Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 ( unit-cell parameters a = b = 136.83, c = 99.82 angstrom, gamma = 120 degrees). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 angstrom resolution using the laboratory X-ray source and are suitable for crystal structure determination.

Solution structure and novel insights into the determinants of the receptor specificity of human relaxin-3

Relaxin- 3 is the most recently discovered member of the relaxin family of peptide hormones. In contrast to relaxin- 1 and - 2, whose main functions are associated with pregnancy, relaxin- 3 is involved in neuropeptide signaling in the brain. Here, we report the solution structure of human relaxin- 3, the first structure of a relaxin family member to be solved by NMR methods. Overall, relaxin- 3 adopts an insulin- like fold, but the structure differs crucially from the crystal structure of human relaxin- 2 near the B- chain terminus. In particular, the B- chain C terminus folds back, allowing Trp(B27) to interact with the hydrophobic-core. This interaction partly blocks the conserved RXXXRXXI motif identified as a determinant for the interaction with the relaxin receptor LGR7 and may account for the lower affinity of relaxin- 3 relative to relaxin for this receptor. This structural feature is likely important for the activation of its endogenous receptor, GPCR135.

Molecular cloning and characterization of the mouse Na+ sulfate cotransporter gene (Slc13a4): structure and expression

Sulfate is an essential ion required for numerous functions in mammalian physiology. Due to its hydrophilic nature, cells require sulfate transporters on their plasma membranes to allow entry of sulfate into cells. In this study, we identified a new mouse Na+-sulfate cotransporter (mNaS2), characterized its tissue distribution and determined its cDNA and gene (Slc13a4) structures. mNaS2 mRNA was expressed in placenta, brain, lung, eye, heart, testis, thymus and liver. The mouse NaS2 cDNA spans 3384 nucleotides and its open frame encodes a protein of 624 amino acids. Slc13a4 maps to mouse chromosome 6131 and contains 16 exons, spanning over 40 kb in length. Its 5'-flanking region contains CART- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, MTF-1, STAT6 and HNF4 consensus sequences. This is the first study to define the tissue distribution of mNaS2 and resolve its cDNA and gene structures, which will allow us to investigate mNaS2 gene expression in vivo and determine its role in mammalian physiology.

myo-inositol pentakisphosphates. Structure, biological occurrence and phosphorylation to myo-inositol hexakisphosphate

1. Standard and high-performance anion-exchange-chromatographic techniques have been used to purify myo-[3H]inositol pentakisphosphates from various myo-[3H]inositol-prelabelled cells. Slime mould (Dictyostelium discoideum) contained 8 microM-myo-[3H]inositol 1,3,4,5,6-pentakisphosphate 16 microM-myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and 36 microM-D-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate [calculated intracellular concentrations; Stephens & Irvine (1990) Nature (London) 346 580-583]; germinating mung-bean (Phaseolus aureus) seedlings contained both D- and L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate (which was characterized by 31P and two-dimensional proton n.m.r.) and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate; HL60 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 500-fold excess over the other species), myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate; and NG-115-401L-C3 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 100-fold excess over the other species), D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate, myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate. 2. Multiple soluble ATP-dependent myo-inositol pentakisphosphate kinase activities have been detected in slime mould, rat brain and germinating mung-bean seedling homogenates. In slime-mould cytosolic fractions, the three myo-inositol pentakisphosphates that were present in intact slime moulds could be phosphorylated to myo-[3H]inositol hexakisphosphate: the relative first-order rate constants for these reactions were, in the order listed above, 1:8:31 respectively (with first-order rate constants in the intact cell of 0.1, 0.8 and 3.1 s-1, assuming a cytosolic protein concentration of 50 mg/ml), and the Km values of the activities for their respective inositol phosphate substrates (in the presence of 5 mM-ATP) were 1.6 microM, 3.8 microM and 1.4 microM. At least two forms of myo-inositol pentakisphosphate kinase activity could be resolved from a slime-mould cytosolic fraction by both pharmacological and chromatographic criteria. Rat brain cytosol and a soluble fraction derived from germinating mung-bean seedlings could phosphorylate myo-inositol D/L-1,2,4,5,6-, D/L-1,2,3,4,5-, 1,2,3,4,6- and 1,3,4,5,6-pentakisphosphates to myo-inositol hexakisphosphate: the relative first-order rate constants were 57:27:77:1 respectively for brain cytosol (with first-order rate constants in the intact cell of 0.0041, 0.0019, 0.0056 and 0.000073 s-1 respectively, assuming a cytosolic protein concentration of 50 mg/ml) and 1:11:12:33 respectively for mung-bean cytosol (with first-order rate constants in a supernatant fraction with a protein concentration of 10 mg/ml of 0.0002, 0.0022, 0.0024 and 0.0066 s-1 respectively).

