Methods and computational tools to study eukaryotic cell migration in vitro.

Cellular movement is essential for many vital biological functions where it plays a pivotal role both at the single cell level, such as during division or differentiation, and at the macroscopic level within tissues, where coordinated migration is crucial for proper morphogenesis. It also has an impact on various pathological processes, one for all, cancer spreading. Cell migration is a complex phenomenon and diverse experimental methods have been developed aimed at dissecting and analysing its distinct facets independently. In parallel, corresponding analytical procedures and tools have been devised to gain deep insight and interpret experimental results. Here we review established experimental techniques designed to investigate specific aspects of cell migration and present a broad collection of historical as well as cutting-edge computational tools used in quantitative analysis of cell motion.

N2FXm, a method for joint nuclear and cytoplasmic volume measurements, unravels the osmo-mechanical regulation of nuclear volume in mammalian cells.

In eukaryotes, cytoplasmic and nuclear volumes are tightly regulated to ensure proper cell homeostasis. However, current methods to measure cytoplasmic and nuclear volumes, including confocal 3D reconstruction, have limitations, such as relying on two-dimensional projections or poor vertical resolution. Here, to overcome these limitations, we describe a method, N2FXm, to jointly measure cytoplasmic and nuclear volumes in single cultured adhering human cells, in real time, and across cell cycles. We find that this method accurately provides joint size over dynamic measurements and at different time resolutions. Moreover, by combining several experimental perturbations and analyzing a mathematical model including osmotic effects and tension, we show that N2FXm can give relevant insights on how mechanical forces exerted by the cytoskeleton on the nuclear envelope can affect the growth of nucleus volume by biasing nuclear import. Our method, by allowing for accurate joint nuclear and cytoplasmic volume dynamic measurements at different time resolutions, highlights the non-constancy of the nucleus/cytoplasm ratio along the cell cycle.

PIP4K2B is mechanoresponsive and controls heterochromatin-driven nuclear softening through UHRF1.

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4,5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localisation. Here, we unveil the role of PIP4K2B as a mechanoresponsive enzyme. PIP4K2B protein level strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to softness, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.

Even without changing the bone mineral density, alcohol consumption decreases the percentage of collagen, the thickness of bone trabeculae, and increases bone fragility.

Excessive alcohol consumption is considered a risk factor for bone health, as it causes a reduction in mass and increases the risk of fracture. However, the determination of bone mineral density (BMD) has not always been an adequate predictor of bone fragility. Thus, the hypothesis arises that chronic alcohol consumption interferes with collagen synthesis and the quality of bone trabeculae, with consequent bone fragility. Groups: Control (n = 6; water intake only during the entire study period); Ethanol (n = 6; ingestion of ethyl alcohol according to the protocol for inducing chronic alcohol consumption). The chronic alcohol consumption model did not cause a significant change in BMD, but there was a significant reduction of 20% in the thickness of the bone trabeculae and of 1.56% in the collagen located in the neck region of immature rat femurs. Although there was no significant change in the mineral matrix, the changes in the organic matrix were able to provide a significant reduction in bone strength. The results suggest harmful effects of alcohol intake on the bone quality of young adult animals and draw attention to the need to also consider methods for the diagnosis of collagen as an element of bone fragility.

The transcription factor PREP1(PKNOX1) regulates nuclear stiffness, the expression of LINC complex proteins and mechanotransduction.

Mechanosignaling, initiated by extracellular forces and propagated through the intracellular cytoskeletal network, triggers signaling cascades employed in processes as embryogenesis, tissue maintenance and disease development. While signal transduction by transcription factors occurs downstream of cellular mechanosensing, little is known about the cell intrinsic mechanisms that can regulate mechanosignaling. Here we show that transcription factor PREP1 (PKNOX1) regulates the stiffness of the nucleus, the expression of LINC complex proteins and mechanotransduction of YAP-TAZ. PREP1 depletion upsets the nuclear membrane protein stoichiometry and renders nuclei soft. Intriguingly, these cells display fortified actomyosin network with bigger focal adhesion complexes resulting in greater traction forces at the substratum. Despite the high traction, YAP-TAZ translocation is impaired indicating disrupted mechanotransduction. Our data demonstrate mechanosignaling upstream of YAP-TAZ and suggest the existence of a transcriptional mechanism actively regulating nuclear membrane homeostasis and signal transduction through the active engagement/disengagement of the cell from the extracellular matrix.

Short-term outcome following significant trauma: increasing age per se has only a relatively low impact.

