Biological activity differences between TGF-β1 and TGF-β3 correlate with differences in the rigidity and arrangement of their component monomers

[Image: see text] TGF-β1, -β2, and -β3 are small, secreted signaling proteins. They share 71–80% sequence identity and signal through the same receptors, yet the isoform-specific null mice have distinctive phenotypes and are inviable. The replacement of the coding sequence of TGF-β1 with TGF-β3 and TGF-β3 with TGF-β1 led to only partial rescue of the mutant phenotypes, suggesting that intrinsic differences between them contribute to the requirement of each in vivo. Here, we investigated whether the previously reported differences in the flexibility of the interfacial helix and arrangement of monomers was responsible for the differences in activity by generating two chimeric proteins in which residues 54–75 in the homodimer interface were swapped. Structural analysis of these using NMR and functional analysis using a dermal fibroblast migration assay showed that swapping the interfacial region swapped both the conformational preferences and activity. Conformational and activity differences were also observed between TGF-β3 and a variant with four helix-stabilizing residues from TGF-β1, suggesting that the observed changes were due to increased helical stability and the altered conformation, as proposed. Surface plasmon resonance analysis showed that TGF-β1, TGF-β3, and variants bound the type II signaling receptor, TβRII, nearly identically, but had small differences in the dissociation rate constant for recruitment of the type I signaling receptor, TβRI. However, the latter did not correlate with conformational preference or activity. Hence, the difference in activity arises from differences in their conformations, not their manner of receptor binding, suggesting that a matrix protein that differentially binds them might determine their distinct activities

Discovery of Yttrium, Zirconium, Niobium, Technetium, and Ruthenium Isotopes

Currently, thirty-four yttrium, thirty-five zirconium, thirty-four niobium, thirty-five technetium, and thirty-eight ruthenium isotopes have been observed and the discovery of these isotopes is discussed here. For each isotope a brief synopsis of the first refereed publication, including the production and identification method, is presented.Comment: To be published in Atomic Data and Nuclear Data Table

Faecal Matrix Metalloprotease-9 Is a More Sensitive Marker For Diagnosing Pouchitis Than Faecal Calprotectin – Results From a Pilot Study

Background. Potential non-invasive markers of pouchitis would have a great deal of significance within clinical practice. This study is aimed at assessing the diagnostic accuracy of faecal calprotectin and matrix metalloprotease-9 as potential markers in patients both with and without pouchitis. Patients and methods. Stool and blood samples were collected from 33 ileal pouch-anal anastomosis patients before a follow-up pouchoscopy. Biopsy samples were taken for histological purposes. The presence of cuffitis and stenosis was evaluated with an endoscopy. Calprotectin and matrix metalloprotease-9 were quantified with an enzyme-linked immunosorbent assay. Results. Pouchitis was detected in 30.3% of the patients. The levels of faecal calprotectin and matrix metalloprotease-9 increased significantly in patients with pouchitis. The sensitivity and specificity of matrix metalloprotease-9 was higher than that of faecal calprotectin. Only matrix metalloprotease-9 correlated significantly with the severity of pouchitis. Conclusions. Faecal matrix metalloprotease-9 has a high specificity in the diagnosis of pouchitis

A Frameshift in CSF2RB Predominant Among Ashkenazi Jews Increases Risk for Crohn's Disease and Reduces Monocyte Signaling via GMCSF