The triune brain in antiquity: Plato, Aristotle, Erasistratus

Tripartite neuropsychologies have featured through two and half millennia of Western thought. They received a modern airing in Paul MacLean's well-known text The Triune Brain. This paper examines the origin of these triune psychophysiologies. It is argued that the first such psychophysiology was developed in the fifth century BCE in the Republic and its Pythagorean sequel, the Timaeus. Aristotle, Plato's pupil and colleague, developed a somewhat similar theory, though this time based on his exhaustive biological researches. Finally, a generation later, Herophilus and Erasistratus at the Alexandrian Museum put together a more anatomically informed tripartite theory that, somewhat modified by Galen in the second century AD, remained the prevailing orthodoxy for nearly fifteen hundred years until it was overturned by the great figures of the Renaissance. Nonetheless, as already mentioned, the notion that human neuropsychology is somehow best thought of as having a tripartite structure has remained remarkably resilient and has reappeared time and again in modern and early modern times. This paper investigates its origins and suggests that it is perhaps now time to move on.

The human hippocampus:structure, function and its significance in neuropsychology and disease

This article discusses the structure, anatomical connections, and functions of the hippocampus (HC) of the human brain and its significance in neuropsychology and disease. The HC is concerned with the analysis of highly abstract data derived from all sensory systems but its specific role remains controversial. Hence, there have been three major theories concerning its function, viz., the memory theory, the spatial theory, and the behavioral inhibition system (BIS) theory. The memory theory has its origin in the surgical destruction of the HC, which results in severe anterograde and partial retrograde amnesia. The spatial theory has its origin in the observation that neurons in the HC of animals show activity related to their location within the environment. By contrast, the behavioral inhibition theory suggests that the HC acts as a 'comparator', i.e., it compares current sensory events with expected or predicted events. If a set of expectations continues to be verified then no alteration of behavior occurs. If, however, a 'mismatch' is detected then the HC intervenes by initiating appropriate action by active inhibition of current motor programs and initiation of new data gathering. Understanding the anatomical connections of the hippocampus may lead to a greater understanding of memory, spatial orientation, and states of anxiety in humans. In addition, HC damage is a feature of neurodegenerative diseases such as Alzheimer's disease (AD), dementia with Lewy bodies (DLB), Pick's disease (PiD), and Creutzfeldt-Jakob disease (CJD) and understanding HC function may help to explain the development of clinical dementia in these disorders.

Fractal descriptors for quantifying brain complexity in MRI

Magnetic Resonance Imaging (MRI) is the in vivo technique most commonly employed to characterize changes in brain structures. The conventional MRI-derived morphological indices are able to capture only partial aspects of brain structural complexity. Fractal geometry and its most popular index, the fractal dimension (FD), can characterize self-similar structures including grey matter (GM) and white matter (WM). Previous literature shows the need for a definition of the so-called fractal scaling window, within which each structure manifests self-similarity. This justifies the existence of fractal properties and confirms Mandelbrot’s assertion that "fractals are not a panacea; they are not everywhere". In this work, we propose a new approach to automatically determine the fractal scaling window, computing two new fractal descriptors, i.e., the minimal and maximal fractal scales (mfs and Mfs). Our method was implemented in a software package, validated on phantoms and applied on large datasets of structural MR images. We demonstrated that the FD is a useful marker of morphological complexity changes that occurred during brain development and aging and, using ultra-high magnetic field (7T) examinations, we showed that the cerebral GM has fractal properties also below the spatial scale of 1 mm. We applied our methodology in two neurological diseases. We observed the reduction of the brain structural complexity in SCA2 patients and, using a machine learning approach, proved that the cerebral WM FD is a consistent feature in predicting cognitive decline in patients with small vessel disease and mild cognitive impairment. Finally, we showed that the FD of the WM skeletons derived from diffusion MRI provides complementary information to those obtained from the FD of the WM general structure in T1-weighted images. In conclusion, the fractal descriptors of structural brain complexity are candidate biomarkers to detect subtle morphological changes during development, aging and in neurological diseases.

