Studies of coat protein-mediated resistance to tobacco mosaic tobamovirus: correlation between assembly of mutant coat proteins and resistance.

Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants. The mutation T42-->W increased protein aggregation and T28-->W abolished aggregation and assembly, while the mutations T28-->W plus T42-->W and T89-->W altered normal CP subunit-subunit interactions. The mutant T28W was unable to assemble virus-like particles (VLPs) during infection and in transgenic plants failed to aggregate; this protein conferred no protection against challenge of transgenic plants by TMV. The mutant T42W had strong CP subunit-subunit interactions and formed VLPs but not infectious virions. Transgenic lines with this protein exhibited stronger protection against TMV infection than transgenic plants that contained the wild-type (wt) CP. It is proposed that increased resistance conferred by the T42W mutant results from strong interaction between transgenic CP subunits and challenge virus CP subunits. CP carrying the mutation T89-->W formed flexuous and unstable VLPs whereas the double mutant T28W:T42W formed open helical structures that accumulated as paracrystalline arrays. In transgenic plants, T89W and the double mutant CPs showed reduced ability to aggregate and provided lower protection against TMV infection than wt CP. A strong correlation between normal CP subunit-subunit interactions and CP-MR is observed, and a model for CP-MR involving interactions between the transgenic CP and the CP of the challenge virus as well as interference with virus movement is discussed.     

Tumor-Derived Microvesicles Induce,Expand and Up-Regulate Biological Activities of Human Regulatory T Cells (Treg)

Background

Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4⁺CD25^highFOXP3⁺ Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/expansion/and activation of human Treg.

Methodology/Principal Findings

TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4⁺CD25^neg T cells into CD4⁺CD25^highFOXP3⁺ Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-β1, CTLA-4, granzyme B and perforin expression (p<0.05) and mediated stronger suppression of responder cell (RC) proliferation (p<0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-β1 and/or IL-10 significantly inhibited TMV ability to expand Treg.

Conclusions/Significance

This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV with Treg represent a newly-defined mechanism that might be involved in regulating peripheral tolerance by tumors and in supporting immune evasion of human cancers.     

NMR Structure of Integrin α4 Cytosolic Tail and Its Interactions with Paxillin

Background

Integrins are a group of transmembrane signaling proteins that are important in biological processes such as cell adhesion, proliferation and migration. Integrins are α/β hetero-dimers and there are 24 different integrins formed by specific combinations of 18 α and 8 β subunits in humans. Generally, each of these subunits has a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). CTs of integrins are important in bidirectional signal transduction and they associate with a large number of intracellular proteins.

Principal Findings

Using NMR spectroscopy, we determined the 3-D structure of the full-length α4 CT (Lys968-Asp999) and characterize its interactions with the adaptor protein paxillin. The α4 CT assumes an overall helical structure with a kink in its membrane proximal region. Residues Gln981-Asn997 formed a continuous helical conformation that may be sustained by potential ionic and/or hydrogen bond interactions and packing of aromatic-aliphatic side-chains. ¹⁵N-¹H HSQC NMR experiments reveal interactions of the α4 CT C-terminal region with a fragment of paxillin (residues G139-K277) that encompassed LD2-LD4 repeats. Residues of these LD repeats including their adjoining linkers showed α4 CT binding-induced chemical shift changes. Furthermore, NMR studies using LD-containing peptides showed predominant interactions between LD3 and LD4 of paxillin and α4 CT. Docked structures of the α4 CT with these LD repeats suggest possible polar and/or salt-bridge and non-polar packing interactions.

Significance

The current study provides molecular insights into the structural diversity of α CTs of integrins and interactions of integrin α4 CT with the adaptor protein paxillin.     

Critical Structural and Functional Roles for the N-Terminal Insertion Sequence in Surfactant Protein B Analogs

Background

Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., ∼residues 8–25 and 63–78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1–7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.

Methodology/Results

FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary α-helix and secondary β-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a “saposin-like” fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.

Conclusion

Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.     

Appearance of “β-Like” Circular Dichroism Spectra on Protein Aggregation That Is not Accompanied by Transition to β-Structure

CD spectra in the 200 to 250 nm spectral region for small ordered aggregates (trimers-pentamers) of tobacco mosaic virus (TMV) coat protein (CP) and for long virus-like helical aggregates of TMV CP were compared. It was found that small (4S) TMV CP aggregates have a CD spectrum typical of a protein with high -helix content, which agrees well with results of X-ray diffraction studies. But in the long helical aggregates (and in the TMV virions) TMV CP gives -like CD spectra similar to those of many other aggregated proteins. From X-ray diffraction data, it is well known that TMV CP subunits do not change their secondary or tertiary structure on assembly into virions or the helical repolymerized protein. Thus, the change in the shape of 200 to 250 nm CD spectra cannot be employed as the sole criterion of the conversion of a protein to -structure in the course of aggregation.     

