Alpha1-acid glycoprotein post-translational modifications: a comparative two dimensional electrophoresis based analysis

Alpha1-acid glycoprotein (AGP) is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. A proteomic approach based on two dimensional electrophoresis, immunoblotting and staining of 2DE gels with dyes specific for post-translational modifications (PTMs) such as glycosylation and phosphorylation has been used to evaluate the differential interspecific protein expression of AGP purified from human, bovine and ovine sera. By means of these techniques, several isoforms have been identified in the investigated species: they have been found to change both with regard to the number of isoforms expressed under physiological condition and with regard to the quality of PTMs (i.e. different oligosaccharidic chains, presence/absence of phosphorilations). In particular, it is suggested that bovine serum AGP may have one of the most complex pattern of PTMs among serum proteins of mammals studied so far

Randomized crossover comparison of proportional assist ventilation and patient-triggered ventilation in extremely low birth weight infants with evolving chronic lung disease

Background: Refinement of ventilatory techniques remains a challenge given the persistence of chronic lung disease of preterm infants. Objective: To test the hypothesis that proportional assist ventilation ( PAV) will allow to lower the ventilator pressure at equivalent fractions of inspiratory oxygen (FiO(2)) and arterial hemoglobin oxygen saturation in ventilator-dependent extremely low birth weight infants in comparison with standard patient-triggered ventilation ( PTV). Methods: Design: Randomized crossover design. Setting: Two level-3 university perinatal centers. Patients: 22 infants ( mean (SD): birth weight, 705 g ( 215); gestational age, 25.6 weeks ( 2.0); age at study, 22.9 days ( 15.6)). Interventions: One 4- hour period of PAV was applied on each of 2 consecutive days and compared with epochs of standard PTV. Results: Mean airway pressure was 5.64 ( SD, 0.81) cm H2O during PAV and 6.59 ( SD, 1.26) cm H2O during PTV ( p < 0.0001), the mean peak inspiratory pressure was 10.3 ( SD, 2.48) cm H2O and 15.1 ( SD, 3.64) cm H2O ( p < 0.001), respectively. The FiO(2) ( 0.34 (0.13) vs. 0.34 ( 0.14)) and pulse oximetry readings were not significantly different. The incidence of arterial oxygen desaturations was not different ( 3.48 ( 3.2) vs. 3.34 ( 3.0) episodes/ h) but desaturations lasted longer during PAV ( 2.60 ( 2.8) vs. 1.85 ( 2.2) min of desaturation/ h, p = 0.049). PaCO2 measured transcutaneously in a subgroup of 12 infants was similar. One infant met prespecified PAV failure criteria. No adverse events occurred during the 164 cumulative hours of PAV application. Conclusions: PAV safely maintains gas exchange at lower mean airway pressures compared with PTV without adverse effects in this population. Backup conventional ventilation breaths must be provided to prevent apnea-related desaturations. Copyright (c) 2007 S. Karger AG, Base

Active focal segmental glomerulosclerosis is associated with massive oxidation of plasma albumin

The basic mechanism for idiopathic FSGS still is obscure. Indirect evidence in humans and generation of FSGS by oxidants in experimental models suggest a role of free radicals. In vitro studies demonstrate a main role of plasma albumin as antioxidant, its modification representing a chemical marker of oxidative stress. With the use of complementary liquid chromatography electron spray ionization tandem mass spectrometry (LC-ESI-MS/MS) and biochemical methods, plasma albumin was characterized in 34 patients with FSGS; 18 had received a renal transplant, and 17 had IgM mesangial deposition. Patients with FSGS that was in remission or without recurrence after transplantation had normal plasma albumin, and the same occurred in patients with primary and secondary nephrites and with chronic renal failure. In contrast, patients with active FSGS or with posttransplantation recurrence had oxidized plasma albumin. This finding was based on the characterization of albumin Cys 34 with an mass-to-charge ratio of 511.71 in triple charge that was consistent with the formation of a cysteic acid carrying a sulfonic group (alb-SO3-). The exact mass of albumin was increased accordingly (+48 Da) for incorporation of three oxygen radicals. Direct titration of the free sulfhydryl group 34 of plasma albumin and electrophoretic titration curves confirmed loss of free sulfhydryl group and formation of a fast-moving isoform in all cases with disease activity. This is the first demonstration of in vivo plasma albumin oxidation that was obtained with an adequate structural approach. Albumin oxidation seems to be specific for FSGS, suggesting some pathogenetic implications. Free radical involvement in FSGS may lead to specific therapeutic interventions

