A model of human neural networks reveals NPTX2 pathology in ALS and FTLD.

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1, which involve human-specific mechanisms2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors6. Single-cell transcriptomics and comparison to independent neural stem cells7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids8. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.

Mechanical stimulation and electrophysiological monitoring at subcellular resolution reveals differential mechanosensation of neurons within networks.

A growing consensus that the brain is a mechanosensitive organ is driving the need for tools that mechanically stimulate and simultaneously record the electrophysiological response of neurons within neuronal networks. Here we introduce a synchronized combination of atomic force microscopy, high-density microelectrode array and fluorescence microscopy to monitor neuronal networks and to mechanically characterize and stimulate individual neurons at piconewton force sensitivity and nanometre precision while monitoring their electrophysiological activity at subcellular spatial and millisecond temporal resolution. No correlation is found between mechanical stiffness and electrophysiological activity of neuronal compartments. Furthermore, spontaneously active neurons show exceptional functional resilience to static mechanical compression of their soma. However, application of fast transient (∼500 ms) mechanical stimuli to the neuronal soma can evoke action potentials, which depend on the anchoring of neuronal membrane and actin cytoskeleton. Neurons show higher responsivity, including bursts of action potentials, to slower transient mechanical stimuli (∼60 s). Moreover, transient and repetitive application of the same compression modulates the neuronal firing rate. Seemingly, neuronal networks can differentiate and respond to specific characteristics of mechanical stimulation. Ultimately, the developed multiparametric tool opens the door to explore manifold nanomechanobiological responses of neuronal systems and new ways of mechanical control.

DeePhys: A machine learning-assisted platform for electrophysiological phenotyping of human neuronal networks.

Reproducible functional assays to study in vitro neuronal networks represent an important cornerstone in the quest to develop physiologically relevant cellular models of human diseases. Here, we introduce DeePhys, a MATLAB-based analysis tool for data-driven functional phenotyping of in vitro neuronal cultures recorded by high-density microelectrode arrays. DeePhys is a modular workflow that offers a range of techniques to extract features from spike-sorted data, allowing for the examination of functional phenotypes both at the individual cell and network levels, as well as across development. In addition, DeePhys incorporates the capability to integrate novel features and to use machine-learning-assisted approaches, which facilitates a comprehensive evaluation of pharmacological interventions. To illustrate its practical application, we apply DeePhys to human induced pluripotent stem cell-derived dopaminergic neurons obtained from both patients and healthy individuals and showcase how DeePhys enables phenotypic screenings.

A CMOS-based highly scalable flexible neural electrode interface.

Perception, thoughts, and actions are encoded by the coordinated activity of large neuronal populations spread over large areas. However, existing electrophysiological devices are limited by their scalability in capturing this cortex-wide activity. Here, we developed an electrode connector based on an ultra-conformable thin-film electrode array that self-assembles onto silicon microelectrode arrays enabling multithousand channel counts at a millimeter scale. The interconnects are formed using microfabricated electrode pads suspended by thin support arms, termed Flex2Chip. Capillary-assisted assembly drives the pads to deform toward the chip surface, and van der Waals forces maintain this deformation, establishing Ohmic contact. Flex2Chip arrays successfully measured extracellular action potentials ex vivo and resolved micrometer scale seizure propagation trajectories in epileptic mice. We find that seizure dynamics in absence epilepsy in the Scn8a+/- model do not have constant propagation trajectories.

Management of distal femoral fractures with metaphyseal and articular comminution (AO/OTA 33C) using nail and plate fixation: a technical note and case series of 14 patients.