Interaction of C-Terminal Truncated Human αA-Crystallins with Target Proteins

Background

Significant portion of αA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH) and βL-crystallin as target proteins, was increased in αA_1–172 and decreased in αA_1–168 and αA_1–162. The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.

Methodology/Principal Findings

Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET) utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k) for ADH and αA_1–172 was nearly the same as that of ADH and αA-wt, αA_1–168 had lower and αA_1–162 had the lowest k values. When βL-crystallin was used as the target protein, αA_1–172 had slightly higher k value than αA-wt and αA_1–168 and αA_1–162 had lower k values. As expected from earlier studies, the chaperone activity of αA_1–172 was slightly better than that of αA-wt, the chaperone activity of αA_1–168 was similar to that of αA-wt and αA_1–162 had substantially decreased chaperone activity.

Conclusions/Significance

Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.     

Mutation of the LXCXE Binding Cleft of pRb Facilitates Transformation by ras In Vitro but Does Not Promote Tumorigenesis In Vivo

Background

The Retinoblastoma protein (pRB) is a key tumor suppressor that is functionally inactivated in most cancers. pRB regulates the cell division cycle and cell cycle exit through protein–protein interactions mediated by its multiple binding interfaces. The LXCXE binding cleft region of pRB mediates interactions with cellular proteins that have chromatin regulatory functions. Chromatin regulation mediated by pRB is required for a stress responsive cell cycle arrest, including oncogene induced senescence. The in vivo role of chromatin regulation by pRB during senescence, and its relevance to cancer is not clear.

Methodology/Principal Findings

Using gene-targeted mice, uniquely defective for pRB mediated chromatin regulation, we investigated its role during transformation and tumor progression in response to activation of oncogenic ras. We report that the pRB^∆L mutation confers susceptibility to escape from HrasV12 induced senescence and allows transformation in vitro, although these cells possess high levels of DNA damage. Intriguingly, LSL-Kras, Rb1 ^∆L/∆L mice show delayed lung tumor formation compared to controls. This is likely due to the increased apoptosis seen in the early hyperplastic lesions shortly following ras activation that inhibits tumor progression. Furthermore, DMBA treatment to induce sporadic ras mutations in other tissues also failed to reveal greater susceptibility to cancer in Rb1 ^∆L/∆L mice.

Conclusions/Significance

Our data suggests that chromatin regulation by pRB can function to limit proliferation, but its loss fails to contribute to cancer susceptibility in ras driven tumor models because of elevated levels of DNA damage and apoptosis.     

Interaction between Alcohol Consumption and CYP 2C19 Gene Polymorphism in Relation to Oesophageal Squamous Cell Carcinoma

Objectives

The purpose of this study is to explore the relationship between the interactions of CYP2C19 gene polymorphisms and several environmental factors and oesophageal squamous cell carcinoma (OSCC).

Methods

In a case-control study of OSCC patients (n = 350) and healthy controls (n = 350), we investigated the roles of polymorphism in the CYP2C19 gene by the use of polymerase chain reaction - restriction fragment length polymorphism (PCR – RFLP) analysis.

Results

The CYP2C19^*3 AG+AA genotype was significantly more prevalent in OSCC patients (10.0% versus 3.43%; P<0.01). Multiple logistic regression analysis showed drinking (OR: 5.603, 95% CI: 3.431–11.112; P = 0.005) and smoking (OR: 4.341, 95% CI: 3.425–10.241; P = 0.001) was the independent risk factor of OSCC respectively, and there were significant interaction between CYP2C19^*3 and drinking (OR: 8.747, 95% CI: 6.321–18.122; P = 0.009).

Conclusions

The CYP2C19^*3 polymorphism and OSCC were synergistically and significantly associated in Chinese Han patients.     

Kinetics of thermal aggregation of tobacco mosaic virus coat protein

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52°C in a wide range of protein concentrations, [P]₀. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52°C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]₀ (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42°C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]₀ and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates {A/A _lim; t} (t is time and A _lim is the limiting value of A at t ). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.     

