Functional gradients reveal cortical hierarchy changes in multiple sclerosis.

Functional gradient (FG) analysis represents an increasingly popular methodological perspective for investigating brain hierarchical organization but whether and how network hierarchy changes concomitant with functional connectivity alterations in multiple sclerosis (MS) has remained elusive. Here, we analyzed FG components to uncover possible alterations in cortical hierarchy using resting-state functional MRI (rs-fMRI) data acquired in 122 MS patients and 97 healthy control (HC) subjects. Cortical hierarchy was assessed by deriving regional FG scores from rs-fMRI connectivity matrices using a functional parcellation of the cerebral cortex. The FG analysis identified a primary (visual-to-sensorimotor) and a secondary (sensory-to-transmodal) component. Results showed a significant alteration in cortical hierarchy as indexed by regional changes in FG scores in MS patients within the sensorimotor network and a compression (i.e., a reduced standard deviation across all cortical parcels) of the sensory-transmodal gradient axis, suggesting disrupted segregation between sensory and cognitive processing. Moreover, FG scores within limbic and default mode networks were significantly correlated ( ρ = 0.30 $$ \rho =0.30 $$ , p < .005 after Bonferroni correction for both) with the symbol digit modality test (SDMT) score, a measure of information processing speed commonly used in MS neuropsychological assessments. Finally, leveraging supervised machine learning, we tested the predictive value of network-level FG features, highlighting the prominent role of the FG scores within the default mode network in the accurate prediction of SDMT scores in MS patients (average mean absolute error of 1.22 ± 0.07 points on a hold-out set of 24 patients). Our work provides a comprehensive evaluation of FG alterations in MS, shedding light on the hierarchical organization of the MS brain and suggesting that FG connectivity analysis can be regarded as a valuable approach in rs-fMRI studies across different MS populations.

AurkA/TPX2 co-overexpression in nontransformed cells promotes genome instability through induction of chromosome mis-segregation and attenuation of the p53 signalling pathway.

The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells.

HuD (ELAVL4) gain-of-function impairs neuromuscular junctions and induces apoptosis in in vitro and in vivo models of amyotrophic lateral sclerosis.

Early defects at the neuromuscular junction (NMJ) are among the first hallmarks of the progressive neurodegenerative disease amyotrophic lateral sclerosis (ALS). According to the "dying back" hypothesis, disruption of the NMJ not only precedes, but is also a trigger for the subsequent degeneration of the motoneuron in both sporadic and familial ALS, including ALS caused by the severe FUS pathogenic variant P525L. However, the mechanisms linking genetic and environmental factors to NMJ defects remain elusive. By taking advantage of co-cultures of motoneurons and skeletal muscle derived from human induced pluripotent stem cells (iPSCs), we show that the neural RNA binding protein HuD (ELAVL4) may underlie NMJ defects and apoptosis in FUS-ALS. HuD overexpression in motoneurons phenocopies the severe FUSP525L mutation, while HuD knockdown in FUSP525L co-cultures produces phenotypic rescue. We validated these findings in vivo in a Drosophila FUS-ALS model. Neuronal-restricted overexpression of the HuD-related gene, elav, produces per se a motor phenotype, while neuronal-restricted elav knockdown significantly rescues motor dysfunction caused by FUS. Finally, we show that HuD levels increase upon oxidative stress in human motoneurons and in sporadic ALS patients with an oxidative stress signature. On these bases, we propose HuD as an important player downstream of FUS mutation in familial ALS, with potential implications for sporadic ALS related to oxidative stress.

Cortical neurons obtained from patient-derived iPSCs with GNAO1 p.G203R variant show altered differentiation and functional properties.