“Nanoglial interfaces: nanostructured materials, interfaces and devices to unveil the role of astrocytes in brain function and dysfunction”

The role of non-neuronal brain cells, called astrocytes, is emerging as crucial in brain function and dysfunction, encompassing the neurocentric concept that was envisioning glia as passive components. Ion and water channels and calcium signalling, expressed in functional micro and nano domains, underpin astrocytes’ homeostatic function, synaptic transmission, neurovascular coupling acting either locally and globally. In this respect, a major issue arises on the mechanism through which astrocytes can control processes across scales. Finally, astrocytes can sense and react to extracellular stimuli such as chemical, physical, mechanical, electrical, photonic ones at the nanoscale. Given their emerging importance and their sensing properties, my PhD research program had the general goal to validate nanomaterials, interfaces and devices approaches that were developed ad-hoc to study astrocytes. The results achieved are reported in the form of collection of papers. Specifically, we demonstrated that i) electrospun nanofibers made of polycaprolactone and polyaniline conductive composites can shape primary astrocytes’ morphology, without affecting their function ii) gold coated silicon nanowires devices enable extracellular recording of unprecedented slow wave in primary differentiated astrocytes iii) colloidal hydrotalcites films allow to get insight in cell volume regulation process in differentiated astrocytes and to describe novel cytoskeletal actin dynamics iv) gold nanoclusters represent nanoprobe to trigger astrocytes structure and function v) nanopillars of photoexcitable organic polymer are potential tool to achieve nanoscale photostimulation of astrocytes. The results were achieved by a multidisciplinary team working with national and international collaborators that are listed and acknowledged in the text. Collectively, the results showed that astrocytes represent a novel opportunity and target for Nanoscience, and that Nanoglial interface might help to unveil clues on brain function or represent novel therapeutic approach to treat brain dysfunctions.

Synthesis and characterization of novel selective NKCC1 inhibitors for the treatment of down syndrome and brain disorders characterized by depolarizing GABAergic transmission.

Proper GABAergic transmission through Cl-permeable GABAA receptors is fundamental for physiological brain development and function. Indeed, defective GABAergic signaling – due to a high NKCC1/KCC2 expression ratio – has been implicated in several neurodevelopmental disorders (e.g., Down syndrome, DS, Autism spectrum disorders, ASD). Interestingly, NKCC1 inhibition by the FDA-approved diuretic drug bumetanide reverts cognitive deficits in the TS65Dn mouse models of DS and core symptoms in other models of brain disorders. However, the required chronic treatment with bumetanide is burdened by its diuretic side effects caused by the antagonization of the kidney Cl importer NKCC2. This may lead to hypokalemia, while jeopardizing drug compliance. Crucially, these issues would be solved by selective NKCC1 inhibitors, thus devoid of the diuretic effect of bumetanide. To this aim, starting from bumetanide’s structure, we applied a ligand-based computational approach to design new molecular entities that we tested in vitro for their capacity to selectively block NKCC1. Extensive synthetic efforts and structure-activity relationships analyses allowed us to improve in vitro potency and overall drug-like properties of the initially identified chemical hits. As a result, we identified a new highly potent NKCC1 inhibitor (ARN23746) that displayed excellent solubility, metabolic stability, and no significant effect on NKCC2 in vitro. Moreover, this novel and selective NKCC1 inhibitor was able to rescue cognitive deficits in DS mice and social/repetitive behaviors in ASD mice, with no diuretic effect and no overt toxicity upon chronic treatment in adult animals. Thus, ARN23746 a selective NKCC1 inhibitor devoid of the diuretic effect – represents a suitable and solid therapeutic strategy for the treatment of Down syndrome and all the brain neurological disorders characterized by depolarizing GABAergic transmission.

A study of brain functional connectivity and cognitive processes in long COVID-19 patients

In this work, a prospective study conducted at the IRCCS Istituto delle Scienze Neurologiche di Bologna is presented. The aim was to investigate the brain functional connectivity of a cohort of patients (N=23) suffering from persistent olfactory dysfunction after SARS-CoV-2 infection (Post-COVID-19 syndrome), as compared to a matching group of healthy controls (N=26). In particular, starting from individual resting state functional-MRI data, different analytical approaches were adopted in order to find potential alterations in the connectivity patterns of patients’ brains. Analyses were conducted both at a whole-brain level and with a special focus on brain regions involved in the processing of olfactory stimuli (Olfactory Network). Statistical correlations between functional connectivity alterations and the results of olfactory and neuropsychological tests were investigated, to explore the associations with cognitive processes. The three approaches implemented for the analysis were the seed-based correlation analysis, the group-level Independent Component analysis and a graph-theoretical analysis of brain connectivity. Due to the relative novelty of such approaches, many implementation details and methodologies are not standardized yet and represent active research fields. Seed-based and group-ICA analyses’ results showed no statistically significant differences between groups, while relevant alterations emerged from those of the graph-based analysis. In particular, patients’ olfactory sub-graph appeared to have a less pronounced modular structure compared to the control group; locally, a hyper-connectivity of the right thalamus was observed in patients, with significant involvement of the right insula and hippocampus. Results of an exploratory correlation analysis showed a positive correlation between the graphs global modularity and the scores obtained in olfactory tests and negative correlations between the thalamus hyper-connectivity and memory tests scores.

Changes In Chromatin Structure In Nih 3t3 Cells Induced By Valproic Acid And Trichostatin A.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA.