PURPOSE: Ongoing demographic changes go hand in hand with an increasing number of elderly injured. Given the conflicting literature we wanted to know how much age per se, apart from other factors, actually explains the outcome for elderly trauma patients. METHODS: Retrospective analysis of prospectively collected data on all significantly injured (new injury severity score, NISS ≥ 8) adult patients treated at a Swiss trauma center between 01.01.2010 and 31.12.2017. The association of age and other demographic, trauma or treatment-related variables on parameters of short-term outcome was examined using uni- and multivariate analyses (mean ± SD; R2; p < 0.05). RESULTS: 2692 consecutive patients (33.4% female; mean age 58.1 ± 21.7; hospital mortality 10.1%) were studied. Detailed analysis of quinquennial age groups demonstrated a significant decline in outcome with regard to mortality or return-to-home rate following hospital discharge after the age of 60 years (p < 0.001). In univariate analysis, age explained 4.6% and the number of years ≥ 60 5.9% of hospital mortality. In multivariate analysis, the investigated demographic, trauma or treatment-related parameters contributed at 36.5% to prediction of mortality, age added another 1.5% and number of years ≥ 60 another 2.1% (R2). CONCLUSION: This monocenter evaluation showed a significant decline in short-term outcome and an increase in hospital resource requirements by the trauma patients investigated after the age of 60 years. Even so, after controlling for demographic, injury and treatment variables, age per se only added less than 2% to the prediction of hospital mortality.

Preventive antibiotic therapy in acute stroke patients: A systematic review and meta-analysis of individual patient data of randomized controlled trials.

Introduction: Infection after stroke is associated with unfavorable outcome. Randomized controlled studies did not show benefit of preventive antibiotics in stroke but lacked power for subgroup analyses. Aim of this study is to assess whether preventive antibiotic therapy after stroke improves functional outcome for specific patient groups in an individual patient data meta-analysis. Patients and methods: We searched MEDLINE (1946-7 May 2021), Embase (1947-7 May 2021), CENTRAL (17th September 2021), trial registries, cross-checked references and contacted researchers for randomized controlled trials of preventive antibiotic therapy versus placebo or standard care in ischemic or hemorrhagic stroke patients. Meta-analysis was performed by a one-step and two-step approach. Primary outcome was functional outcome adjusted for age and stroke severity. Secondary outcomes were infections and mortality. Results: 4197 patients from nine trials were included. Preventive antibiotic therapy was not associated with a shift in functional outcome (mRS) at 3 months (OR1.13, 95%CI 0.98-1.31) or unfavorable functional outcome (mRS 3-6) (OR0.85, 95%CI 0.60-1.19). Preventive antibiotics did not improve functional outcome in pre-defined subgroups (age, stroke severity, timing and type of antibiotic therapy, pneumonia prediction scores, dysphagia, type of stroke, and type of trial). Preventive antibiotics reduced infections (276/2066 (13.4%) in the preventive antibiotic group vs. 417/2059 (20.3%) in the control group, OR 0.60, 95% CI 0.51-0.71, p < 0.001), but not pneumonia (191/2066 (9.2%) in the preventive antibiotic group vs. 205/2061 (9.9%) in the control group (OR 0.92 (0.75-1.14), p = 0.450). Discussion and conclusion: Preventive antibiotic therapy did not benefit any subgroup of patients with acute stroke and currently cannot be recommended.

A 13.56 MHz Rectifier Based on Fully Inkjet Printed Organic Diodes.

The increasing diffusion of portable and wearable technologies results in a growing interest in electronic devices having features such as flexibility, lightness-in-weight, transparency, and wireless operation. Organic electronics is proposed as a potential candidate to fulfill such needs, in particular targeting pervasive radio-frequency (RF) applications. Still, limitations in terms of device performances at RF, particularly severe when large-area and scalable fabrication techniques are employed, have largely precluded the achievement of such an appealing scenario. In this work, the rectification of an electromagnetic wave at 13.56 MHz with a fully inkjet printed polymer diode is demonstrated. The rectifier, a key enabling component of future pervasive wireless systems, is fabricated through scalable large-area methods on plastic. To provide a proof-of-principle demonstration of its future applicability, its adoption in powering a printed integrated polymer circuit is presented. The possibility of harvesting electrical power from RF waves and delivering it to a cheap flexible substrate through a simple printed circuitry paves the way to a plethora of appealing distributed electronic applications.

Antidepressant effect of vagal nerve stimulation in epilepsy patients: a systematic review.