Pathogenic variants in the GNAO1 gene, encoding the alpha subunit of an inhibitory heterotrimeric guanine nucleotide-binding protein (Go) highly expressed in the mammalian brain, have been linked to encephalopathy characterized by different combinations of neurological symptoms, including developmental delay, hypotonia, epilepsy and hyperkinetic movement disorder with life-threatening paroxysmal exacerbations. Currently, there are only symptomatic treatments, and little is known about the pathophysiology of GNAO1-related disorders. Here, we report the characterization of a new in vitro model system based on patient-derived induced pluripotent stem cells (hiPSCs) carrying the recurrent p.G203R amino acid substitution in Gαo, and a CRISPR-Cas9-genetically corrected isogenic control line. RNA-Seq analysis highlighted aberrant cell fate commitment in neuronal progenitor cells carrying the p.G203R pathogenic variant. Upon differentiation into cortical neurons, patients' cells showed reduced expression of early neural genes and increased expression of astrocyte markers, as well as premature and defective differentiation processes leading to aberrant formation of neuronal rosettes. Of note, comparable defects in gene expression and in the morphology of neural rosettes were observed in hiPSCs from an unrelated individual harboring the same GNAO1 variant. Functional characterization showed lower basal intracellular free calcium concentration ([Ca2+]i), reduced frequency of spontaneous activity, and a smaller response to several neurotransmitters in 40- and 50-days differentiated p.G203R neurons compared to control cells. These findings suggest that the GNAO1 pathogenic variant causes a neurodevelopmental phenotype characterized by aberrant differentiation of both neuronal and glial populations leading to a significant alteration of neuronal communication and signal transduction.

Effectiveness and safety of immunosuppressive regimens used as maintenance therapy in kidney transplantation: The CESIT study.

Maintenance immunosuppressive therapy used in kidney transplantation typically involves calcineurin inhibitors, such as tacrolimus or cyclosporine, in combination with mycophenolate or mechanistic target of rapamycin (mTORi) with or without corticosteroids. An Italian retrospective multicentre observational study was conducted to investigate the risk-benefit profile of different immunosuppressive regimens. We identified all subjects who underwent kidney transplant between 2009 and 2019, using healthcare claims data. Patients on cyclosporine and tacrolimus-based therapies were matched 1:1 based on propensity score, and effectiveness and safety outcomes were compared using Cox models (HR; 95%CI). Analyses were also conducted comparing mTORi versus mycophenolate among tacrolimus-treated patients. Patients treated with cyclosporine had a higher risk of rejection or graft loss (HR:1.69; 95%CI:1.16-2.46) and a higher incidence of severe infections (1.25;1.00-1.55), but a lower risk of diabetes (0.66;0.47-0.91) compared to those treated with tacrolimus. Among tacrolimus users, mTORi showed non-inferiority to MMF in terms of mortality (1.01;0.68-1.62), reject/graft loss (0.61;0.36-1.04) and severe infections (0.76;0.56-1.03). In a real-life setting, tacrolimus-based immunosuppressive therapy appeared to be superior to cyclosporine in reducing rejection and severe infections, albeit with an associated increased risk of diabetes. The combination of tacrolimus and mTORi may represent a valid alternative to the combination with mycophenolate, although further studies are needed to confirm this finding.

Selective Reduction of Ca2+ Entry Through the Human NMDA Receptor: a Quantitative Study by Simultaneous Ca2+ and Na+ Imaging.

Excessive Ca2+ influx through N-methyl-D-aspartate type glutamate receptors (NMDAR) is associated with excitotoxicity and neuronal death, but the inhibition of this receptor-channel causes severe adverse effects. Thus, a selective reduction of NMDA-mediated Ca2+ entry, leaving unaltered the Na+ current, could represent a valid neuroprotective strategy. We developed a new two-fluorophore approach to efficiently assess the Ca2+ permeability of ligand-gated ion channels, including NMDARs, in different conditions. This technique was able to discriminate differential Ca2+/Na+ permeation ratio through different receptor channels, and through the same channel in different conditions. With this method, we confirmed that EU1794-4, a negative allosteric modulator of NMDARs, decreased their Ca2+ permeability. Furthermore, we measured for the first time the fractional Ca2+ current (Pf, i.e. the percentage of the total current carried by Ca2+ ions) of human NMDARs in the presence of EU1794-4, exhibiting a 40% reduction in comparison to control conditions. EU1794-4 was also able to reduce NMDA-mediated Ca2+ entry in human neurons derived from induced pluripotent stem cells. This last effect was stronger in the absence of extracellular Mg2+, but still significant in its presence, supporting the hypothesis to use NMDA-selective allosteric modulators to lower Ca2+ influx in human neurons, to prevent Ca2+-dependent excitotoxicity and consequent neurodegeneration.

Immunosuppression with Generics in Liver and Kidney Transplantation: A Real-World Evidence Study.