Feasibility And Reproducibility Of Diffusion-weighted Magnetic Resonance Imaging Of The Fetal Brain In Twin-twin Transfusion Syndrome.

The aim of this study is to test the feasibility and reproducibility of diffusion-weighted magnetic resonance imaging (DW-MRI) evaluations of the fetal brains in cases of twin-twin transfusion syndrome (TTTS). From May 2011 to June 2012, 24 patients with severe TTTS underwent MRI scans for evaluation of the fetal brains. Datasets were analyzed offline on axial DW images and apparent diffusion coefficient (ADC) maps by two radiologists. The subjective evaluation was described as the absence or presence of water diffusion restriction. The objective evaluation was performed by the placement of 20-mm(2) circular regions of interest on the DW image and ADC maps. Subjective interobserver agreement was assessed by the kappa correlation coefficient. Objective intraobserver and interobserver agreements were assessed by proportionate Bland-Altman tests. Seventy-four DW-MRI scans were performed. Sixty of them (81.1%) were considered to be of good quality. Agreement between the radiologists was 100% for the absence or presence of diffusion restriction of water. For both intraobserver and interobserver agreement of ADC measurements, proportionate Bland-Altman tests showed average percentage differences of less than 1.5% and 95% CI of less than 18% for all sites evaluated. Our data demonstrate that DW-MRI evaluation of the fetal brain in TTTS is feasible and reproducible.

Evidences Of Endocytosis Via Caveolae Following Blood-brain Barrier Breakdown By Phoneutria Nigriventer Spider Venom.

Spider venoms contain neurotoxic peptides aimed at paralyzing prey or for defense against predators; that is why they represent valuable tools for studies in neuroscience field. The present study aimed at identifying the process of internalization that occurs during the increased trafficking of vesicles caused by Phoneutria nigriventer spider venom (PNV)-induced blood-brain barrier (BBB) breakdown. Herein, we found that caveolin-1α is up-regulated in the cerebellar capillaries and Purkinje neurons of PNV-administered P14 (neonate) and 8- to 10-week-old (adult) rats. The white matter and granular layers were regions where caveolin-1α showed major upregulation. The variable age played a role in this effect. Caveolin-1 is the central protein that controls caveolae formation. Caveolar-specialized cholesterol- and sphingolipid-rich membrane sub-domains are involved in endocytosis, transcytosis, mechano-sensing, synapse formation and stabilization, signal transduction, intercellular communication, apoptosis, and various signaling events, including those related to calcium handling. PNV is extremely rich in neurotoxic peptides that affect glutamate handling and interferes with ion channels physiology. We suggest that the PNV-induced BBB opening is associated with a high expression of caveolae frame-forming caveolin-1α, and therefore in the process of internalization and enhanced transcytosis. Caveolin-1α up-regulation in Purkinje neurons could be related to a way of neurons to preserve, restore, and enhance function following PNV-induced excitotoxicity. The findings disclose interesting perspectives for further molecular studies of the interaction between PNV and caveolar specialized membrane domains. It proves PNV to be excellent tool for studies of transcytosis, the most common form of BBB-enhanced permeability.

Matrix Metalloproteinases And Left Ventricular Function And Structure In Spinal Cord Injured Subjects.

Subjects with spinal cord injury (SCI) exhibit impaired left ventricular (LV) diastolic function, which has been reported to be attenuated by regular physical activity. This study investigated the relationship between circulating matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) and echocardiographic parameters in SCI subjects and the role of physical activity in this regard. Forty-two men with SCI [19 sedentary (S-SCI) and 23 physically-active (PA-SCI)] were evaluated by clinical, anthropometric, laboratory, and echocardiographic analysis. Plasmatic pro-MMP-2, MMP-2, MMP-8, pro-MMP-9, MMP-9, TIMP-1 and TIMP-2 levels were determined by enzyme-linked immunosorbent assay and zymography. PA-SCI subjects presented lower pro-MMP-2 and pro-MMP-2/TIMP-2 levels and improved markers of LV diastolic function (lower E/Em and higher Em and E/A values) than S-SCI ones. Bivariate analysis showed that pro-MMP-2 correlated inversely with Em and directly with E/Em, while MMP-9 correlated directly with LV mass index and LV end-diastolic diameter in the whole sample. Following multiple regression analysis, pro-MMP-2, but not physical activity, remained associated with Em, while MMP-9 was associated with LV mass index in the whole sample. These findings suggest differing roles for MMPs in LV structure and function regulation and an interaction among pro-MMP-2, diastolic function and physical activity in SCI subjects.

Growth Factor Stimulation Improves The Structure And Properties Of Scaffold-free Engineered Auricular Cartilage Constructs.

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.