BACKGROUND & AIMS: Crohn's disease (CD) has the highest prevalence in Ashkenazi Jewish populations. We sought to identify rare, CD-associated frameshift variants of high functional and statistical effects. METHODS: We performed exome-sequencing and array-based genotype analyses of 1477 Ashkenazi Jewish individuals with CD and 2614 Ashkenazi Jewish individuals without CD (controls). To validate our findings, we performed genotype analyses of an additional 1515 CD cases and 7052 controls for frameshift mutations in the colony stimulating factor 2 receptor beta common subunit gene (CSF2RB). Intestinal tissues and blood samples were collected from patients with CD; lamina propria leukocytes were isolated and expression of CSF2RB and GMCSF-responsive cells were defined by mass cytometry (CyTOF analysis). Variants of CSF2RB were transfected into HEK293 cells and expression and functions of gene products were compared. RESULTS: In the discovery cohort, we associated CD with a frameshift mutation in CSF2RB (P=8.52x10-4); the finding was validated in the replication cohort (combined P=3.42x10-6). Incubation of intestinal lamina propria leukocytes with GMCSF resulted in high levels of phosphorylation of STAT5 and lesser increases in phosphorylation of ERK and AKT. Cells co-transfected with full-length and mutant forms of CSF2RB had reduced pSTAT5 following stimulation with GMCSF, compared to cells transfected with control CSF2RB, indicating a dominant negative effect of the mutant gene. Monocytes from patients with CD who were heterozygous for the frameshift mutation (6% of CD cases analyzed) had reduced responses to GMCSF and markedly decreased activity of aldehyde dehydrogenase; activity of this enzyme has been associated with immune tolerance. CONCLUSIONS: In a genetic analysis of Ashkenazi Jewish individuals, we associated CD with a frameshift mutation in CSF2RB. Intestinal monocytes from carriers of this mutation had reduced responses to GMCSF, providing an additional mechanism for alterations to the innate immune response in individuals with CD

A trematode parasite derived growth factor binds and exerts influences on host immune functions via host cytokine receptor complexes

The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allow- ing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-β RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-β RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory recep- tor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is depen- dent on TGF-β RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody- dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages—again dependent on TGF-β RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for damp- ened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced effector response targetingjuvenile parasites which we demonstrate extends to an abrogation of the ADCC response. Thus suggesting that FhTLM is a stage specific evasion molecule that utilises host cytokine receptors. These findings are the first to clearly demonstrate the interaction of a helminth cytokine with a host receptor complex resulting in immune modifications that facilitate the non-protective chronic immune response which is characteristic of F. hepatica infection

Hemodynamic Effects of a High-Frequency Oscillatory Ventilation Open-Lung Strategy in Critically Ill Children With Acquired or Congenital Cardiac Disease

OBJECTIVES: To study the hemodynamic consequences of an open-lung high-frequency oscillatory ventilation (HFOV) strategy in patients with an underlying cardiac anomaly with or without intracardiac shunt or primary pulmonary hypertension with severe lung injury.DESIGN: Secondary analysis of prospectively collected data.SETTING: Medical-surgical PICU.PATIENTS: Children less than 18 years old with cardiac anomalies (± intracardiac shunt) or primary pulmonary hypertension.INTERVENTIONS: None.MEASUREMENTS AND MAIN RESULTS: Data from 52 subjects were analyzed, of whom 39 of 52 with cardiac anomaly (23/39 with intracardiac shunt) and 13 of 52 with primary pulmonary hypertension. Fourteen patients were admitted postoperatively, and 26 patients were admitted with acute respiratory failure. Five subjects (9.6%) were canulated for ECMO (of whom four for worsening respiratory status). Ten patients (19.2%) died during PICU stay. Median conventional mechanical ventilation settings prior to HFOV were peak inspiratory pressure 30 cm H 2 O (27-33 cm H 2 O), positive end-expiratory pressure 8 cm H 2 O (6-10 cm H 2 O), and F io2 0.72 (0.56-0.94). After transitioning to HFOV, there was no negative effect on mean arterial blood pressure, central venous pressure, or arterial lactate. Heart rate decreased significantly over time ( p &lt; 0.0001), without group differences. The percentage of subjects receiving a fluid bolus decreased over time ( p = 0.003), especially in those with primary pulmonary hypertension ( p = 0.0155) and without intracardiac shunt ( p = 0.0328). There were no significant differences in the cumulative number of daily boluses over time. Vasoactive Infusion Score did not increase over time. Pa co2 decreased ( p &lt; 0.0002) and arterial pH significantly improved ( p &lt; 0.0001) over time in the whole cohort. Neuromuscular blocking agents were used in all subjects switched to HFOV. Daily cumulative sedative doses were unchanged, and no clinically apparent barotrauma was found.CONCLUSIONS: No negative hemodynamic consequences occurred with an individualized, physiology-based open-lung HFOV approach in patients with cardiac anomalies or primary pulmonary hypertension suffering from severe lung injury.</p