Vesicular glutamate release from feeder-free hiPSC-derived neurons

Human-induced pluripotent stem cells (hiPSCs) represent one of the main and powerful tools for the in vitro modeling of neurological diseases. Standard hiPSC-based protocols make use of animal-derived feeder systems to better support the neuronal differentiation process. Despite their efficiency, such protocols may not be appropriate to dissect neuronal specific properties or to avoid interspecies contaminations, hindering their future translation into clinical and drug discovery approaches. In this work, we focused on the optimization of a reproducible protocol in feeder-free conditions able to generate functional glutamatergic neurons. This protocol is based on a generation of neuroprecursor cells differentiated into human neurons with the administration in the culture medium of specific neurotrophins in a Geltrex-coated substrate. We confirmed the efficiency of this protocol through molecular analysis (upregulation of neuronal markers and neurotransmitter receptors assessed by gene expression profiling and expression of the neuronal markers at the protein level), morphological analysis, and immunfluorescence detection of pre-synaptic and post-synaptic markers at synaptic boutons. The hiPSC-derived neurons acquired Ca2+-dependent glutamate release properties as a hallmark of neuronal maturation. In conclusion, our study describes a new methodological approach to achieve feeder-free neuronal differentiation from hiPSC and adds a new tool for functional characterization of hiPSC-derived neurons

Characterization of Newcastle Disease Viruses Isolated from Cormorant and Gull Species in the United States in 2010

Newcastle disease virus (NDV), a member of the genus Avulavirus of the family Paramyxoviridae, is the causative agent of Newcastle disease (ND), a highly contagious disease that affects many species of birds and which frequently causes significant economic losses to the poultry industry worldwide. Virulent NDV (vNDV) is exotic in poultry in the United States; however, the virus has been frequently associated with outbreaks of ND in cormorants, which poses a significant threat to poultry species. Here, we present the characterization of 13 NDV isolates obtained from outbreaks of ND affecting cormorants and gulls in the states of Minnesota, Massachusetts, Maine, New Hampshire, and Maryland in 2010. All 2010 isolates are closely related to the viruses that caused the ND outbreaks in Minnesota in 2008, following the new evolutionary trend observed in cormorant NDV isolates since 2005. Similar to the results obtained with the 2008 isolates, the standard United States Department of Agriculture F-gene real-time reverse-transcription PCR (RRT-PCR) assay failed to detect the 2010 cormorant viruses, whereas all viruses were detected by a cormorant-specific F-gene RRTPCR assay. Notably, NDV-positive gulls were captured on the eastern shore of Maryland, which represents a significant geographic expansion of the virus since its emergence in North America. This is the first report of vNDV originating from cormorants isolated from wild birds in Maryland and, notably, the first time that genotype V vNDV has been isolated from multiple wild bird species in the United States. These findings highlight the need for constant epidemiologic surveillance for NDV in wild bird populations and for consistent biosecurity measures to prevent the introduction of the agent into domestic poultry flocks

Life events and hemodynamic stress reactivity in the middle-aged and elderly

Recent versions of the reactivity hypothesis, which consider it to be the product of stress exposure and exaggerated haemodynamic reactions to stress that confers cardiovascular disease risk, assume that reactivity is independent of the experience of stressful life events. This assumption was tested in two substantial cohorts, one middle-aged and one elderly. Participants had to indicate from a list of major stressful life events up to six they had experienced in the previous two years. They were also asked to rate how disruptive and stressful they were, at the time of occurrence and now. Blood pressure and pulse rate were measured at rest and in response to acute mental stress. Those who rated the events as highly disruptive at the time of exposure and currently exhibited blunted systolic blood pressure reactions to acute stress. The present results suggest that acute stress reactivity may not be independent of stressful life events experience

Sources of inter-individual variability leading to significant changes in anti-PD-1 and anti-PD-L1 efficacy identified in mouse tumor models using a QSP framework

While anti-PD-1 and anti-PD-L1 [anti-PD-(L)1] monotherapies are effective treatments for many types of cancer, high variability in patient responses is observed in clinical trials. Understanding the sources of response variability can help prospectively identify potential responsive patient populations. Preclinical data may offer insights to this point and, in combination with modeling, may be predictive of sources of variability and their impact on efficacy. Herein, a quantitative systems pharmacology (QSP) model of anti-PD-(L)1 was developed to account for the known pharmacokinetic properties of anti-PD-(L)1 antibodies, their impact on CD8+ T cell activation and influx into the tumor microenvironment, and subsequent anti-tumor effects in CT26 tumor syngeneic mouse model. The QSP model was sufficient to describe the variability inherent in the anti-tumor responses post anti-PD-(L)1 treatments. Local sensitivity analysis identified tumor cell proliferation rate, PD-1 expression on CD8+ T cells, PD-L1 expression on tumor cells, and the binding affinity of PD-1:PD-L1 as strong influencers of tumor growth. It also suggested that treatment-mediated tumor growth inhibition is sensitive to T cell properties including the CD8+ T cell proliferation half-life, CD8+ T cell half-life, cytotoxic T-lymphocyte (CTL)-mediated tumor cell killing rate, and maximum rate of CD8+ T cell influx into the tumor microenvironment. Each of these parameters alone could not predict anti-PD-(L)1 treatment response but they could shift an individual mouse’s treatment response when perturbed. The presented preclinical QSP modeling framework provides a path to incorporate potential sources of response variability in human translation modeling of anti-PD-(L)1

