Molecular-scale substrate anisotropy, crowding and division drive collective behaviours in cell monolayers.

The ability of cells to reorganize in response to external stimuli is important in areas ranging from morphogenesis to tissue engineering. While nematic order is common in biological tissues, it typically only extends to small regions of cells interacting via steric repulsion. On isotropic substrates, elongated cells can co-align due to steric effects, forming ordered but randomly oriented finite-size domains. However, we have discovered that flat substrates with nematic order can induce global nematic alignment of dense, spindle-like cells, thereby influencing cell organization and collective motion and driving alignment on the scale of the entire tissue. Remarkably, single cells are not sensitive to the substrate's anisotropy. Rather, the emergence of global nematic order is a collective phenomenon that requires both steric effects and molecular-scale anisotropy of the substrate. To quantify the rich set of behaviours afforded by this system, we analyse velocity, positional and orientational correlations for several thousand cells over days. The establishment of global order is facilitated by enhanced cell division along the substrate's nematic axis, and associated extensile stresses that restructure the cells' actomyosin networks. Our work provides a new understanding of the dynamics of cellular remodelling and organization among weakly interacting cells.

GABAergic miR-34a regulates Dorsal Raphè inhibitory transmission in response to aversive, but not rewarding, stimuli.

The brain employs distinct circuitries to encode positive and negative valence stimuli, and dysfunctions of these neuronal circuits have a key role in the etiopathogenesis of many psychiatric disorders. The Dorsal Raphè Nucleus (DRN) is involved in various behaviors and drives the emotional response to rewarding and aversive experiences. Whether specific subpopulations of neurons within the DRN encode these behaviors with different valence is still unknown. Notably, microRNA expression in the mammalian brain is characterized by tissue and neuronal specificity, suggesting that it might play a role in cell and circuit functionality. However, this specificity has not been fully exploited. Here, we demonstrate that microRNA-34a (miR-34a) is selectively expressed in a subpopulation of GABAergic neurons of the ventrolateral DRN. Moreover, we report that acute exposure to both aversive (restraint stress) and rewarding (chocolate) stimuli reduces GABA release in the DRN, an effect prevented by the inactivation of DRN miR-34a or its genetic deletion in GABAergic neurons in aversive but not rewarding conditions. Finally, miR-34a inhibition selectively reduced passive coping with severe stressors. These data support a role of miR-34a in regulating GABAergic neurotransmitter activity and behavior in a context-dependent manner and suggest that microRNAs could represent a functional signature of specific neuronal subpopulations with valence-specific activity in the brain.

On-Site Inspection Form in Veterinary Cases: The Parma Veterinary Form.

The on-site inspection of the scene of an animal cadaver is crucial for a correct interpretation of the autopsy results, to determine the manner, method, and cause of death. This information plays a crucial role in the control of public health including the prevention of zoonoses. It is also fundamental for the recognition and the contrast of crimes against animals and to animal abuse phenomena, considered an alert sign of an anti-social or violent behavior of humans. Today the best veterinary procedure requires an accurate collection of the evidence at the scene that can be then handed to experts belonging to other forensic disciplines for further evaluation and data interpretation. In this paper authors suggest a form aiming to facilitate either the on-site and the autopsy activities, as a guarantee of the quality of the forensic process starting from the discovery scene up to the reconstruction of the case. Essential is training of non-medical personnel who often represent the first responder to be present on the scene. The form is inspired by the interdisciplinary form developed by the European Council of Legal Medicine and represents an initial tool to stimulate a multidisciplinary activity in close synergy with other forensic experts.

Lamotrigine rescues neuronal alterations and prevents seizure-induced memory decline in an Alzheimer's disease mouse model.

