Prevalence of second-hand tobacco smoke in relation to smoke-free legislation in the European Union.

Exposure to second-hand smoke (SHS) is an important public health problem. We assessed SHS exposure in restaurants and bars across the European Union (EU) and studied associations with country-level smoke-free legislation. Data of Eurobarometer surveys 2014 and 2017 were used to estimate country-specific prevalence of observing smoking in restaurants and bars, which can be considered a marker of SHS exposure. Additionally, we used multilevel logistic regression models to study associations with comprehensiveness of national smoke-free regulations in restaurants and bars, which were derived from the Tobacco Control Scale. In total, 44,809 people from all 28 EU member states were included in the analysis. The results of the multilevel logistic analysis show that in countries with complete and extensive bans, respondents were less likely to have observed people smoking inside restaurants than in countries with partial bans, which represented the lowest level of smoke-free policy implementation (OR 0.24, 95%CI 0.10-0.57 for complete ban and OR 0.23, 95%CI 0.10-0.54 for incomplete but extensive ban). Also, the prevalence of seeing people smoking in a bar was lower in the countries with an extensive ban (OR 0.23 95%CI 0.11-0.45) and with a complete ban (OR 0.20 95%CI 0.10-0.40). Between 2014 and 2017, SHS exposure in restaurants and bars decreased significantly. Our results confirm that in countries with extensive or complete smoking bans, people were less exposed to SHS in restaurants and bars; and that partial bans are less effective in reducing SHS exposure.

IAPs regulate the plasticity of cell migration by directly targeting Rac1 for degradation.

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.

Integrin-linked kinase regulates process extension in oligodendrocytes via control of actin cytoskeletal dynamics.

Integrin-linked kinase (ILK) is a major structural adaptor protein governing signaling complex formation and cytoskeletal dynamics. Here, through the use of conditional knock-out mice, we demonstrate a requirement for ILK in oligodendrocyte differentiation and axonal myelination in vivo. In conjunction, ILK-deficient primary oligodendrocytes are defined by a failure in process extension and an inability to form myelin membrane upon axonal contact. Surprisingly, phosphorylation of the canonical downstream targets Akt and GSK3ß is unaffected following ILK loss. Rather, the defects are due in part to actin cytoskeleton dysregulation with a correspondent increase in active RhoA levels. Morphological rescue is possible following Rho kinase inhibition in an oligodendrocyte subset. Furthermore, phenotypic severity correlates with environmental complexity; oligodendrocytes are severely malformed in vitro (a relatively simple environment), but undergo phenotypic recovery in the context of the whole animal. Together, our work demonstrates ILK as necessary for normal oligodendrocyte development, reinforces its role as a bridge between the actin cytoskeleton and cell membrane, and highlights the overarching compensatory capacity of oligodendrocytes in response to cellular milieu.

Use of imaged capillary isoelectric focusing technique in development of diphtheria toxin mutant CRM197.

Polysaccharide based-vaccines have been successful in providing protection in adults from bacterial infections, however they are not as effective in infants or young children. To enhance the immune response in these high risk groups, the polysaccharide is conjugated with a carrier protein such as cross-reacting material 197 (CRM197). The CRM197 protein has been well-characterized biochemically and biophysically using various analytical techniques however, none of these have been CE-based methods. Of the various CE techniques, imaged capillary isoelectric focusing (icIEF) is a method that has been used extensively in the field of protein-based drug development as a tool for product identification, stability monitoring, and characterization. Applications of icIEF technique using Convergent Bioscience icIEF instrumentation with whole-field imaging technology are presented and discussed in this paper. These applications include rapid method development to establish a CRM197 identity test for product release, a concentration assay for upstream and downstream in-process product development, and CRM197 stability with respect to its charge heterogeneity under accelerated temperature stress. The data presented demonstrates the utility of the icIEF method as a multifunctional assay because it can screen for better product candidates during early stage clonal selection as well as support in-process and final product characterization throughout CRM197 development.

Propyne: Determination of Physical Properties and Unit Cell Parameters under Titan-Relevant Conditions.

With its large size, dense atmosphere, methane-based hydrological-like cycle, and diverse surface features, the Saturnian moon Titan is one of the most unique of the outer Solar System satellites. Study of the photochemically produced molecules in Titan's atmosphere is critical in order to understand the mechanics of the atmosphere and, by extension, the interactions between atmosphere, surface, and subsurface water ocean. One example is propyne vapor, a photochemically produced species in Titan's upper atmosphere expected to condense in Titan's stratosphere at lower altitudes. Propyne may also be a trace species in Titan's stratospheric co-condensed ice clouds detected by the Cassini Composite InfraRed Spectrometer. Bulk structural characterization of propyne ice is currently incomplete and is lacking in published laboratory Raman spectra and X-ray diffraction data. Here, we present a laboratory characterization of propyne ice, including the first published X-ray diffraction and Raman spectroscopy results for propyne ice.