PURPOSE: To describe the surgical technique and the outcome of a case series of comminuted intra-articular distal femur fractures (AO/OTA 33C) treated with a nail-plate combination (NPC) implant. METHODS: We retrospectively analyzed a case series of 14 patients with comminuted intra-articular distal femur fractures (DFF) treated with an intramedullary retrograde nail in combination with a lateral low-contact condylar locking plate, in a period between June 2020 and January 2023 at a Level 1 trauma center. Baseline demographic and clinical data were recorded. Time to bone healing, function using Schatzker Lambert Score, and complications were documented. RESULTS: Fourteen patients, 8 males and 6 females, with a total of 15 NPC implants, were included in this study. Eight out of 14 patients had open fractures, all with a Gustilo Anderson type IIIA exposure. The mean age was 48.5 ± 18.05 years. The median follow-up was 392 days, with only one patient lost to follow-up. 11 out of 15 implants achieved complete radiographic consolidation during follow-up, at a mean of 5.40 ± 1.07 months. At the 12-month follow-up, all patients could fully bear weight painlessly or with mild pain. Schatzker Lambert Score was excellent for 4 patients, good for 2 patients, fair for 5 patients, and failure for 2 patients. The main postoperative complications were rigidity (3 cases), limb shortening (2 cases), and septic non-union (1 case). CONCLUSION: This study suggests that the nail-plate combination (NPC) may provide a more effective surgical technique for addressing the challenges associated with comminuted intra-articular distal femur fractures (AO/OTA 33C).

Predicting in vitro single-neuron firing rates upon pharmacological perturbation using Graph Neural Networks.

Modern Graph Neural Networks (GNNs) provide opportunities to study the determinants underlying the complex activity patterns of biological neuronal networks. In this study, we applied GNNs to a large-scale electrophysiological dataset of rodent primary neuronal networks obtained by means of high-density microelectrode arrays (HD-MEAs). HD-MEAs allow for long-term recording of extracellular spiking activity of individual neurons and networks and enable the extraction of physiologically relevant features at the single-neuron and population level. We employed established GNNs to generate a combined representation of single-neuron and connectivity features obtained from HD-MEA data, with the ultimate goal of predicting changes in single-neuron firing rate induced by a pharmacological perturbation. The aim of the main prediction task was to assess whether single-neuron and functional connectivity features, inferred under baseline conditions, were informative for predicting changes in neuronal activity in response to a perturbation with Bicuculline, a GABA A receptor antagonist. Our results suggest that the joint representation of node features and functional connectivity, extracted from a baseline recording, was informative for predicting firing rate changes of individual neurons after the perturbation. Specifically, our implementation of a GNN model with inductive learning capability (GraphSAGE) outperformed other prediction models that relied only on single-neuron features. We tested the generalizability of the results on two additional datasets of HD-MEA recordings-a second dataset with cultures perturbed with Bicuculline and a dataset perturbed with the GABA A receptor antagonist Gabazine. GraphSAGE models showed improved prediction accuracy over other prediction models. Our results demonstrate the added value of taking into account the functional connectivity between neurons and the potential of GNNs to study complex interactions between neurons.

2,3-Carbamate mannosamine glycosyl donors in glycosylation reactions of diacetone-d-glucose. An experimental and theoretical study.

The role of the cyclic 2,3-N,O-carbamate protecting group in directing the selectivity of mannosylation reactions of diacetone-d-glucose, promoted by BSP/Tf2O via α-triflate intermediates, has been investigated through a combined computational and experimental approach. DFT calculations were used to locate the transition states leading to the α or ß anomers. These data indicate the preferential formation of the ß-adduct with mannosyl donors either equipped with the 4,6-O-benzylidene protection or without it. The synthetic results confirmed this preference, showing in both cases an α/ß selectivity of 4:6. This highlights a role for the 2,3-N,O-carbamate in sharp contrast with what described in the case of 2,3-O-carbonate mannosyl donors.

Electrophysiological Phenotype Characterization of Human iPSC-Derived Neuronal Cell Lines by Means of High-Density Microelectrode Arrays.