Epilepsy is a comorbidity associated with Alzheimer's disease (AD), often starting many years earlier than memory decline. Investigating this association in the early pre-symptomatic stages of AD can unveil new mechanisms of the pathology as well as guide the use of antiepileptic drugs to prevent or delay hyperexcitability-related pathological effects of AD. We investigated the impact of repeated seizures on hippocampal memory and amyloid-ß (Aß) load in pre-symptomatic Tg2576 mice, a transgenic model of AD. Seizure induction caused memory deficits and an increase in oligomeric Aß42 and fibrillary species selectively in pre-symptomatic transgenic mice, and not in their wildtype littermates. Electrophysiological patch-clamp recordings in ex vivo CA1 pyramidal neurons and immunoblots were carried out to investigate the neuronal alterations associated with the behavioral outcomes of Tg2576 mice. CA1 pyramidal neurons exhibited increased intrinsic excitability and lower hyperpolarization-activated Ih current. CA1 also displayed lower expression of the hyperpolarization-activated cyclic nucleotide-gated HCN1 subunit, a protein already identified as downregulated in the AD human proteome. The antiepileptic drug lamotrigine restored electrophysiological alterations and prevented both memory deficits and the increase in extracellular Aß induced by seizures. Thus our study provides evidence of pre-symptomatic hippocampal neuronal alterations leading to hyperexcitability and associated with both higher susceptibility to seizures and to AD-specific seizure-induced memory impairment. Our findings also provide a basis for the use of the antiepileptic drug lamotrigine as a way to counteract acceleration of AD induced by seizures in the early phases of the pathology.

Impaired synaptic plasticity in an animal model of autism exhibiting early hippocampal GABAergic-BDNF/TrkB signaling alterations.

In Neurodevelopmental Disorders, alterations of synaptic plasticity may trigger structural changes in neuronal circuits involved in cognitive functions. This hypothesis was tested in mice carrying the human R451C mutation of Nlgn3 gene (NLG3R451C KI), found in some families with autistic children. To this aim, the spike time dependent plasticity (STDP) protocol was applied to immature GABAergic Mossy Fibers (MF)-CA3 connections in hippocampal slices from NLG3R451C KI mice. These animals failed to exhibit STD-LTP, an effect that persisted in adulthood when these synapses became glutamatergic. Similar results were obtained in mice lacking the Nlgn3 gene (NLG3 KO mice), suggesting a loss of function. The loss of STD-LTP was associated with a premature shift of GABA from the depolarizing to the hyperpolarizing direction, a reduced BDNF availability and TrkB phosphorylation at potentiated synapses. These effects may constitute a general mechanism underlying cognitive deficits in those forms of Autism caused by synaptic dysfunctions.

Use of the international classification of diseases (ICD)-11 method applied to veterinary forensic pathology for coding the cause and manner of death in wildlife.

The growth of human population has led, in recent years, to increasingly frequent contacts with the wild animals with which we share the territory, sometimes leading to negative interactions with them. The purpose of the study is to apply the codes contained in the 11th Revision of the International Classification of Diseases (ICD-11) method to investigate the cause and the manner of death, also to entrust the veterinarian with the task of recognizing and describing a suspected animal abuse as a sentinel indicator of violence toward humans and non-humans, thus expanding the concept of "One Health" from a forensic investigation perspective. The subjects recruited are wild mammals submitted for autopsy to the Pathology Unit of the Department of Veterinary Science, University of Parma, Italy, from 2015 to 2018. The manner and the cause of death of 167 wild animals of 16 different species have been investigated. When possible, an on-site inspection where the corpse was found was performed. Injuries were classified according to the on-line 11th Revision of the International Classification of Diseases method. Section 22 (Injury, poisoning or certain other consequences of external causes) was used to record the "immediate cause of death" (cause of death) and Section 23 (External causes of morbidity or mortality) was used to record the "underlying cause of death" (manner of death) for each animal. In most cases, death occurred as a result of road trauma but in some cases, abuse and voluntary killing were investigated. The recognition of non-accidental injuries is particularly important for both the defense in court of animals and for the connection between crimes committed against animals and against humans, known as "The Link". The use of the ICD-11 method, as a sort of summary of the autopsy report, was confirmed to be of great value for the clarity and simplicity of processing the data collected also by veterinary pathologists. The veterinary pathologists can use this evidence-based method with the aim of creating a national register and therefore, to understand the real extent of the human impact on wildlife and document it in a scientific and statistically usable way.

Flow around topological defects in active nematic films.