Rho-kinase inactivation prolongs survival of an intermediate SMA mouse model.

Spinal muscular atrophy (SMA) is an inherited disease resulting in the highest mortality of children under the age of two. SMA is caused by mutations or deletions in the survival motor neuron 1 (SMN1) gene, leading to aberrant neuromuscular junction (NMJ) development and the loss of spinal cord alpha-motor neurons. Here, we show that Smn depletion leads to increased activation of RhoA, a major regulator of actin dynamics, in the spinal cord of an intermediate SMA mouse model. Treating these mice with Y-27632, which inhibits ROCK, a direct downstream effector of RhoA, dramatically improves their survival. This lifespan rescue is independent of Smn expression and is accompanied by an improvement in the maturation of the NMJs and an increase in muscle fiber size in the SMA mice. Our study presents evidence linking disruption of actin cytoskeletal dynamics to SMA pathogenesis and, for the first time, identifies RhoA effectors as viable targets for therapeutic intervention in the disease.

Fasudil improves survival and promotes skeletal muscle development in a mouse model of spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is the leading genetic cause of infant death. It is caused by mutations/deletions of the survival motor neuron 1 (SMN1) gene and is typified by the loss of spinal cord motor neurons, muscular atrophy, and in severe cases, death. The SMN protein is ubiquitously expressed and various cellular- and tissue-specific functions have been investigated to explain the specific motor neuron loss in SMA. We have previously shown that the RhoA/Rho kinase (ROCK) pathway is misregulated in cellular and animal SMA models, and that inhibition of ROCK with the chemical Y-27632 significantly increased the lifespan of a mouse model of SMA. In the present study, we evaluated the therapeutic potential of the clinically approved ROCK inhibitor fasudil. METHODS: Fasudil was administered by oral gavage from post-natal day 3 to 21 at a concentration of 30 mg/kg twice daily. The effects of fasudil on lifespan and SMA pathological hallmarks of the SMA mice were assessed and compared to vehicle-treated mice. For the Kaplan-Meier survival analysis, the log-rank test was used and survival curves were considered significantly different at P < 0.05. For the remaining analyses, the Student's two-tail t test for paired variables and one-way analysis of variance (ANOVA) were used to test for differences between samples and data were considered significantly different at P < 0.05. RESULTS: Fasudil significantly improves survival of SMA mice. This dramatic phenotypic improvement is not mediated by an up-regulation of Smn protein or via preservation of motor neurons. However, fasudil administration results in a significant increase in muscle fiber and postsynaptic endplate size, and restores normal expression of markers of skeletal muscle development, suggesting that the beneficial effects of fasudil could be muscle-specific. CONCLUSIONS: Our work underscores the importance of muscle as a therapeutic target in SMA and highlights the beneficial potential of ROCK inhibitors as a therapeutic strategy for SMA and for other degenerative diseases characterized by muscular atrophy and postsynaptic immaturity.

Applications of imaged capillary isoelectric focussing technique in development of biopharmaceutical glycoprotein-based products.

CE-based methods have increasingly been applied to the analysis of a variety of different type proteins. One of those techniques is imaged capillary isoelectric focusing (icIEF), a method that has been used extensively in the field of protein-based drug development as a tool for product identification, stability monitoring, and characterization. It offers many advantages over the traditional labor-intensive IEF slab gel method and even standard cIEF with on-line detection technologies with regard to method development, reproducibility, robustness, and speed. Here, specific examples are provided for biopharmaceutical glycoprotein products such as mAbs, erythropoietin (EPO), and recombinant Fc-fusion proteins, though the technique can be adapted for many other therapeutic proteins. Applications of iCIEF using a Convergent Bioscience instrument (Toronto, Canada) with whole-field imaging technology are presented and discussed. These include a quick method to establish an identity test for many protein-based products, product release, and stability evaluation of glycoproteins with respect to charge heterogeneity under accelerated temperature stress, different pH conditions, and in different formulations. Finally, characterization of glycoproteins using this iCIEF technology is discussed with respect to biosimilar development, clone selection, and antigen binding. The data presented provide a "taste'' of what icIEF method can do to support the development of biopharmaceutical glycoprotein products from early clone screening for better product candidates to characterization of the final commercial products.

Monitoring bromide loss in bromoacetyl-derivatized polyribosylribitol polysaccharide in Haemophilus influenzae type b for PedvaxHIB® by capillary electrophoresis and NMR spectroscopy.