Recent advances in the field of cellular reprogramming have opened a route to studying the fundamental mechanisms underlying common neurological disorders. High-density microelectrode-arrays (HD-MEAs) provide unprecedented means to study neuronal physiology at different scales, ranging from network through single-neuron to subcellular features. In this work, HD-MEAs are used in vitro to characterize and compare human induced-pluripotent-stem-cell-derived dopaminergic and motor neurons, including isogenic neuronal lines modeling Parkinson's disease and amyotrophic lateral sclerosis. Reproducible electrophysiological network, single-cell and subcellular metrics are used for phenotype characterization and drug testing. Metrics, such as burst shape and axonal velocity, enable the distinction of healthy and diseased neurons. The HD-MEA metrics can also be used to detect the effects of dosing the drug retigabine to human motor neurons. Finally, it is shown that the ability to detect drug effects and the observed culture-to-culture variability critically depend on the number of available recording electrodes.

[Elderly and surgery: impact of frailty on surgical outcome. A literature review.] / Anziano e chirurgia: impatto della fragilità sugli outcome chirurgici. Una revisione della letteratura.

AIM: Currently more than 40% of surgical procedures are performed on subjects over65 years. this event has brought to the fore the concept of frailty. The aim of this work is to summarize the knowledge available in the literature in order to better understand what are the essential aspects of frailty, which impact it has on surgical outcomes of the elderly and what action we need to apply to prevent it. METHODS: A narrative literature review was conducted using the principal bibliographic biome- dical and nursing databases. RESULTS: Literature analysis revealed that frailty is a medical syndrome with multiple causes characterized by reduced physiological functions and decreased stress resistance, resulting in several adverse outcome following surgery. In the selected studies, frailty has emerged as a reliable indi- cator of increased morbidity and mortality, long-term hospital stay, post-discharge institutionali- zation, and increased healthcare costs. For frailty prevention and treatment, the most important clinical intervention is physical exercise. CONCLUSION: Identifying a patient like frail in the preoperative period allows the health profes- sional to adequately transmit information about individual perioperative risks. It should provide an opportunity to discuss the expectations and wishes of the patient for whom an adverse posto- perative outcome is expected. In addition, for high risk subjects, a clinical pathway with a well- structured discharge program can be anticipated in order to avoid hospital prolongations, thus reducing hospitalization costs.

Single-Cell Electrical Stimulation Using CMOS-Based High-Density Microelectrode Arrays.

Non-invasive electrical stimulation can be used to study and control neural activity in the brain or to alleviate somatosensory dysfunctions. One intriguing prospect is to precisely stimulate individual targeted neurons. Here, we investigated single-neuron current and voltage stimulation in vitro using high-density microelectrode arrays featuring 26,400 bidirectional electrodes at a pitch of 17.5 µm and an electrode area of 5 × 9 µm2. We determined optimal waveforms, amplitudes and durations for both stimulation modes. Owing to the high spatial resolution of our arrays and the close proximity of the electrodes to the respective neurons, we were able to stimulate the axon initial segments (AIS) with charges of less than 2 pC. This resulted in minimal artifact production and reliable readout of stimulation efficiency directly at the soma of the stimulated cell. Stimulation signals as low as 70 mV or 100 nA, with pulse durations as short as 18 µs, yielded measurable action potential initiation and propagation. We found that the required stimulation signal amplitudes decreased with cell growth and development and that stimulation efficiency did not improve at higher electric fields generated by simultaneous multi-electrode stimulation.

Combining habitat requirements of endemic bird species and other ecosystem services may synergistically enhance conservation efforts.