BACKGROUND: Vagal nerve stimulation (VNS) is an effective palliative therapy in drug-resistant epileptic patients and is also approved as a therapy for treatment-resistant depression. Depression is a frequent comorbidity in epilepsy and it affects the quality of life of patients more than the seizure frequency itself. The aim of this systematic review is to analyze the available literature about the VNS effect on depressive symptoms in epileptic patients. MATERIAL AND METHODS: A comprehensive search of PubMed, Medline, Scopus, and Google Scholar was performed, and results were included up to January 2020. All studies concerning depressive symptom assessment in epileptic patients treated with VNS were included. RESULTS: Nine studies were included because they fulfilled inclusion criteria. Six out of nine papers reported a positive effect of VNS on depressive symptoms. Eight out of nine studies did not find any correlation between seizure reduction and depressive symptom amelioration, as induced by VNS. Clinical scales for depression, drug regimens, and age of patients were broadly different among the examined studies. CONCLUSIONS: Reviewed studies strongly suggest that VNS ameliorates depressive symptoms in drug-resistant epileptic patients and that the VNS effect on depression is uncorrelated to seizure response. However, more rigorous studies addressing this issue are encouraged.

Tailoring Cellular Function: The Contribution of the Nucleus in Mechanotransduction.

Cells sense a variety of different mechanochemical stimuli and promptly react to such signals by reshaping their morphology and adapting their structural organization and tensional state. Cell reactions to mechanical stimuli arising from the local microenvironment, mechanotransduction, play a crucial role in many cellular functions in both physiological and pathological conditions. To decipher this complex process, several studies have been undertaken to develop engineered materials and devices as tools to properly control cell mechanical state and evaluate cellular responses. Recent reports highlight how the nucleus serves as an important mechanosensor organelle and governs cell mechanoresponse. In this review, we will introduce the basic mechanisms linking cytoskeleton organization to the nucleus and how this reacts to mechanical properties of the cell microenvironment. We will also discuss how perturbations of nucleus-cytoskeleton connections, affecting mechanotransduction, influence health and disease. Moreover, we will present some of the main technological tools used to characterize and perturb the nuclear mechanical state.

Non-invasive Production of Multi-Compartmental Biodegradable Polymer Microneedles for Controlled Intradermal Drug Release of Labile Molecules.

Transdermal drug delivery represents an appealing alternative to conventional drug administration systems. In fact, due to their high patient compliance, the development of dissolvable and biodegradable polymer microneedles has recently attracted great attention. Although stamp-based procedures guarantee high tip resolution and reproducibility, they have long processing times, low levels of system engineering, are a source of possible contaminants, and thermo-sensitive drugs cannot be used in conjunction with them. In this work, a novel stamp-based microneedle fabrication method is proposed. It provides a rapid room-temperature production of multi-compartmental biodegradable polymeric microneedles for controlled intradermal drug release. Solvent casting was carried out for only a few minutes and produced a short dissolvable tip made of polyvinylpyrrolidone (PVP). The rest of the stamp was then filled with degradable poly(lactide-co-glycolide) (PLGA) microparticles (µPs) quickly compacted with a vapor-assisted plasticization. The outcome was an array of microneedles with tunable release. The ability of the resulting microneedles to indent was assessed using pig cadaver skin. Controlled intradermal delivery was demonstrated by loading both the tip and the body of the microneedles with model therapeutics; POXA1b laccase from Pleurotus ostreatus is a commercial enzyme used for the whitening of skin spots. The action and indentation of the enzyme-loaded microneedle action were assessed in an in vitro skin model and this highlighted their ability to control the kinetic release of the encapsulated compound.

Three-dimensionally Patterned Scaffolds Modulate the Biointerface at the Nanoscale.

The main aim of cell instructive materials is to guide in a controlled way cellular behavior by fine-tuning cell-material crosstalk. In the last decades, several efforts have been spent in elucidating the relations between material cues and cellular fate at the nanoscale and in the development of novel strategies for gaining a superior control over cellular function modulation. In this context, a particular attention has been recently paid to the role played by cellular membrane rearrangement in triggering specific molecular pathways linked to the regulation of different cellular functions. Here, we characterize the effect of linear microtopographies upon cellular behavior in three-dimensional (3D) environments, with particular focus on the relations linking cytoskeleton structuration to membrane rearrangement and internalization tuning. The performed analysis shown that, by altering the cellular adhesion processes at the micro- and nanoscale, it is possible to alter the membrane physical state and cellular internalization capability. More specifically, our findings pointed out that an increased cytoskeletal structuration influences the formation of nanoinvagination membrane process at the cell-material interface and the expression of clathrin and caveolin, two of the main proteins involved in the endocytosis regulation. Moreover, we proved that such topographies enhance the engulfment of inert polystyrene nanoparticles attached on 3D patterned surfaces. Our results could give new guidelines for the design of innovative and more efficient 3D cell culture systems usable for diagnostic, therapeutic, and tissue engineering purposes.