Lack of replication of interactions between polymorphisms in rheumatoid arthritis susceptibility: case–control study

Introduction

Approximately 100 loci have been definitively associated with rheumatoid arthritis (RA) susceptibility. However, they explain only a fraction of RA heritability. Interactions between polymorphisms could explain part of the remaining heritability. Multiple interactions have been reported, but only the shared epitope (SE) × protein tyrosine phosphatase nonreceptor type 22 (PTPN22) interaction has been replicated convincingly. Two recent studies deserve attention because of their quality, including their replication in a second sample collection. In one of them, researchers identified interactions between PTPN22 and seven single-nucleotide polymorphisms (SNPs). The other showed interactions between the SE and the null genotype of glutathione S-transferase Mu 1 (GSTM1) in the anti–cyclic citrullinated peptide–positive (anti-CCP⁺) patients. In the present study, we aimed to replicate association with RA susceptibility of interactions described in these two high-quality studies.

Methods

A total of 1,744 patients with RA and 1,650 healthy controls of Spanish ancestry were studied. Polymorphisms were genotyped by single-base extension. SE genotypes of 736 patients were available from previous studies. Interaction analysis was done using multiple methods, including those originally reported and the most powerful methods described.

Results

Genotypes of one of the SNPs (rs4695888) failed quality control tests. The call rate for the other eight polymorphisms was 99.9%. The frequencies of the polymorphisms were similar in RA patients and controls, except for PTPN22 SNP. None of the interactions between PTPN22 SNPs and the six SNPs that met quality control tests was replicated as a significant interaction term—the originally reported finding—or with any of the other methods. Nor was the interaction between GSTM1 and the SE replicated as a departure from additivity in anti-CCP⁺ patients or with any of the other methods.

Conclusions

None of the interactions tested were replicated in spite of sufficient power and assessment with different assays. These negative results indicate that whether interactions are significant contributors to RA susceptibility remains unknown and that strict standards need to be applied to claim that an interaction exists.     

Pathways to injury in chronic pancreatitis: decoding the role of the high-risk SPINK1 N34S haplotype using meta-analysis

Background

The complex interactions between recurrent trypsin-mediated pancreatic injury, alcohol-associated pancreatic injury and SPINK1 polymorphisms in chronic pancreatitis (CP) are undefined. We hypothesize that CP occurs as a result of multiple pathological mechanisms (pathways) that are initiated by different metabolic or environmental factors (etiologies) and may be influenced differentially by downstream genetic risk factors. We tested this hypothesis by evaluating the differences in effect size of the high risk SPINK1 N34S haplotype on CP from multiple etiologies after combining clinical reports of SPINK1 N34S frequency using meta-analysis.

Methods and Findings

The Pubmed and the Embase databases were reviewed. We studied 24 reports of SPINK1 N34S in CP (2,421 cases, 4,857 controls) using reported etiological factors as surrogates for pathways and multiple meta-analyses to determine the differential effects of SPINK1 N34S between alcoholic and non-alcoholic etiologies. Using estimates of between-study heterogeneity, we sub-classified our 24 studies into four specific clusters. We found that SPINK1 N34S is strongly associated with CP overall (OR 11.00; 95% CI: 7.59–15.93), but the effect of SPINK1 N34S in alcoholic CP (OR 4.98, 95% CI: 3.16–7.85) was significantly smaller than in idiopathic CP (OR 14.97, 95% C.I. = 9.09–24.67) or tropical CP (OR 19.15, 95% C.I. = 8.83–41.56). Studies analyzing familial CP showed very high heterogeneity suggestive of a complex etiology with an I² = 80.95%.

Conclusion

The small effect of SPINK1 N34S in alcoholic subjects suggests that CP is driven through a different pathway that is largely trypsin-independent. The results also suggest that large effect sizes of SPINK1 N34S in small candidate gene studies in CP may be related to a mixture of multiple etiologic pathways leading to the same clinical endpoint.     

Impact of Obesity on the Clinical Profile of a Population-Based Sample with Chronic Obstructive Pulmonary Disease

Aims

To characterize the distribution of BMI in a population-based sample of COPD patients and to evaluate the impact of obesity on their health status, exercise tolerance, systemic inflammation and comorbidity.

Methods

A population-based sample of 3,797 subjects aged 40–80 years from the EPI-SCAN study was selected. Subjects were categorized according their body mass index (BMI) as underweight (<18.5 kg/m²), normal weight (18.5–24.9 kg/m²), overweight (25.0–29.9 kg/m²) or obese (BMI≥30.0 kg/m²). Subjects were evaluated with post-bronchodilator spirometry and 6-minute walk tests. Smoking habits, respiratory symptoms, generic and specific quality of life, daily physical activities, comorbidities and systemic inflammatory biomarkers were recorded.