Purpose: This study evaluates the use, benefit-risk profile, and economic impact of generic immunosuppressants (tacrolimus-TAC, cyclosporine-CsA, and mycophenolate-MYC) in kidney and liver transplant recipients compared to brand-name drugs. Patients and Methods: A retrospective multicentre observational study, involving four Italian regions, was conducted based on the national transplant Information system and regional healthcare claims data. The analysis focused on incident patients who received kidney and liver transplants between 2013 and 2019 and evaluated the use of generic of CsA, TAC, and MYC during the 30-day period following discharge. For each type of transplant and immunosuppressive agent, the benefit-risk profile of generic vs branded drugs in a two-year window was estimated by multivariate Cox models (HR; 95% CI). Furthermore, the potential cost savings per person associated with one year of treatment using generics were calculated. Results: The utilization of generic drugs showed a significant increase; over the study years, the proportion of users among kidney recipients ranged from 14.2% to 40.5% for TAC, from 36.9% to 56.7% for MYC, and from 18.2% to 94.7% for CsA. A great variability in generic uptake for region was found. A comparable risk-benefit profile between generic and branded formulations was shown for all immunosuppressors considered. Choosing generic immunosuppressants during maintenance could result in yearly savings of around 2000 euros per person for each therapy ingredient. Conclusion: The study shows an increasing proportion of patients using generic immunosuppressive drugs over time suggesting a growing acceptance of generics within the transplant community and reveals comparable risk-benefit profiles between the generic and branded formulations of TAC, CsA, and MYC. A significant variability in the use of generics immunosuppressive agents was found both at the regional level and among transplant centers and future research should delve into regional prescribing variations.

The long noncoding RNA nHOTAIRM1 is necessary for differentiation and activity of iPSC-derived spinal motor neurons.

The mammalian nervous system is made up of an extraordinary array of diverse cells that form intricate functional connections. The programs underlying cell lineage specification, identity and function of the neuronal subtypes are managed by regulatory proteins and RNAs, which coordinate the succession of steps in a stereotyped temporal order. In the central nervous system (CNS), motor neurons (MNs) are responsible for controlling essential functions such as movement, breathing, and swallowing by integrating signal transmission from the cortex, brainstem, and spinal cord (SC) towards peripheral muscles. A prime role in guiding the progression of progenitor cells towards the MN fate has been largely attributed to protein factors. More recently, the relevance of a class of regulatory RNAs abundantly expressed in the CNS - the long noncoding RNAs (lncRNAs) - has emerged overwhelmingly. LncRNA-driven gene expression control is key to regulating any step of MN differentiation and function, and its derangement profoundly impacts neuronal pathophysiology. Here, we uncover a novel function for the neuronal isoform of HOTAIRM1 (nHOTAIRM1), a lncRNA specifically expressed in the SC. Using a model system that recapitulates spinal MN (spMN) differentiation, we show that nHOTAIRM1 intervenes in the binary cell fate decision between MNs and interneurons, acting as a pro-MN factor. Furthermore, human iPSC-derived spMNs without nHOTAIRM1 display altered neurite outgrowth, with a significant reduction of both branch and junction numbers. Finally, the expression of genes essential for synaptic connectivity and neurotransmission is also profoundly impaired when nHOTAIRM1 is absent in spMNs. Mechanistically, nHOTAIRM1 establishes both direct and indirect interactions with a number of target genes in the cytoplasm, being a novel post-transcriptional regulator of MN biology. Overall, our results indicate that the lncRNA nHOTAIRM1 is essential for the specification of MN identity and the acquisition of proper morphology and synaptic activity of post-mitotic MNs.

Dose escalation of adalimumab as a strategy to overcome anti-drug antibodies: A case report of infantile-onset inflammatory bowel disease.

BACKGROUND: Treatment of infantile-onset inflammatory bowel disease (IO-IBD) is often challenging due to its aggressive disease course and failure of standard therapies with a need for biologics. Secondary loss of response is frequently caused by the production of anti-drug antibodies, a well-known problem in IBD patients on biologic treatment. We present a case of IO-IBD treated with therapeutic drug monitoring (TDM)-guided high-dose anti-tumor necrosis factor therapy, in which dose escalation monitoring was used as a strategy to overcome anti-drug antibodies. CASE SUMMARY: A 5-mo-old boy presented with a history of persistent hematochezia from the 10th d of life, as well as relapsing perianal abscess and growth failure. Hypoalbuminemia, anemia, and elevated inflammatory markers were also present. Endoscopic assessment revealed skip lesions with deep colic ulcerations, inflammatory anal sub-stenosis, and deep fissures with persistent abscess. A diagnosis of IO-IBD Crohn-like was made. The patient was initially treated with oral steroids and fistulotomy. After the perianal abscess healed, adalimumab (ADA) was administered with concomitant gradual tapering of steroids. Clinical and biochemical steroid-free remission was achieved with good trough levels. After 3 mo, antibodies to ADA (ATA) were found with undetectable trough levels; therefore, we optimized the therapy schedule, first administering 10 mg weekly and subsequently up to 20 mg weekly (2.8 mg/kg/dose). After 2 mo of high-dose treatment, ATA disappeared, with concomitant high trough levels and stable clinical and biochemical remission of the disease. CONCLUSION: TDM-guided high-dose ADA treatment as a monotherapy overcame ATA production. This strategy could be a good alternative to combination therapy, especially in very young patients.