We study the active flow around isolated defects and the self-propulsion velocity of + 1 / 2 defects in an active nematic film with both viscous dissipation (with viscosity η ) and frictional damping Γ with a substrate. The interplay between these two dissipation mechanisms is controlled by the hydrodynamic dissipation length ℓ d = η / Γ that screens the flows. For an isolated defect, in the absence of screening from other defects, the size of the shear vorticity around the defect is controlled by the system size R . In the presence of friction that leads to a finite value of ℓ d , the vorticity field decays to zero on the lengthscales larger than ℓ d . We show that the self-propulsion velocity of + 1 / 2 defects grows with R in small systems where R < ℓ d , while in the infinite system limit or when R ≫ ℓ d , it approaches a constant value determined by ℓ d .

Systemic delivery of a specific antibody targeting the pathological N-terminal truncated tau peptide reduces retinal degeneration in a mouse model of Alzheimer's Disease.

Retina and optic nerve are sites of extra-cerebral manifestations of Alzheimer's Disease (AD). Amyloid-ß (Aß) plaques and neurofibrillary tangles of hyperphosphorylated tau protein are detected in eyes from AD patients and transgenic animals in correlation with inflammation, reduction of synapses, visual deficits, loss of retinal cells and nerve fiber. However, neither the pathological relevance of other post-translational tau modifications-such as truncation with generation of toxic fragments-nor the potential neuroprotective action induced by their in vivo clearance have been investigated in the context of AD retinal degeneration. We have recently developed a monoclonal tau antibody (12A12mAb) which selectively targets the neurotoxic 20-22 kDa NH2-derived peptide generated from pathological truncation at the N-terminal domain of tau without cross-reacting with its full-length normal protein. Previous studies have shown that 12A12mAb, when intravenously (i.v.)-injected into 6-month-old Tg2576 animals, markedly improves their AD-like, behavioural and neuropathological syndrome. By taking advantage of this well-established tau-directed immunization regimen, we found that 12A12mAb administration also exerts a beneficial action on biochemical, morphological and metabolic parameters (i.e. APP/Aß processing, tau hyperphosphorylation, neuroinflammation, synaptic proteins, microtubule stability, mitochondria-based energy production, neuronal death) associated with ocular injury in the AD phenotype. These findings prospect translational implications in the AD field by: (1) showing for the first time that cleavage of tau takes part in several pathological changes occurring in vivo in affected retinas and vitreous bodies and that its deleterious effects are successfully antagonized by administration of the specific 12A12mAb; (2) shedding further insights on the tight connections between neurosensory retina and brain, in particular following tau-based immunotherapy. In our view, the parallel response we detected in this preclinical animal model, both in the eye and in the hippocampus, following i.v. 12A12mAb injection opens novel diagnostic and therapeutic avenues for the clinical management of cerebral and extracerebral AD signs in human beings.

Diatom extraction: A new technique with heated H2O2. A technical note.

The best method of diatom identification in animal and human tissues is still an important discussion topic, in terms of effectiveness and reliability. In this technical note, authors propose a new method of extraction of diatoms using heated hydrogen peroxide from animal and human tissue samples. This method has been compared with the traditional method of digestion with acids. The results of the comparison show that heated hydrogen peroxide extraction is more efficient in terms of reduction of sediment, extraction of the material and preservation of diatoms proving to be a viable alternative to conventional approaches with acids in terms of costs and operator safety.

Profile of gamma-delta (γδ) T lymphocytes in the peripheral blood of crossbreed dogs during stages of life and implication in aging.

BACKGROUND: Data on gamma-delta (γδ) T lymphocytes in the peripheral blood of dogs are scant, related only to healthy pure breed dogs and limited to a restricted age range. The aim of the study was to investigate the modulation of the γδ T lymphocyte (TCRγδ+) subpopulation in peripheral blood of crossbreed healthy dogs according to five identified stages of life: Puppy, Junior, Adult, Mature, Senior and to determine its implication in aging. A rigorous method of recruitment was used to minimize the influence of internal or external pressure on the immune response. Twenty-three intact female and twenty-four intact male dogs were enrolled. Blood samples were collected and immunophenotyping of peripheral blood T lymphocytes and γδ T cell subpopulations was performed. RESULTS: The percentage of γδ T cells in peripheral blood lymphocytes was comparable with the value of 2.5% published by Faldyna and co-workers (2001), despite the percentage reported was investigated in less arranged age range groups and coming from four different dog pure breeds, whereas our data were recorded on wider age range groups and coming from crossbreed dogs. Therefore, the γδ T cell percentage (2.5%) is consistent and points out that such value is breed-independent. Statistical analysis highlighted differences in both percentage and absolute γδ T cells according to the stage of life. γδ T cells decreased significantly in the peripheral blood of elder dogs (Senior group) in comparison with previous stages of life (Puppy, Junior, and Adult groups). Differences in γδ T cells are significant and they are reported, for the first time, related to dog aging. CONCLUSIONS: The study confirms dogs to be among the animals with a low TCRγδ+ cell profile. A decrease of the TCRγδ+ subpopulation percentage was observed in elder dogs. TCRγδ+ cells of group S were different from those of groups P, J, and A. The differences are reported for the first time in dog aging. Identifying the stage of life when the decrease of γδ T lymphocytes starts can be useful for providing a rationale for drafting a wellness plan trial to support thymus immune functions and mitigate its functional exhaustion.

