affected patients using peripheral or skin-derived cell cultures. This work was supported by an educational grant from CUCC

Morphological characterization of a single knock out double transgenic mouse model of spinal muscle atrophy

Spinal muscular atrophy (SMA) is a neurogenetic autosomal recessive disorder characterized by degeneration of lower motor neurons associated with muscle atrophy and paralysis. Due to a lack of an in depth knowledge on the molecular mechanisms and fine neuropathology of SMA, validation of appropriate animal models is key in fostering SMA research. Recent studies set up an animal model showing long survival and slow disease progression. This model is knocked out for mouse SMN (Smn−/−) gene and carries a human mutation of the SMN1 gene (SMN1A2G), along with human SMN2 gene. In the present study we used this knockout double transgenic mouse as a SMA III model, to characterize the spinal cord pathology along with motor deficit at prolonged survival times (18 months). This long time interval (i.e. up to 535 days) was never analyzed before especially concerning specific motor tasks. We found that the delayed disease progression was likely to maintain fair motor activity despite a dramatic loss of large motor neurons (44.77%). At this stage, spared motor neurons showed significant cell body enlargement. Moreover, similar to what was described in patients affected by SMA we found neuronal heterotopy in the anterior white matter. Motor neuron degeneration was accompanied by the loss of SMN protein in the spinal cord. In summary, the present study validates over a long time period a SMA III mouse model showing neuropathology reminiscent of human patients and provide a useful experimental model to probe novel therapeutic strategies

Functionalized aliphatic polyketones with germicide activity

The COVID-19 pandemic has further confirmed to the community that direct contact with contaminated surfaces and objects represents an important source of pathogen spreading among humans. Therefore, it is of paramount importance to design effective germicidal paints to ensure a rapid and potent disinfectant action of surfaces. In this work, we design novel germicide polymeric coatings by inserting quaternary ammonium and sugar groups on the macromolecular backbone, thus respectively endowing the polymer with germicide features and hydrophilicity to interact with the surfaces. An aliphatic polyketone was selected as a starting polymer matrix that was functionalized with primary amine derivatives via the Paal–Knorr reaction. The resulting polymers were deposited on cellulose filter papers and checkboard charts with excellent coating yield and substrate coverage as determined by scanning electron microscopy for cellulose. Remarkably, the substrates coated by the functional polymers bearing quaternary ammonium compounds showed excellent bactericide properties with antibacterial rate of 99% and logarithmic reduction of >3. Notably, the polymers with higher hydrophobicity showed better retention on the substrate after being treated with water at neutral pH

SARS-CoV-2 vaccination modelling for safe surgery to save lives : data from an international prospective cohort study

Background: Preoperative SARS-CoV-2 vaccination could support safer elective surgery. Vaccine numbers are limited so this study aimed to inform their prioritization by modelling. Methods: The primary outcome was the number needed to vaccinate (NNV) to prevent one COVID-19-related death in 1 year. NNVs were based on postoperative SARS-CoV-2 rates and mortality in an international cohort study (surgical patients), and community SARS-CoV-2 incidence and case fatality data (general population). NNV estimates were stratified by age (18-49, 50-69, 70 or more years) and type of surgery. Best- and worst-case scenarios were used to describe uncertainty. Results: NNVs were more favourable in surgical patients than the general population. The most favourable NNVs were in patients aged 70 years or more needing cancer surgery (351; best case 196, worst case 816) or non-cancer surgery (733; best case 407, worst case 1664). Both exceeded the NNV in the general population (1840; best case 1196, worst case 3066). NNVs for surgical patients remained favourable at a range of SARS-CoV-2 incidence rates in sensitivity analysis modelling. Globally, prioritizing preoperative vaccination of patients needing elective surgery ahead of the general population could prevent an additional 58 687 (best case 115 007, worst case 20 177) COVID-19-related deaths in 1 year. Conclusion: As global roll out of SARS-CoV-2 vaccination proceeds, patients needing elective surgery should be prioritized ahead of the general population.Peer reviewe

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research