Determinants of immunosuppressive therapy in renal transplant recipients: an Italian observational study (the CESIT project).

BACKGROUND: Very scanty evidence is available on factors influencing the choice of immunosuppressive drug therapy after kidney transplantation. METHODS: An Italian multiregional real-world study was conducted integrating national transplant information system and claims data. All patients undergoing kidney transplantation for the first time during 2009-2019 (incident patients) were considered. Multilevel logistic models were used to estimate Odds Ratio (OR) and corresponding 95% Confidence intervals. Factors with statistically significance were identified as characteristics associated with treatment regimens: cyclosporin-CsA vs tacrolimus-Tac and, within the latter group, mTOR inhibitors vs mycophenolate-MMF. RESULTS: We identified 3,622 kidney patients undergoing transplantation in 17 hospitals located in 4 Italian regions, 78.3% was treated with TAC-based therapy, of which 78% and 22% in combination with MMF and mTOR, respectively. For both comparison groups, the choice of immunosuppressive regimens was mostly guided by standard hospital practices. Only few recipient and donor characteristics were found associated with specific regimen (donor/receipt age, immunological risk and diabetes). CONCLUSIONS: The choice of post-renal transplant immunosuppressive therapy seems to be mostly driven by standard Centre practices, while only partially based on patient's characteristics and recognized international guidelines.

Patient Blood Management in Microsurgical Procedures for Reconstructive Surgery.

Introduction: The main purpose of reconstructive surgery (RS) is to restore the integrity of soft tissues damaged by trauma, surgery, congenital deformity, burns, or infection. Microsurgical techniques consist of harvesting tissues that are separated from the vascular sources of the donor site and anastomosed to the vessels of the recipient site. In these procedures, there are some preoperative modifiable factors that have the potential to influence the outcome of the flap transfer and its anastomosis. The management of anemia, which is always present in the postoperative period and plays a decisive role in the implantation of the flap, covers significant importance, and is associated with clinical and laboratory settings of chronic inflammation. Methods: Chronic inflammatory anemia (ACD) is a constant condition in patients who have undergone RS and correlates with the perfusion of the free flap. The aim of this treatment protocol is to reduce the transfusion rate by maintaining both a good organ perfusion and correction of the patient's anemic state. From January 2017 to September 2019, we studied 16 patients (16 males, mean age 38 years) who underwent microsurgical procedures for RS. Their hemoglobin (Hb) levels, corpuscular indexes, transferrin saturation (TSAT) ferritin concentrations and creatinine clearance were measured the first day after surgery (T0), after the first week (T1), and after five weeks (T2). At T0, all the patients showed low hemoglobin levels (average 7.4 g/dL, STD 0.71 range 6.2-7.4 g dL-1), with an MCV of 72, MCH of 28, MCHC of 33, RDW of 16, serum iron of 35, ferritin of 28, Ret% of 1.36, TRF of 277, creatinine clearance of 119 and high ferritin levels (range 320-560 ng mL-1) with TSAT less than 20%. All the patients were assessed for their clinical status, medical history and comorbidities before the beginning of the therapy. Results: A collaboration between the two departments (Department of Transfusion Medicine and Department of Reconstructive Surgery) resulted in the application of a therapeutic protocol with erythropoietic stimulating agents (ESAs) (Binocrit 6000 UI/week) and intravenous iron every other day, starting the second day after surgery. Thirteen patients received ESAs and FCM (ferric carboxymaltose, 500-1000 mg per session), three patients received ESAs and iron gluconate (one vial every other day). No patients received blood transfusions. No side effects were observed, and most importantly, no limb or flap rejection occurred. Conclusions: Preliminary data from our protocol show an optimal therapeutic response, notwithstanding the very limited scientific literature and data available in this specific surgical field. The enrollment of further patients will allow us to validate this therapeutic protocol with statistically sound data.