Regulation of Innate Lymphoid Cells in Acute Kidney Injury: Crosstalk between Cannabidiol and GILZ.

Innate lymphoid cells (ILCs) have emerged as largely tissue-resident archetypal cells of the immune system. We tested the hypotheses that renal ischemia-reperfusion injury (IRI) is a contributing factor to polarization of ILCs and that glucocorticoid-induced leucine zipper (GILZ) and cannabidiol regulate them in this condition. Mice subjected to unilateral renal IRI were treated with the following agents before restoration of renal blood flow: cannabidiol, DMSO, transactivator of transcription- (TAT-) GILZ, or the TAT peptide. Thereafter, kidney cells were prepared for flow cytometry analyses. Sham kidneys treated with either cannabidiol or TAT-GILZ displayed similar frequencies of each subset of ILCs compared to DMSO or TAT, respectively. Renal IRI increased ILC1s and ILC3s but reduced ILC2s compared to the sham group. Cannabidiol or TAT-GILZ treatment of IRI kidneys reversed this pattern as evidenced by reduced ILC1s and ILC3s but increased ILC2s compared to their DMSO- or TAT-treated counterparts. While TAT-GILZ treatment did not significantly affect cells positive for cannabinoid receptors subtype 2 (CB2+), cannabidiol treatment increased frequency of both CB2+ and GILZ-positive (GILZ+) cells of IRI kidneys. Subsequent studies showed that IRI reduced GILZ+ subsets of ILCs, an effect less marked for ILC2s. Treatment with cannabidiol increased frequencies of each subset of GILZ+ ILCs, but the effect was more marked for ILC2s. Indeed, cannabidiol treatment increased CB2+ GILZ+ ILC2s. Collectively, the results indicate that both cannabidiol and GILZ regulate ILC frequency and phenotype, in acute kidney injury, and that the effects of cannabidiol likely relate to modulation of endogenous GILZ.

Synaptic Correlates of Anterograde Amnesia and Intact Retrograde Memory in a Mouse Model of Alzheimer's Disease.

BACKGROUND: Clinical evidence indicates that patients affected by Alzheimer's Disease (AD) fail to form new memories although their memories for old events are intact. This amnesic pattern depends on the selective vulnerability to AD-neurodegeneration of the hippocampus, the brain region that sustains the formation of new memories, while cortical regions that store remote memories are spared. OBJECTIVE: To identify the cellular mechanisms underlying impaired recent memories and intact remote memories in a mouse model of AD. METHODS: Glutamatergic synaptic currents were recorded by patch-clamp in acute hippocampal and anterior Cingulate Cortical (aCC) slices of AD-like Tg2576 mice and Wild-type (Wt) littermates subjected to the Contextual Fear Conditioning (CFC) task or in naïve conditions. RESULTS: We identified a deficit in the formation of recent memories, but not in the recall of remote ones, in Tg2576 mice. With electrophysiological recordings, we detected CFC-induced modifications of the AMPA/NMDA ratio in CA1 pyramidal cells of Wt, but not Tg2576, mice one day after training. CFC-induced changes in the AMPA/NMDA ratio were also detected in the aCC of both Wt and Tg2576 mice 8 days after training. CONCLUSION: Our data suggest that in the early AD stages synaptic plasticity of CA1 synapses, crucial to form new memories, is lost, while plasticity of aCC synapses is intact and contributes to the persistence of long-term memories.