Mutations in PTRH2 cause novel infantile-onset multisystem disease with intellectual disability, microcephaly, progressive ataxia, and muscle weakness

OBJECTIVE: To identify the cause of a so-far unreported phenotype of infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD). METHODS: We characterized a consanguineous family of Yazidian-Turkish descent with IMNEPD. The two affected children suffer from intellectual disability, postnatal microcephaly, growth retardation, progressive ataxia, distal muscle weakness, peripheral demyelinating sensorimotor neuropathy, sensorineural deafness, exocrine pancreas insufficiency, hypothyroidism, and show signs of liver fibrosis. We performed whole-exome sequencing followed by bioinformatic analysis and Sanger sequencing on affected and unaffected family members. The effect of mutations in the candidate gene was studied in wild-type and mutant mice and in patient and control fibroblasts. RESULTS: In a consanguineous family with two individuals with IMNEPD, we identified a homozygous frameshift mutation in the previously not disease-associated peptidyl-tRNA hydrolase 2 (PTRH2) gene. PTRH2 encodes a primarily mitochondrial protein involved in integrin-mediated cell survival and apoptosis signaling. We show that PTRH2 is highly expressed in the developing brain and is a key determinant in maintaining cell survival during human tissue development. Moreover, we link PTRH2 to the mTOR pathway and thus the control of cell size. The pathology suggested by the human phenotype and neuroimaging studies is supported by analysis of mutant mice and patient fibroblasts. INTERPRETATION: We report a novel disease phenotype, show that the genetic cause is a homozygous mutation in the PTRH2 gene, and demonstrate functional effects in mouse and human tissues. Mutations in PTRH2 should be considered in patients with undiagnosed multisystem neurologic, endocrine, and pancreatic disease

Expanding Phenotype of Poirier\u2013Bienvenu Syndrome: New Evidence from an Italian Multicentrical Cohort of Patients

Background: Poirier\u2013Bienvenu Neurodevelopmental Syndrome (POBINDS) is a rare disease linked to mutations of the CSNK2B gene, which encodes for a subunit of caseinkinase CK2 involved in neuronal growth and synaptic transmission. Its main features include early-onset epilepsy and intellectual disability. Despite the lack of cases described, it appears that POBINDS could manifest with a wide range of phenotypes, possibly related to the different mutations of CSNK2B. Methods: Our multicentric, retrospective study recruited nine patients with POBINDS, detected using next-generation sequencing panels and whole-exome sequencing. Clinical, laboratory, and neuroimaging data were reported for each patient in order to assess the severity of phenotype, and eventually, a correlation with the type of CSNK2B mutation. Results: We reported nine unrelated patients with heterozygous de novo mutations of the CSNK2B gene. All cases presented epilepsy, and eight patients were associated with a different degree of intellectual disability. Other features detected included endocrinological and vascular abnormalities and dysmorphisms. Genetic analysis revealed six new variants of CSNK2B that have not been reported previously. Conclusion: Although it was not possible to assess a genotype\u2013phenotype correlation in our patients, our research further expands the phenotype spectrum of POBINDS patients, identifying new mutations occurring in the CSNK2B gene

Genetics of intellectual disability in consanguineous families

Autosomal recessive (AR) gene defects are the leading genetic cause of intellectual disability (ID) in countries with frequent parental consanguinity, which account for about 1/7th of the world population. Yet, compared to autosomal dominant de novo mutations, which are the predominant cause of ID in Western countries, the identification of AR-ID genes has lagged behind. Here, we report on whole exome and whole genome sequencing in 404 consanguineous predominantly Iranian families with two or more affected offspring. In 219 of these, we found likely causative variants, involving 77 known and 77 novel AR-ID (candidate) genes, 21 X-linked genes, as well as 9 genes previously implicated in diseases other than ID. This study, the largest of its kind published to date, illustrates that high-throughput DNA sequencing in consanguineous families is a superior strategy for elucidating the thousands of hitherto unknown gene defects underlying AR-ID, and it sheds light on their prevalence