Biodiversity conservation and the optimisation of other ecosystem service delivery as a contribution to human well-being are often tackled as mutually alternative targets. Modern agriculture is a great challenge for the fulfilment of both. Here, we explore the potential benefits of integrating biodiversity conservation and the preservation of wider ecosystem services, considering the conservation of an endemic species (Moltoni's warbler Sylvia subalpina; Aves: Sylvidae) and soil erosion control (a final ecosystem service) in intensive vineyards in Italy. We modelled factors affecting warbler occurrence and abundance at 71 study plots by means of N-mixture models, and estimated soil erosion at the same plots by means of the Universal Soil Loss Equation. Shrub cover had positive effects on both warbler abundance and soil retention, whereas higher slopes promote warbler abundance as well as soil erosion. Creating shrub patches over sloping sites would be at the same time particularly suited for warblers and for soil retention. We simulated three alternative conservation strategies: exclusive focus on warbler conservation (1), exclusive focus on soil preservation (2), integration of the two targets (3). Strategies assumed the creation of 1.5-ha shrub patches over 5% of the total area covered by plots and targeted either at wildlife or soil conservation. The exclusive strategies would allow an increase of 105 individuals and the preservation of 783 tons ha-1year-1, respectively. Each individual strategy would ensure benefits for the other target corresponding to 61-64% of the above totals. The integrated strategy would allow for the achievement of 91-93% of the benefits (96 warblers and 729 tons ha-1year-1) of the individual strategies. The integration of the two approaches could provide important synergies, allowing to broaden the effects of conservation strategies, such as agri-environmental schemes that could be drawn from our results (and which are particularly urgent for intensive permanent crops).

The park-view effect: Residential development is higher at the boundaries of protected areas.

Land-use changes in the surrounding of protected areas might compromise their conservation efficacy, and thus the potential attractive effect that protected areas may exert on urban development is particularly concerning. We investigated whether the proximity to protected areas in a region (Lombardy, Italy) with a high density of urban areas and parks may increase the likelihood of residential development. The main change around protected areas was the loss of open areas (mostly due to development processes at the expense of arable land), which was higher in proximity of parks. Changes in residential discontinuous development were significantly and negatively related to proximity to parks, whereas changes in productive developments were unrelated to park proximity. The higher likelihood of residential development in proximity to parks is likely due to the attraction exerted by parks. The severe loss of open areas and increase of residential development around parks heavily impacted on habitat availability for a declining bird species (Alauda arvensis). Conservation policies considering also what happens around protected areas are urgently needed.

HER2 targeting as a two-sided strategy for breast cancer diagnosis and treatment: Outlook and recent implications in nanomedical approaches.

At present, mammary carcinoma is the second most common type of malignant tumor in adult women after lung cancer, as more than one million women are diagnosed with breast cancer every year. Despite advances in diagnosis and treatment, which have resulted in a decrease in mortality in recent decades, breast cancer remains a major public health problem. One of the most significant unresolved clinical and scientific problems is the occurrence of resistance to clinical treatments and their toxicity (and how to predict, prevent and overcome them). However, the heterogeneity of human breast cancer in terms of genetic features, molecular profiles and clinical behavior represents a constraint obstructing the discovery of a solution to the disease. It is currently considered that the chances of success of therapy may increase if the tumor cells are selectively removed before they can evolve to their mature stages up to metastases production. Therefore, novel and more sensitive diagnostic tools are being developed, with the aim of improving the early and noninvasive detection of rising malignancies and the accuracy of tumor tissue localization. Meanwhile, there is an emerging use of targeted therapies in oncology, depending on the expression of specific proteins or genes present in tumor cells. Among the molecular targets considered for the treatment of breast cancer cells so far, we chose to focus on examples involving overexpression and/or gene amplification of "Human Epidermal growth factor Receptor 2" (HER2) protein. In current studies, various types of nanoparticles conjugated with the anti-HER2 monoclonal antibody, the so-called "trastuzumab", are investigated extensively due to promising results in biological and preclinical applications aimed at improving the treatment of breast cancer. In this paper, we present a critical review of the preparation and use of different kinds of trastuzumab-functionalized nanoparticles, with an emphasis on the therapeutic and diagnostic (theranostic) potential of this generation of hybrid nanoparticles, exploiting the multifaceted mechanisms of action of trastuzumab against malignant cells.

Synthesis, molecular dynamics simulations, and biology of a carba-analogue of the trisaccharide repeating unit of Streptococcus pneumoniae 19F capsular polysaccharide.