Hypertension, seizures, and epilepsy: a review on pathophysiology and management.

BACKGROUND: Epilepsy and hypertension are common chronic conditions, both showing high prevalence in older age groups. This review outlines current experimental and clinical evidence on both direct and indirect role of hypertension in epileptogenesis and discusses the principles of drug treatment in patients with hypertension and epilepsy. METHODS: We selected English-written articles on epilepsy, hypertension, stroke, and cerebrovascular disease until December, 2018. RESULTS: Renin-angiotensin system might play a central role in the direct interaction between hypertension and epilepsy, but other mechanisms may be contemplated. Large-artery stroke, small vessel disease and posterior reversible leukoencephalopathy syndrome are hypertension-related brain lesions able to determine epilepsy by indirect mechanisms. The role of hypertension as an independent risk factor for post-stroke epilepsy has not been demonstrated. The role of hypertension-related small vessel disease in adult-onset epilepsy has been demonstrated. Posterior reversible encephalopathy syndrome is an acute condition, often caused by a hypertensive crisis, associated with the occurrence of acute symptomatic seizures. Chronic antiepileptic treatment should consider the risk of drug-drug interactions with antihypertensives. CONCLUSIONS: Current evidence from preclinical and clinical studies supports the vision that hypertension may be a cause of seizures and epilepsy through direct or indirect mechanisms. In both post-stroke epilepsy and small vessel disease-associated epilepsy, chronic antiepileptic treatment is recommended. In posterior reversible encephalopathy syndrome blood pressure must be rapidly lowered and prompt antiepileptic treatment should be initiated.

Electron Microscopy for 3D Scaffolds-Cell Biointerface Characterization.

Cell fate is largely determined by interactions that occur at the interface between cells and their surrounding microenvironment. For this reason, especially in the field of tissue-engineering, there is a growing interest in developing techniques that allow evaluating cell-material interaction at the nanoscale, particularly focusing on cell adhesion processes. While for 2D culturing systems a consolidated series of tools already satisfy this need, in 3D environments, more closely recapitulating complex in vivo structures, there is still a lack of procedures furthering the comprehension of cell-material interactions. Here, the use of scanning electron microscopy coupled with a focused ion beam (SEM/FIB) for the characterization of cell interactions with 3D scaffolds obtained by different fabrication techniques is reported for the first time. The results clearly show the capability of the developed approach to preserve and finely resolve scaffold-cell interfaces highlighting details such as plasma membrane arrangement, extracellular matrix architecture and composition, and cellular structures playing a role in cell adhesion to the surface. It is anticipated that the developed approach will be relevant for the design of efficient cell-instructive platforms in the study of cellular guidance strategies for tissue-engineering applications as well as for in vitro 3D models.

Controlling the orientation of a cell-synthesized extracellular matrix by using engineered gelatin-based building blocks.

In tissue engineering there is growing interest in fabricating highly engineered platforms designed to instruct cells towards the synthesis of tissues that reproduce their natural counterpart. In this context, a fundamental factor to take into account is the control over the final tissue orientation, especially for what concerns the replication of load-bearing tissues whose functions are strictly related to their microstructural organization. Starting from this point, in this work we have engineered a gelatin-based hydrogel in order to be patterned by 2-photon polymerization (2PP) lithography for the fabrication of instructive free standing building blocks designed to produce anisotropic collagen-based µtissues. Biological results clearly highlighted the strong relationship between µtissue orientation and such topographies, which resulted in a crucial element in the production of highly anisotropic µtissues.

Three-Dimensional Microstructured Azobenzene-Containing Gelatin as a Photoactuable Cell Confining System.

