The impact of blood contamination on the proteome of cerebrospinal fluid

Human cerebrospinal fluid (CSF) is in direct contact with the brain extracellular space. Beside the secretion of CSF by the choroid plexus the fluid also derives directly from the brain by the ependymal lining of the ventricular system and the glial membrane and from blood vessels in the arachnoid. Therefore, biochemical change in the brain may be reflected in the CSF. CSF is a potential source of protein molecular indices of central nervous system function and pathology. However, various amounts of blood contamination in CSF may arise during sample acquisition. The concentration of protein in the CSF is only 0.2 to 0.5% that of blood. Minor contamination of CSF with blood during collection of the fluid may dramatically alter the protein profile confounding the identification of potential biomarkers. We have analyzed CSF and CSF spiked with increasing amounts of whole blood using proteomic techniques. We detected at least four blood specific highly abundant proteins: hemoglobin, catalase, peroxiredoxin and carbonic anhydrase I. These proteins can be used as blood contamination markers for proteomic analysis of CSF. Proteins in blood contaminated CSF samples were less stable compared to neat CSF at 37 degrees C suggesting that blood borne protease may induce protein degradation in CSF during sample acquisition. This analysis was aimed at identification of proteins found primarily in CSF, those found primarily in blood and assessment of the impact of blood contamination on those proteins found in both fluids.     

Novel Approach to Simulate Sleep Apnea Patients for Evaluating Positive Pressure Therapy Devices

Bench testing is a useful method to characterize the response of different automatic positive airway pressure (APAP) devices under well-controlled conditions. However, previous models did not consider the diversity of obstructive sleep apnea (OSA) patients’ characteristics and phenotypes. The objective of this proof-of-concept study was to design a new bench test for realistically simulating an OSA patient’s night, and to implement a one-night example of a typical female phenotype for comparing responses to several currently-available APAP devices. We developed a novel approach aimed at replicating a typical night of sleep which includes different disturbed breathing events, disease severities, sleep/wake phases, body postures and respiratory artefacts. The simulated female OSA patient example that we implemented included periods of wake, light sleep and deep sleep with positional changes and was connected to ten different APAP devices. Flow and pressure readings were recorded; each device was tested twice. The new approach for simulating female OSA patients effectively combined a wide variety of disturbed breathing patterns to mimic the response of a predefined patient type. There were marked differences in response between devices; only three were able to overcome flow limitation to normalize breathing, and only five devices were associated with a residual apnea-hypopnea index of <5/h. In conclusion, bench tests can be designed to simulate specific patient characteristics, and typical stages of sleep, body position, and wake. Each APAP device behaved differently when exposed to this controlled model of a female OSA patient, and should lead to further understanding of OSA treatment.     

Leptin-mediated ion channel regulation: PI3K pathways,physiological role,and therapeutic potential

Leptin is produced by adipose tissue and identified as a “satiety signal,” informing the brain when the body has consumed enough food. Specific areas of the hypothalamus express leptin receptors (LEPRs) and are the primary site of leptin action for body weight regulation. In response to leptin, appetite is suppressed and energy expenditure allowed. Beside this hypothalamic action, leptin targets other brain areas in addition to neuroendocrine cells. LEPRs are expressed also in the hippocampus, neocortex, cerebellum, substantia nigra, pancreatic β-cells, and chromaffin cells of the adrenal gland. It is intriguing how leptin is able to activate different ionic conductances, thus affecting excitability, synaptic plasticity and neurotransmitter release, depending on the target cell. Most of the intracellular pathways activated by leptin and directed to ion channels involve PI3K, which in turn phosphorylates different downstream substrates, although parallel pathways involve AMPK and MAPK. In this review we will describe the effects of leptin on BK, K_ATP, K_V, Ca_V, TRPC, NMDAR and AMPAR channels and clarify the landscape of pathways involved. Given the ability of leptin to influence neuronal excitability and synaptic plasticity by modulating ion channels activity, we also provide a short overview of the growing potentiality of leptin as therapeutic agent for treating neurological disorders.     

Antagonism of the Prokineticin System Prevents and Reverses Allodynia and Inflammation in a Mouse Model of Diabetes

Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease.     

Structure-selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern

Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to affect the quality of the antibody response focusing it to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and three variants of concern (VOC), B.1.351, B.1.617.2, and BA.1. We demonstrate that immunogens built on structure selection can be used to influence the quality of the antibody response by focusing it to a conserved site of vulnerability shared between wildtype virus and VOCs, resulting in neutralizing antibodies across variants.     

Comprehensive label-free method for the relative quantification of proteins from biological samples

Pharmaceutical companies and regulatory agencies are broadly pursuing biomarkers as a means to increase the productivity of drug development. Quantifying differential levels of proteins from complex biological samples such as plasma or cerebrospinal fluid is one specific approach being used to identify markers of drug action, efficacy, toxicity, etc. We have developed a comprehensive, fully automated, and label-free approach to relative protein quantification from LC-MS/MS experiments of proteolytic protein digests including: de-noising, mass and charge state estimation, chromatographic alignment, and peptide quantification via integration of extracted ion chromatograms. Results from a variance components study of the entire method indicate that most of the variability is attributable to the LC-MS injection, with a median peptide LC-MS injection coefficient of variation of 8% on a ThermoFinnigan LTQ mass spectrometer. Spiked recovery results suggest a quantifiable range of approximately 32-fold for a sample protein.     

Intersession reliability of a posturo-stabilometric test,using a force platform

Aim of the studyTo evaluate the intersession reliability of a posturo-stabilometric examination.MethodsSingle blind clinical trial conducted in two sessions over two weeks.44 healthy volunteers free from postural and temporomandibular disorders. All the subjects complied with the criteria for completing the study.All the subjects underwent two sessions of posturo-stabilometric examinations in different visual and mandibular conditions.Sway area, sway length and the coordinates of the center of pressure were evaluated and statistically analyzed using the Intraclass correlation coefficient (ICC).ResultsAll the posturo-stabilometric parameters seemed to have an excellent reproducibility with overall ICCs higher than 70% and good confidence intervals except for the sway area (ICC 0.422 with CI 0.283–0.560 with open eyes and ICC 0.554 with CI 0.424–0.683 with closed eyes).ConclusionsThe posturo-stabilometric examination carried out using a force platform has a good intrasession and intersession reliability, especially considering sway velocity, COP X and COP Y parameters. The force platform usefulness in analyzing static posture is confirmed in any medical field.     

Thrombin binding aptamer analogues containing inversion of polarity sites endowed with antiproliferative and anti-motility properties against Calu-6 cells

Background

Although the thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative properties, it is possible to reduce the first and enhance the second one by suitable chemical modifications.