Glucocorticoid-Induced Leucine Zipper Promotes Neutrophil and T-Cell Polarization with Protective Effects in Acute Kidney Injury.

The glucocorticoid-induced leucine zipper (GILZ) mediates anti-inflammatory effects of glucocorticoids. Acute kidney injury (AKI) mobilizes immune/inflammatory mechanisms, causing tissue injury, but the impact of GILZ in AKI is not known. Neutrophils play context-specific proinflammatory [type 1 neutrophil (N1)] and anti-inflammatory [type 2 neutrophil (N2)] functional roles. Also, regulatory T lymphocytes (Tregs) and regulatory T-17 (Treg17) cells exert counterinflammatory effects, including the suppression of effector T lymphocytes [e.g., T-helper (Th) 17 cells]. Thus, utilizing cell preparations of mice kidneys subjected to AKI or sham operation, we determined the effects of GILZ on T cells and neutrophil subtypes in the context of its renoprotective effect; these studies used the transactivator of transcription (TAT)-GILZ or the TAT peptide. AKI increased N1 and Th-17 cells but reduced N2, Tregs, and Treg17 cells in association with increased interleukin (IL)-17+ but reduced IL-10+ cells accompanied with the disruption of mitochondrial membrane potential (ψ m) and increased apoptosis/necrosis compared with sham kidneys. TAT-GILZ, compared with TAT, treatment reduced N1 and Th-17 cells but increased N2 and Tregs, without affecting Treg17 cells, in association with a reduction in IL-17+ cells but an increase in IL-10+ cells; TAT-GILZ caused less disruption of ψ m and reduced cell death in AKI. Importantly, TAT-GILZ increased perfusion of the ischemic-reperfused kidney but reduced tissue edema compared with TAT. Utilizing splenic T cells and bone marrow-derived neutrophils, we further showed marked reduction in the proliferation of Th cells in response to TAT-GILZ compared with response to TAT. Collectively, the results indicate that GILZ exerts renoprotection accompanied by the upregulation of the regulatory/suppressive arm of immunity in AKI, likely via regulating cross talk between T cells and neutrophils.

Copeptin levels and kidney function in ADPKD: case-control study.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease. Studies have suggested a possible prognostic role of copeptin in determining the rate of progressive kidney function decline in ADPKD patients. However, it remains unresolved whether the changes in copeptin levels are specific for ADPKD or merely reflect a decline in glomerular filtration rate (GFR) regardless of the etiology of chronic kidney disease (CKD). METHODS: We performed a case-control study in ADPKD and non-ADPKD (control) patients. Patients were categorized based on the GFR-category (G-stage, KDIGO). We evaluated urea, creatinine, cystatin C, and copeptin in plasma and correlated these levels with estimated glomerular filtration rate (eGFR) (CKD-EPI). All p-values were two sided, and p < 0.05 was considered as statistically significant. RESULTS: We enrolled 112 ADPKD and 112 control patients. The median copeptin level was 10.72 (interquartile range (IQR) 5.21 - 26.21) pmol/L in the ADPKD group and 12.32 (IQR 4.47 - 30.73) pmol/L in the control group. The median copeptin level increased according to the G-stage in a progressive fashion and remained statistically significant across all G-stages and in both groups. Copeptin levels were not significantly different between ADPKD and control groups. We found a significant inverse correlation between copeptin level and eGFR (Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)) in the ADPKD, r = -0.81 (p < 0.001), and in the control group, r = -0.76 (p < 0.001). CONCLUSIONS: Copeptin levels seem to be strongly correlated with renal function rather than the presence of ADPKD. Further prospective studies need to evaluate its role as a prognostic marker in the early stage of CKD for ADPKD progression.

A General Procedure to Study Subcellular Models of Transsynaptic Signaling at Inhibitory Synapses.