In materials science, there is a considerable interest in the fabrication of highly engineered biomaterials that can interact with cells and control their shape. In particular, from the literature, the role played by physical cell confinement in cellular structural organization and thus in the regulation of its functions has been well-established. In this context, the addition of a dynamic feature to physically confining platforms aiming at reproducing in vitro the well-known dynamic interaction between the cells and their microenvironment would be highly desirable. To this aim, we have developed an advanced gelatin-based hydrogel that can be finely micropatterned by two-photon polymerization and stimulated in a controlled way by light irradiation thanks to the presence of an azobenzene cross-linker. Light-triggered expansion of gelatin microstructures induced an in-plane nuclear deformation of physically confined NIH-3T3 cells. The microfabricated photoactuable gelatin shown in this work paves the way to new "dynamic" caging culture systems that can find applications, for example, as "engineered stem cell niches".

Effect of Feed Additives on Productivity and Campylobacter spp. Loads in Broilers Reared under Free Range Conditions.

The poultry reservoir, especially broiler meat, is generally recognized as one of the most-important sources for human Campylobacteriosis. The measures to control Campylobacter targeted essentially the primary production level. The aim of this work was to evaluate the effectiveness of different treatments against natural Campylobacter colonization in a French experimental farm of free-range broilers during the whole rearing period. Five commercial products and a combination of two of them were tested and all the products were added to feed or to water at the dose recommended by the suppliers. Campylobacter loads in caeca and on carcasses of broilers at the slaughter were determined by culture methods. Natural contamination of the flock occurred at the end of the indoor rearing period between day 35 and day 42. At day 42, the multispecies probiotic added to the feed reduced the contamination of 0.55 log10 CFU/g (p = 0.02) but was not significant (p > 0.05) at the end of rearing at day 78. However, another treatment, a combination of a cation exchange clay-based product in feed and an organic acid mixture (formic acid, sodium formate, lactic acid, propionic acid) in water, led to a slight but significant reduction of 0.82 ± 0.25 log10 CFU/g (p = 0.02) compared to the control group at day 78. Testing this combination in field conditions in several flocks is needed to determine if it is biologically relevant and if it could be a valuable measure to reduce Campylobacter in broiler flocks.

[Palliative care in neurology]. / La dimensione delle cure palliative in neurologia.

Palliative care in neurology is characterized by the need of taking into account some distinguishing features which supplement and often differ from the general palliative approach to cancer or to severe organ failures. Such position is emphasized by a new concept of palliative assistance which is not limited to the "end of life" stage, as it was the traditional one, but is applied along the entire course of progressive, life-limiting, and disabling conditions. There are various reasons accounting for a differentiation of palliative care in neurology and for the development of specific expertise; the long duration of the advanced stages of many neurological diseases and the distinguishing features of some clinical problems (cognitive disorders, psychic disorders, etc.), in addition to the deterioration of some general aspects (nutrition, etc.), make the general criteria adopted for cancer, severe respiratory, hepatic or renal failures and heart failure inadequate. The neurological diseases which could benefit from the development of a specific palliative approach are dementia, cerebrovascular diseases, movement disorders, neuromuscular diseases, severe traumatic brain injury, brain cancers and multiple sclerosis, as well as less frequent conditions. The growing literature on palliative care in neurology provides evidence of the neurological community's increasing interest in taking care of the advanced and terminal stages of nervous system diseases, thus encouraging research, training and updating in such direction. This document aims to underline the specific neurological requirements concerning the palliative assistance.

Need for neurology specialists to be dedicated to hospital care in Italy.

Any patients admitted to healthcare facilities with a neurological diseases deserves to be managed by a neurologist. This is particularly important for acute onset neurological disorders, because of their severity and the requirement of early and appropriate diagnostic-therapeutic approach. In addition, this may reduce both unnecessary admissions and length of stay, with a significant saving of resources for the National Health System (NHS). To ensure this, it is important to evaluate predictable needs for hospital neurologists in Italy. The hospital discharges for neurological medical diseases of the Major Disease Category (MDC) 1 were 455,132 in 2009 and 491,836 in 2008, more than 50 % of which were acute neurological disorders. Stroke and transient ischemic attacks (TIA) accounted for about 170,000 per year. Currently available neurologists in Italian healthcare facilities are largely insufficient to assist such a large number of patients. The 270 Neurological Care Units in Italy (of which 243 open to the emergency care) are equipped with an average number of 7.7 neurologists per unit, inadequate to ensure on duty care 24 h a day, 7 days a week. In addition, the mean age of hospital neurologists is quite high, with provision for large retirement. It is therefore required to increase in the next few years the number of neurologists by at least 30 %, with an increase of 562 units. To meet the need for neurology specialists committed to hospital care in Italy is also necessary to increase the number of scholarships for residents in post-graduate schools.