Materials in particulate form for tissue engineering. 2 Applications in bone

Materials in particulate form have been the subjects of intensive research in view of their use as drug delivery systems. While within this application there are still issues to be addressed, these systems are now being regarded as having a great potential for tissue engineering applications. Bone repair is a very demanding task, due to the specific characteristics of skeletal tissues, and the design of scaffolds for bone tissue engineering presents several difficulties. Materials in particulate form are now seen as a means of achieving higher control over parameters such as porosity, pore size, surface area and the mechanical properties of the scaffold. These materials also have the potential to incorporate biologically active molecules for release and to serve as carriers for cells. It is believed that the combination of these features would create a more efficient approach towards regeneration. This review focuses on the application ofmaterials in particulate formfor bone tissue engineering. A brief overview of bone biology and the healing process is also provided in order to place the application in its broader context. An original compilation of molecules with a documented role in bone tissue biology is listed, as they have the potential to be used in bone tissue engineering strategies. To sum up this review, examples of works addressing the above aspects are presented

Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease

Crohn’s disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of nonsynonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2066 CD cases and 3633 healthy controls. We detected association signals in the LRRK2 gene that conferred risk for CD (N2081D variant, P = 9.5 × 10−10) or protection from CD (N551K variant, tagging R1398H-associated haplotype, P = 3.3 × 10−8). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson’s disease (PD), and healthy controls revealed extensive pleiotropy, with shared genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant was associated with increased kinase activity, whereas neither N551K nor R1398H variants on the protective haplotype altered kinase activity. We also confirmed that R1398H, but not N551K, increased guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases

Early Neuromuscular Blockade in Moderate-to-Severe Pediatric Acute Respiratory Distress Syndrome

Objectives: The use of neuromuscular blocking agents (NMBAs) in pediatric acute respiratory distress syndrome (PARDS) is common but unsupported by efficacy data. We sought to compare the outcomes between patients with moderate-to-severe PARDS receiving continuous NMBA during the first 48 hours of endotracheal intubation (early NMBA) and those without. Design: Secondary analysis of data from the Randomized Evaluation of Sedation Titration for Respiratory Failure (RESTORE) clinical trial, a pediatric multicenter cluster randomized trial of sedation. Setting: Thirty-one PICUs in the United States. Patients: Children 2 weeks to 17 years receiving invasive mechanical ventilation (MV) for moderate-to-severe PARDS (i.e., oxygenation index >= 8 and bilateral infiltrates on chest radiograph on days 0-1 of endotracheal intubation). Interventions: NMBA for the entire duration of days 1 and 2 after intubation. Measurements and Main Results: Among 1,182 RESTORE patients with moderate-to-severe PARDS, 196 (17%) received early NMBA for a median of 50.0% ventilator days (interquartile range, 33.3-60.7%). The propensity score model predicting the probability of receiving early NMBA included high-frequency oscillatory ventilation on days 0-2 (odds ratio [OR], 7.61; 95% CI, 4.75-12.21) and severe PARDS on days 0-1 (OR, 2.16; 95% CI, 1.50-3.12). After adjusting for risk category, early use of NMBA was associated with a longer duration of MV (hazard ratio, 0.57; 95% CI, 0.48-0.68; p < 0.0001), but not with mortality (OR, 1.62; 95% CI, 0.92-2.85; p = 0.096) compared with no early use of NMBA. Other outcomes including cognitive, functional, and physical impairment at 6 months post-PICU discharge were similar. Outcomes did not differ when comparing high versus low NMBA usage sites or when patients were stratified by baseline PaO2/FIO2 less than 150. Conclusions: Early NMBA use was associated with a longer duration of MV. This propensity score analysis underscores the need for a randomized controlled trial in pediatrics