Computational modeling of brain circuits requires the definition of many parameters that are difficult to determine from experimental findings. One way to help interpret these data is to fit them using a particular kinetic model. In this paper, we propose a general procedure to fit individual synaptic events recorded from voltage clamp experiments. Starting from any given model description (mod file) in the NEURON simulation environment, the procedure exploits user-defined constraints, dependencies, and rules for the parameters of the model to fit the time course of individual spontaneous synaptic events that are recorded experimentally. The procedure, implemented in NEURON, is currently available in ModelDB. A Python version is installed, and will be soon available for public use, as a standalone task in the Collaboratory Portal of the Human Brain Project. To illustrate the potential application of the procedure, we tested its use with various sets of experimental data on GABAergic synapses; gephyrin and gephyrin-dependent pathways were chosen as a suitable example of a kinetic model of synaptic transmission. For individual spontaneous inhibitory events in hippocampal pyramidal CA1 neurons, we found that gephyrin-dependent subcellular pathways may shape synaptic events at different levels, and can be correlated with cell- or event-specific activity history and/or pathological conditions.

State of the art of aldosterone immunoassays. A multicenter collaborative study on the behalf of the Cardiovascular Biomarkers Study Group of the Italian Section of European Society of Ligand Assay (ELAS) and Società Italiana di Biochimica Clinica (SIBIOC).

BACKGROUND: Two new immunoassay methods for aldosterone assay using automated platforms recently became available into market. The main aim of the present study is to evaluate the analytical performance of these automated direct immunoassay methods, and also to compare their analytical characteristics to those of the most popular RIA and EIA methods used in an Italian External Quality Assessment (EQA) study. METHODS: In this study analytical performances of two aldosterone immunoassays using the IDS iSYS and DiaSorin LIAISON fully automated platforms, were evaluated. Results obtained with the two platforms in EDTA plasma samples of healthy subjects and patients were compared with those obtained by RIA and EIA methods used in the Italian EQA scheme, named Immunocheck study. RESULTS: The two automated methods showed similar analytical performances: LoD 83.9 vs 92.2 pmol/L, LoQ 104.4 vs 111.1 pmol/L, respectively; moreover, the within-run and total imprecision values showed CV% between 8.1 and 14.1 for samples with 180.8 and 387.2 pmol/L concentration for both methods. There was a close linear regression between methods, however we found a significant proportional bias between LIAISON and iSYS methods. The EQA samples results obtained with these two methods were highly correlated to the consensus mean values. CONCLUSIONS: Our data indicate that aldosterone values measured with the two automated methods actually show better reproducibility, shorter laboratory Turn Around Time (TAT) and require less "hands on labor" compared to RIA and EIA immunoassays. However, in our study significant bias was observed in result comparison, this means that translating aldosterone concentration in clinical information an appropriate definition of reference ranges for each method is mandatory.

The novel partnership of L-GILZ and p53: a new affair in cancer?

A recent report from our laboratory reveals how long glucocorticoid-induced leucine zipper (L-Gilz) protein binds to p53 and mouse double minute 2 homolog (Mdm2), thus dissociating the p53/Mdm2 complex and activating p53 with subsequent activation of downstream genes p21 and p53 upregulated modulator of apoptosis (Puma). p53 activation appears to be the mechanism by which both basal and glucocorticoid (GC)-induced L-Gilz inhibits proliferation and induces antioncogenic activity in human cancer.

Effects of increasing CREB-dependent transcription on the storage and recall processes in a hippocampal CA1 microcircuit.

The involvement of the hippocampus in learning processes and major brain diseases makes it an ideal candidate to investigate possible ways to devise effective therapies for memory-related pathologies like Alzheimer's Disease (AD). It has been previously reported that augmenting CREB activity increases the synaptic Long-Term Potentiation (LTP) magnitude in CA1 pyramidal neurons and their intrinsic excitability in healthy rodents. It has also been suggested that hippocampal CREB signaling is likely to be down-regulated during AD, possibly degrading memory functions. Therefore, the concept of CREB-based memory enhancers, i.e. drugs that would boost memory by activation of CREB, has emerged. Here, using a model of a CA1 microcircuit, we investigate whether hippocampal CA1 pyramidal neuron properties altered by increasing CREB activity may contribute to improve memory storage and recall. With a set of patterns presented to a network, we find that the pattern recall quality under AD-like conditions is significantly better when boosting CREB function with respect to control. The results are robust and consistent upon increasing the synaptic damage expected by AD progression, supporting the idea that the use of CREB-based therapies could provide a new approach to treat AD.