Effect of cultivation conditions on the structure and morphological properties of BNC biomaterials with a focus on vascular grafts

20 New materials that are not thrombogenic and have mechanical properties that mimic the native blood vessel are in very great demand. Nanocellulose produced by the bacteria Gluconacetobacter xylinus is a biomaterial that has gained interest in the field of tissue engineering because of its unique properties, such as great mechanical strength, high water content (around 99%), and the ability to be shaped into three-dimensional structures during biosynthesis. The fabrication process of bacterial nanocellulose (BNC) vascular grafts is very unique because the material synthesis and product formation takes place simultaneously. The bio mechanical performance, which includes rupture pressure and compliance along with biological response (endothelialization, blood compatibility, etc.), is dependent on the morphology of a fibrillar network. The network formation is affected by cellulose assembly and bacteria motion, proliferation rate, and other factors. An understanding of the effects of cultivation conditions on BNC network formation is therefore of great importance

Treatment temperature and insult severity influence the neuroprotective effects of therapeutic hypothermia

Therapeutic hypothermia (HT) is standard care for moderate and severe neonatal hypoxic-ischaemic encephalopathy (HIE), the leading cause of permanent brain injury in term newborns. However, the optimal temperature for HT is still unknown, and few preclinical studies have compared multiple HT treatment temperatures. Additionally, HT may not benefit infants with severe encephalopathy. In a neonatal rat model of unilateral hypoxia-ischaemia (HI), the effect of five different HT temperatures was investigated after either moderate or severe injury. At postnatal-day seven, rat pups underwent moderate or severe HI followed by 5 h at normothermia (37 °C), or one of five HT temperatures: 33.5 °C, 32 °C, 30 °C, 26 °C, and 18 °C. One week after treatment, neuropathological analysis of hemispheric and hippocampal area loss, and CA1 hippocampal pyramidal neuron count, was performed. After moderate injury, a significant reduction in hemispheric and hippocampal loss on the injured side, and preservation of CA1 pyramidal neurons, was seen in the 33.5 °C, 32 °C, and 30 °C groups. Cooling below 33.5 °C did not provide additional neuroprotection. Regardless of treatment temperature, HT was not neuroprotective in the severe HI model. Based on these findings, and previous experience translating preclinical studies into clinical application, we propose that milder cooling should be considered for future clinical trials

HAMLET Interacts with Lipid Membranes and Perturbs Their Structure and Integrity

Background Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. Methodology/Principal Findings We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. Conclusions/Significance The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death

Navigating the Murine Brain: Toward Best Practices for Determining and Documenting Neuroanatomical Locations in Experimental Studies

In experimental neuroscientific research, anatomical location is a key attribute of experimental observations and critical for interpretation of results, replication of findings, and comparison of data across studies. With steadily rising numbers of publications reporting basic experimental results, there is an increasing need for integration and synthesis of data. Since comparison of data relies on consistently defined anatomical locations, it is a major concern that practices and precision in the reporting of location of observations from different types of experimental studies seem to vary considerably. To elucidate and possibly meet this challenge, we have evaluated and compared current practices for interpreting and documenting the anatomical location of measurements acquired from murine brains with different experimental methods. Our observations show substantial differences in approach, interpretation and reproducibility of anatomical locations among reports of different categories of experimental research, and strongly indicate that ambiguous reports of anatomical location can be attributed to missing descriptions. Based on these findings, we suggest a set of minimum requirements for documentation of anatomical location in experimental murine brain research. We furthermore demonstrate how these requirements have been applied in the EU Human Brain Project to optimize workflows for integration of heterogeneous data in common reference atlases. We propose broad adoption of some straightforward steps for improving the precision of location metadata and thereby facilitating interpretation, reuse and integration of data

Development of proteomic methods for studying cerebrospinal fluid proteins involved in Alzheimer\ub4s disease