Collective behavior and self-organization in neural rosette morphogenesis.

Neural rosettes develop from the self-organization of differentiating human pluripotent stem cells. This process mimics the emergence of the embryonic central nervous system primordium, i.e., the neural tube, whose formation is under close investigation as errors during such process result in severe diseases like spina bifida and anencephaly. While neural tube formation is recognized as an example of self-organization, we still do not understand the fundamental mechanisms guiding the process. Here, we discuss the different theoretical frameworks that have been proposed to explain self-organization in morphogenesis. We show that an explanation based exclusively on stem cell differentiation cannot describe the emergence of spatial organization, and an explanation based on patterning models cannot explain how different groups of cells can collectively migrate and produce the mechanical transformations required to generate the neural tube. We conclude that neural rosette development is a relevant experimental 2D in-vitro model of morphogenesis because it is a multi-scale self-organization process that involves both cell differentiation and tissue development. Ultimately, to understand rosette formation, we first need to fully understand the complex interplay between growth, migration, cytoarchitecture organization, and cell type evolution.

Arterial spin labeling MRI applied to migraine: current insights and future perspectives.

INTRODUCTION: Advanced neuroimaging techniques have extensively contributed to elucidate the complex mechanisms underpinning the pathophysiology of migraine, a neurovascular disorder characterized by episodes of headache associated with a constellation of non-pain symptoms. The present manuscript, summarizing the most recent progresses of the arterial spin labelling (ASL) MRI techniques and the most significant findings from ASL studies conducted in migraine, is aimed to clarify how ASL investigations are contributing to the evolving insight on migraine pathophysiology and their putative role in migraine clinical setting. ASL techniques, allowing to quantitatively demonstrate changes in cerebral blood flow (CBF) both during the attacks and in the course of interictal period, could represent the melting point between advanced neuroimaging investigations, conducted with pure scientific purposes, and conventional neuroimaging approaches, employed in the diagnostic decision-making processes. MAIN BODY: Converging ASL evidences have demonstrated that abnormal CBF, exceeding the boundaries of a single vascular territory, with biphasic trend dominated by an initial hypoperfusion (during the aura phenomenon but also in the first part of the headache phase) followed by hyperperfusion, characterizes migraine with aura attack and can represent a valuable clinical tool in the differential diagnosis from acute ischemic strokes and epileptic seizures. Studies conducted during migraine without aura attacks are converging to highlight the involvement of dorsolateral pons and hypothalamus in migraine pathophysiology, albeit not able to disentangle their role as "migraine generators" from mere attack epiphenomenon. Furthermore, ASL findings tend to support the presence of perfusion abnormalities in brain regions known to be involved in aura ignition and propagation as well as in areas involved in multisensory processing, in both patients with migraine with aura and migraine without aura. CONCLUSION: Although ASL studies have dramatically clarified quality and timing of perfusion abnormalities during migraine with aura attacks, the same cannot be said for perfusion changes during migraine attacks without aura and interictal periods. Future studies with more rigorous methodological approaches in terms of study protocol, ASL technique and sample selection and size are mandatory to exploit the possibility of better understanding migraine pathophysiology and identifying neuroimaging biomarkers of each migraine phase in different migraine phenotypes.

[Epidemiological research and big data. The state of the art.] / Ricerca epidemiologica e big data. Lo stato dell'arte.

Epidemiology, since its birth as a biomedical discipline to the present day, has progressively enriched and refined tools (and methodology) for research and generating evidence, adapting over time and in the context in which evidence is generated. In contemporary times, characterized by technological pervasiveness, increased computing power, and a global pandemic in an interconnected world, the paradigms of epidemiological research are opening up, at varying speeds depending on their real applicability, to a new, broader conception of "data" and more generally of its treatment. In this overview, we aim to take stock of what we know about this moment in epidemiology, where new research strands and "data-driven" analysis techniques are emerging alongside the traditional etiological vocation; a complex, ever-evolving scenario made up of lights, shadows, stimuli, and failures in which issues of method validity, professional training, and patients' right to privacy are increasingly central. The review therefore provides a starting point for reflection on this transition, identifying examples that support both the methodological and academic debate and case studies regarding the impact of big data in real clinical practice and, more generally, in service epidemiology.

CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes.

Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.