The synthesis of a carba-analogue corresponding to the trisaccharide repeating unit of Streptococcus pneumoniae type 19F capsular polysaccharide, where a residue of carba-L-rhamnose has been inserted into the natural trisaccharide in place of L-rhamnose, is described. The conformational properties of the analogue were investigated with the aid of molecular dynamics simulations and were strictly analogous to those of the natural compound. The biological activity of the carba-analogue was comparable to that of the corresponding natural repeating unit, thus suggesting that this compound, more stable to hydrolysis, is a good mimic of the natural structure.

Towards ideal magnetofluorescent nanoparticles for bimodal detection of breast-cancer cells.

An increasing number of novel molecular markers based on nanomaterials for tumor diagnostics have been developed in recent years. Many efforts have focused on the achievement of site-targeted bioconjugated nanoparticles. In contrast, the mechanisms of toxicity, endocytosis, and degradation pathways are still poorly understood, despite their primary importance for clinical translation. In this study, three different model nanoscale magnetofluorescent particle systems (MFNs) are designed and fabricated. These nanoparticles are evaluated in terms of size, morphology, zeta potential, fluorescence efficiency, capability of enhancing T(2) relaxivity of water protons, and stability. Accordingly, two are developed and the mechanism of internalization, the intracellular fate, and the toxicity in MCF-7 adenocarcinoma cells are studied. Besides the well-documented size effect, the anionic charge seems to be a crucial factor for particle internalization, as MFN penetration through the cell membrane could be modulated by surface charge. Ultrastructural analysis of transmission electron micrographs combined with evidence from confocal microscopy reveals that MFNs are internalized by clathrin-mediated endocytosis and macropinocytosis. Moreover, MFNs are found in EEA1-positive endosomes and in lysosomes, indicating that they follow a physiological pathway of endocytosis. Magnetorelaxometric analysis demonstrates that MFNs enable the detection of 5 x 10(5) cells mL(-1) after treatment with particle dosages as low as 30 microg mL(-1). Hence, MFNs appear to be a valuable and safe bimodal contrast agent that can be developed for the noninvasive diagnosis of breast cancer.

Femtomolar detection of autoantibodies by magnetic relaxation nanosensors.

The development of nanosystems applied to rapid and sensitive measurement of biomarkers in fluid samples is a current major goal in diagnostic biomedicine. In this article, we report the accurate and reliable detection of anti-HSA (human serum albumin) antibodies by protein-functionalized magnetic nanospherical probes due to the reversible alteration of their microaggregation state induced by protein antibody-specific interaction, sensed as changes in the T(2) relaxation time of surrounding water molecules. Once the optimal parameters were adjusted, the method proved to be very sensitive, providing concentration- and time-dependent responses. Furthermore, we demonstrate that the developed immunoassay is able to quantitatively determine the biomarker concentration from T(2) linear correlation, thereby supplying a rapid, yet accurate, assay with sensitivity in the femtomolar range. The high susceptibility and stability of these magnetic nanoparticles, as well as their accessible synthetic preparation, make these nanosensors a promising new tool for versatile and effective medical diagnostics.

Synthesis of sulfated galactocerebrosides from an orthogonal beta-D-galactosylceramide scaffold for the study of CD1-antigen interactions.

CD1a protein binds sulfatide (3-O-sulfo-beta-D-galactosylceramide) to form an antigen complex that interacts with T cell receptors and activates T cells. To assess the role of the position of the sulfate in T cell activation, the synthesis of three beta-D-galactosylceramides, variously bearing a sulfate at position 2, 4, or 6 of galactose, has been planned and carried out. The compounds were synthesized by an orthogonal sulfation strategy from a common beta-D-galactosylceramide scaffold, which was in turn obtained through an efficient glycosylation reaction between a fully orthogonally protected galactosyl imidate and 3-O-benzoylazidosphingosine. Immunological evaluation of the three sulfated compounds in CD1a-mediated T cell activation, in comparison with natural sulfatide, provided evidence of the influence of the sulfate position in the recognition event between the antigen, the CD1 protein and the T cell receptor.