Results

The prevalence of obesity or being overweight was higher in the 382 COPD patients than in the subjects without airflow limitation (29.4%, 95%CI 24.8–33.9% vs. 24.3, 95%CI 22.9–25.8; and 44.7%, 95%CI 39.7–49.6% vs. 43.0%, 95%CI 41.3–44.6, respectively; p = 0.020). In the COPD subgroup, obese subjects presented more dyspnea and less chronic cough, chronic bronchitis or chronic phlegm than normal-weight patients, as well as a worse health status. Moreover, reduced exercise tolerance and higher plasmatic C-reactive protein levels were found in the obese patients, who also presented a greater prevalence of cardiovascular disease (adjusted odds ratio 4.796, 95%CI 1.806–12.736, p = 0.002).

Conclusions

In a population-based sample, obesity is more prevalent in COPD patients than in subjects without airflow limitation. Furthermore, obesity affects the clinical manifestations, quality of life and exercise tolerance of COPD patients, and it may contribute to a phenotype characterized by increased systemic inflammation and greater frequency of cardiovascular comorbidity.     

Gliadin Peptides Induce Tissue Transglutaminase Activation and ER-Stress through Ca2+ Mobilization in Caco-2 Cells

Background

Celiac disease (CD) is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG) seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca²⁺ homeostasis and tTG activity.

Methods/Principal Findings

We studied Ca²⁺ homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31–43 and 57–68 rapidly mobilized Ca²⁺ from intracellular stores. Specifically, peptide 31–43 mobilized Ca²⁺ from the endoplasmic reticulum (ER) and mitochondria, whereas peptide 57–68 mobilized Ca²⁺ only from mitochondria. We also found that gliadin peptide-induced Ca²⁺ mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31–43, but not peptide 57–68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31–43, but not of peptide 57–68, induces the expression of both genes.

Conclusions

By inducing Ca²⁺ mobilization from the ER, peptide 31–43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31–43 and 57–68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin–tTG crosslinking in enterocytes and specialized antigen-presenting cells.     

Respiratory Function and Changes in Lung Epithelium Biomarkers after a Short-Training Intervention in Chlorinated vs. Ozone Indoor Pools

Background

Swimming in indoor pools treated with combined chemical treatments (e.g. ozone) may reduce direct exposure to disinfection byproducts and thus have less negative effects on respiratory function compared to chlorinated pools. The aim of this study is to analyze the effects of a short-term training intervention on respiratory function and lung epithelial damage in adults exercising in indoor swimming pool waters treated with different disinfection methods (chlorine vs. ozone with bromine).

Methods

Lung permeability biomakers [surfactant protein D (SP-D) and Clara cell secretory protein (CC16) in plasma] and forced expiratory volumes and flow (FEV1, FVC and FEF_25–75) were measured in 39 healthy adults. Thirteen participants swam during 20 sessions in a chlorinated pool (CP), 13 performed and equivolumic intervention in an ozone pool (OP) and 13 were included in a control group (CG) without exposition.

Results

Median plasma CC16 levels increased in CP swimmers (4.27±3.29 and 6.62±5.51 µg/L, p = 0.01, pre and post intervention respectively) while no significant changes in OP and CG participants were found. No significant changes in median plasma SP-D levels were found in any of the groups after the training period. FVC increased in OP (4.26±0.86 and 4.43±0.92 L, p<0.01) and CP swimmers (4.25±0.86 and 4.35±0.85 L, p<0.01). FEV1 only increased in OP swimmers (3.50±0.65 and 3.59±0.67, p = 0.02) and FEF_25–75 decreased in CP swimmers (3.70±0.87 and 3.37±0.67, p = 0.02).

Conclusion

Despite lung function being similar in both groups, a higher lung permeability in CP compared to OP swimmers was found after a short-term swimming program. Combined chemical treatments for swimming pools such as ozone seem to have less impact on lung epithelial of swimmers compared to chlorinated treated pools.     