Alzheimer\u27s disease (AD) is the most common cause of dementia in western countries. The main neuropathological findings in the AD brain are senile plaques, neurofibrillary tangles and degeneration of neurons and synapses. Although research on AD is progressing fast, the causes and mechanisms of this disease remain to be elucidated and development of new methods is necessary to study neuron-related proteins involved in the pathophysiological mechanisms. Six low-abundance synaptic proteins in human cerebrospinal fluid (CSF), namely rab 3a, synaptotagmin, synapsin, the presynaptic protein GAP-43, the synaptosomal-associated protein 25 and the postsynaptic protein neurogranin, were detected with liquid phase isoelectric focusing and immunoblotting. An ELISA method for quantification of the phosphorylated form (Ser 9) of synapsin I was developed. Increased levels of phosphosynapsin I were demonstrated in AD patients compared to controls. These results are consistent with the hypothesis of impaired protein phosphorylation mechanisms in AD. To purify and characterise proteins in CSF, a new strategy combining two-dimensional liquid phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was developed. Two brain-specific proteins, cystatin C and b2 microglobulin, were isolated from CSF in sufficient quantities for analysis by MALDI-TOF MS. Special attention was needed to make 2D-LPE and mass spectrometry compatible. Chloroform/methanol/water extraction was the most efficient method for SDS removal, allowing the acquisition of good quality MALDI spectra of the tryptic digest of the proteins analysed. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry have been used for clinical screening of disease-influenced CSF proteins in AD. In order to increase the detection of CSF proteins and to improve the separation of protein isoforms, micro-narrow range immobilised pH gradient strips and prefractionation prior to 2-DE of CSF were used. Previously detected protein changes by 2-DE, between AD patients and controls, such as apoliprotein E and apoliprotein A1, were confirmed. Several new protein changes were demonstrated, including kininogen, apoliprotein J, b-trace, 1 b glycoprotein, a 2-HS glycoprotein and a-1 antitrypsin. As shown in this study, different isoforms i.e. different states of glycosylated proteins, are altered in AD. Therefore, the determination of post-translational modifications such as glycosylation and phosphorylation, is of importance for an increased understanding of the neuropathology in AD. The use of complementary strategies in proteome studies of CSF offers new perspectives on the pathology in neurodegenerative diseases and also reveals new potential biomarkers for brain disorders such as AD

In Vitro Electrochemistry of Biological Systems

This article reviews recent work involving electrochemical methods for in vitro analysis of biomolecules, with an emphasis on detection and manipulation at and of single cells and cultures of cells. The techniques discussed include constant potential amperometry, chronoamperometry, cellular electroporation, scanning electrochemical microscopy, and microfluidic platforms integrated with electrochemical detection. The principles of these methods are briefly described, followed in most cases with a short description of an analytical or biological application and its significance. The use of electrochemical methods to examine specific mechanistic issues in exocytosis is highlighted, as a great deal of recent work has been devoted to this application

Validation of a prefractionation method followed by two-dimensional electrophoresis - Applied to cerebrospinal fluid proteins from frontotemporal dementia patients.

BACKGROUND: The aim of this study was firstly, to improve and validate a cerebrospinal fluid (CSF) prefractionation method followed by two-dimensional electrophoresis (2-DE) and secondly, using this strategy to investigate differences between the CSF proteome of frontotemporal dementia (FTD) patients and controls. From each subject three ml of CSF was prefractionated using liquid phase isoelectric focusing prior to 2-DE. RESULTS: With respect to protein recovery and purification potential, ethanol precipitation of the prefractionated CSF sample was found superior, after testing several sample preparation methods.The reproducibility of prefractionated CSF analyzed on 2-D gels was comparable to direct 2-DE analysis of CSF. The protein spots on the prefractionated 2-D gels had an increased intensity, indicating a higher protein concentration, compared to direct 2-D gels. Prefractionated 2-DE analysis of FTD and control CSF showed that 26 protein spots were changed at least two fold. Using mass spectrometry, 13 of these protein spots were identified, including retinol-binding protein, Zn-alpha-2-glycoprotein, proapolipoproteinA1, beta-2-microglobulin, transthyretin, albumin and alloalbumin. CONCLUSION: The results suggest that the prefractionated 2-DE method can be useful for enrichment of CSF proteins and may provide a new tool to investigate the pathology of neurodegenerative diseases. This study confirmed reduced levels of retinol-binding protein and revealed some new biomarker candidates for FTD