Impaired cardiac and peripheral hemodynamic responses to inhaled β2-agonist in cystic fibrosis

Background

Pulmonary system dysfunction is a hallmark of cystic fibrosis (CF) disease. In addition to impaired cystic fibrosis transmembrane conductance regulator protein, dysfunctional β₂-adrenergic receptors (β₂AR) contribute to low airway function in CF. Recent observations suggest CF may also be associated with impaired cardiac function that is demonstrated by attenuated cardiac output (Q), stroke volume (SV), and cardiac power (CP) at both rest and during exercise. However, β₂AR regulation of cardiac and peripheral vascular tissue, in-vivo, is unknown in CF. We have previously demonstrated that the administration of an inhaled β-agonist increases SV and Q while also decreasing SVR in healthy individuals. Therefore, we aimed to assess cardiac and peripheral hemodynamic responses to the selective β₂AR agonist albuterol in individuals with CF.

Methods

18 CF and 30 control (CTL) subjects participated (ages 22 ± 2 versus 27 ± 2 and BSA = 1.7 ± 0.1 versus 1.8 ± 0.0 m², both p < 0.05). We assessed the following at baseline and at 30- and 60-minutes following nebulized albuterol (2.5mg diluted in 3.0mL of normal saline) inhalation: 12-lead ECG for HR, manual sphygmomanometry for systolic and diastolic blood pressure (SBP and DBP, respectively), acetylene rebreathe for Q and SV. We calculated MAP = DBP + 1/3(SBP–DBP); systemic vascular resistance (SVR) = (MAP/Q)•80; CP = Q•MAP; stroke work (SW) = SV•MAP; reserve (%change baseline to 30- or 60-minutes). Hemodynamics were indexed to BSA (Q_I, SV_I, SW_I, CP_I, SVR_I).

Results

At baseline, CF demonstrated lower SV, SV_I, SW, and SW_I but higher HR than CTL (p < 0.05); other measures did not differ. At 30-minutes, CF demonstrated higher HR and SVR_I, but lower Q, SV, SV_I, CP, CP_I, SW, and SW_I versus CTL (p < 0.05). At 60-minutes, CF demonstrated higher HR, SVR, and SVR_I, whereas all cardiac hemodynamics were lower than CTL (p < 0.05). Reserves of CP, SW, and SVR were lower in CF versus CTL at both 30 and 60-minutes (p < 0.05).

Conclusions

Cardiac and peripheral hemodynamic responsiveness to acute β₂AR stimulation via albuterol is attenuated in individuals with CF, suggesting β₂AR located in cardiac and peripheral vascular tissue may be dysfunctional in this population.     

Population-Based Study of Birth Prevalence and Factors Associated with Cleft Lip and/or Palate in Taiwan 2002–2009

Background

Facial cleft deformities, including cleft lip with or without cleft palate (CL/P) and cleft palate (CP), are common congenital birth anomalies, especially in Asia. This study aimed to analyze the prevalence of CL/P and CP and to identify associated factors in Taiwan.

Methods

This population-based epidemiological study retrospectively analyzed birth data obtained from the Department of Health in Taiwan for years 2002–2009. Frequency distribution, percentages and related predictors were investigated, and findings were presented by types of cleft deformities. Logistic regression analysis was performed to identify factors associated with cleft deformities.

Results

Overall prevalence of cleft deformities among 1,705,192 births was 0.1% for CL/P and 0.04% for CP over the 8-year study period. Higher prevalence of CL/P or CP was observed with multiple pregnancies, being male for CL/P, being female for CP, gestational age ≤37 weeks and lower birth weight (<1.5 kg). Both CL/P and CP were significantly associated with gestational age <37 weeks and birth weight<1.5 kg (all P <0.0001). CL/P was significantly associated with multiple parities (P = 0.0004–0.002). Male newborns and female newborns were significantly associated with CL/P and CP, respectively (both P<0.0001).

Conclusions

Overall prevalence for congenital cleft deformities in study subjects was 0.1%, in keeping with high rates in Asia. Results suggest the need for awareness and early identification of those at high risk for cleft deformities, including newborns with gestational age <37 weeks, weighing <1.5 kg at birth and women with multiple parities, as a potential strategy to counter long-term adverse effects on speech and language in this population.     

Clinical and Laboratory Toxicity after Intra-Arterial Radioembolization with 90Y-Microspheres for Unresectable Liver Metastases

Objective

To investigate clinical and laboratory toxicity in patients with unresectable liver metastases, treated with yttrium-90 radioembolization (⁹⁰Y-RE).

Methods

Patients with liver metastases treated with ⁹⁰Y-RE, between February 1^st 2009 and March 31^st 2012, were included in this study. Clinical toxicity assessment was based on the reporting in patient’s charts. Laboratory investigations at baseline and during a four-month follow-up were used to assess laboratory toxicity according to the Common Terminology Criteria for Adverse Events version 4.02. The occurrence of grade 3–4 laboratory toxicity was stratified according to treatment strategy (whole liver treatment in one session versus sequential sessions). Response assessment was performed at the level of target lesions, whole liver and overall response in accordance with RECIST 1.1 at 3- and 6 months post-treatment. Median time to progression (TTP) and overall survival were calculated by Kaplan-Meier analysis.

Results

A total of 59 patients, with liver metastases from colorectal cancer (n = 30), neuroendocrine tumors (NET) (n = 6) and other primary tumors (n = 23) were included. Clinical toxicity after ⁹⁰Y-RE treatment was confined to grade 1–2 events, predominantly post-embolization symptoms. No grade 3–4 clinical toxicity was observed, whereas laboratory toxicity grade 3–4 was observed in 38% of patients. Whole liver treatment in one session was not associated with increased laboratory toxicity. Three-months disease control rates for target lesions, whole liver and overall response were 35%, 21% and 19% respectively. Median TTP was 6.2 months for target lesions, 3.3 months for the whole liver and 3.0 months for overall response. Median overall survival was 8.9 months.

Conclusion

The risk of severe complications or grade 3–4 clinical toxicity in patients with liver metastases of various primary tumors undergoing ⁹⁰Y-RE is low. In contrast, laboratory toxicity grade 3–4 can be expected to occur in more than one-third of patients without any clinical signs of radiation induced liver disease.     

Structure and Binding Interface of the Cytosolic Tails of αXβ2 Integrin

Background

Integrins are signal transducer proteins involved in a number of vital physiological processes including cell adhesion, proliferation and migration. Integrin molecules are hetero-dimers composed of two distinct subunits, α and β. In humans, 18 α and 8 β subunits are combined into 24 different integrin molecules. Each of the subunit comprises a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). The CTs of integrins are vital for bidirectional signal transduction and in maintaining the resting state of the receptors. A large number of intracellular proteins have been found to interact with the CTs of integrins linking integrins to the cytoskeleton.

Methodology/Principal Findings

In this work, we have investigated structure and interactions of CTs of the leukocyte specific integrin αXβ2. We determined the atomic resolution structure of a myristoylated CT of αX in perdeuterated dodecylphosphocholine (DPC) by NMR spectroscopy. Our results reveal that the 35-residue long CT of αX adopts an α-helical conformation for residues F4-N17 at the N-terminal region. The remaining residues located at the C-terminal segment of αX delineate a long loop of irregular conformations. A segment of the loop maintains packing interactions with the helical structure by an extended non-polar surface of the αX CT. Interactions between αX and β2 CTs are demonstrated by ¹⁵N-¹H HSQC NMR experiments. We find that residues constituting the polar face of the helical conformation of αX are involved in interactions with the N-terminal residues of β2 CT. A docked structure of the CT complex indicates that a network of polar and/or salt-bridge interactions may sustain the heteromeric interactions.

Conclusions/Significance

The current study provides important insights into the conservation of interactions and structures among different CTs of integrins.     

Do More Active Children Sleep More? A Repeated Cross-Sectional Analysis Using Accelerometry

Aim

To determine whether levels of daytime physical activity are associated with sleep duration and night waking in children assessed using accelerometry, and if these associations change over time.

Methods

24-hour accelerometry data were obtained from 234 children at 3, 5 and 7 years of age for at least 5 days at each time. Sleep duration was estimated using the Sadeh algorithm. Time spent in sedentary, light and moderate-vigorous (MVPA) activity was established using published cut-points. Appropriate statistical techniques were utilised to account for the closed nature of the data (24-hour periods).

Results

Time spent asleep was related more to sedentary or light activity and not to MVPA. The most active (95^th percentile) children spent 55–84 fewer minutes asleep and 16–19 more minutes awake at night compared to the least active (5^th percentile) children. Children with later bedtimes slept less at night (30–40 minutes) and undertook more sedentary (10–15 minutes) but also more light (18–23 minutes) activity during the day. However, no differences in MVPA were apparent according to bedtime. Children slept slightly less on weekend nights (11 minutes) compared with week-nights, but only at 3 years of age. Most relationships were broadly similar at 3, 5 and 7 years of age.

Conclusion

Children who are more physically active during the day have shorter total sleep time and are more awake at night than less active children. The protective effect of sleep on obesity does not appear to be mediated by increased physical activity.