PedvaxHIB® is an effective pediatric vaccine for protecting infants from invasive gram-negative bacterium Haemophilus influenzae type b. It is a highly purified capsular polysaccharide, polyribosylribitol phosphate that is covalently linked to an outer membrane protein complex of Neisseria meningitidis. PRP is first derivatized with an organic linker, followed by the coupling of a butadiamine group, and then at the end terminal, a bromoacetyl group is attached for conjugation with thiolated OMPC. The stability of the bromide group in derivatized PRP is monitored by two different methods, capillary electrophoresis and NMR spectroscopy. The loss of the bromide group is detected by measuring the amount of free bromide ion liberated using capillary electrophoresis and by observing a change in amide proton peaks near the bromide group using NMR. The two methods give similar rate hydrolysis results, therefore both can be employed as quick stability tools for bromoacetylation PRP content during manufacturing.

Structural elucidation of an α-1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris.

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-ß-Man-(1 → 2)-ß-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple ß-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.

Androgens regulate adenylate cyclase activity and intracellular calcium in stromal cells derived from human prostate.

BACKGROUND: Increased smooth muscle tone is a significant component of benign prostatic hyperplasia, the onset of which correlates with age and declining serum testosterone levels. This study investigates the effects of androgens on key regulators of smooth muscle tone: intracellular calcium ([Ca(2+)](i)) and cyclic adenosine monophosphate (cAMP) in human cultured prostatic stromal cells (HCPSC). METHODS: HCPSC were cultured in the absence or presence of dihydrotestosterone (DHT; 3, 30, and 300 nM) or testosterone (0.3-300 nM) alone or in the presence of flutamide (10 microM). Changes in [Ca(2+)](i) were determined in FURA-2AM (10 microM) loaded cells. Changes in cAMP were determined by Alpha Screen(R) assay. RESULTS: Up to 32% of cultured cells exhibited spontaneous elevations of [Ca(2+)](i). The frequency of these elevations was reduced by nifedipine (10 microM), ryanodine (1 microM), and the adenylate cyclase inhibitor MDL 12,330A (20 microM). Compared to steroid-free cells, a 3-day incubation of cells with testosterone (only 3 nM) elevated basal, but not peak [Ca(2+)](i). In the presence of flutamide, all concentrations of testosterone tested elevated basal, but not peak [Ca(2+)](i). DHT (30 and 300, but not 3 nM) lowered peak and basal [Ca(2+)](i). Increased testosterone concentration dependently decreased resting cell cAMP (pIC(50): 7.64 +/- 0.29 nM). CONCLUSIONS: These findings demonstrate that some HCPSC have the ability to spontaneously and transiently elevate [Ca(2+)](i). The magnitude of these [Ca(2+)](i) peaks, along with resting levels of calcium and cAMP, appear to be regulated by androgens.

SMN, profilin IIa and plastin 3: a link between the deregulation of actin dynamics and SMA pathogenesis.

Spinal muscular atrophy (SMA) is the most common human genetic disease resulting in infant mortality. SMA is caused by mutations or deletions in the ubiquitously expressed survival motor neuron 1 (SMN1) gene. Why SMA specifically affects motor neurons remains poorly understood. We have shown that Smn deficient PC12 cells have increased levels of the neuronal profilin IIa protein, leading to an inappropriate activation of the RhoA/ROCK pathway. This suggests that mis-regulation of neuronal actin dynamics is central to SMA pathogenesis. Here, we demonstrate an increase in profilin IIa and a decrease in plastin 3 protein levels in a SMA mouse model. Furthermore, knock-out of profilin II upregulates plastin 3 expression in a Smn-dependent manner. However, the depletion of profilin II and the restoration of plastin 3 are not sufficient to rescue the SMA phenotype. Our study suggests that additional regulators of actin dynamics must also contribute to SMA pathogenesis.

Global progress in tobacco control: the question of policy compliance.

Background: Currently, about 65% of the world's population is covered by at least one MPOWER tobacco control policy measure. The impact of such policies might rely on policy compliance. Objective: This study aims to describe and compare global trends in legislation and compliance of the following three tobacco control policies between 2009 and 2019: direct advertisement, promotion and sponsorship, and smoke-free environments. Method: Data from the six most recent WHO Tobacco Control (2009-2019) reports were used to show the development of and possible associations between legislated policies and policy compliance. Data pertaining to the three indicators direct advertisement, promotion and sponsorship, and smoke-free environments were collected and analysed per country income category, according to the Organization for Economic Co-operation and Development. For each country, we (i) calculated the legislation describing the situation according to the law as a percentage of fulfilled MPOWER measurements and (ii) present the level of compliance (ranging from 0 to 10) for the corresponding policy. Results: Both tobacco control policy legislation and compliance for direct advertising improved worldwide - between 2009 and 2019 the median increased from 37.5% to 87.5% for policy and from 5 to 8 for compliance. In contrast, promotion and sponsorship restrictions hardly developed since 2011 and are especially weak among low- and middle-income countries. With respect to smoke-free environments, global policy legislation increased steadily over time while the relative compliance hardly increased. In 2019 data did not show significant correlations between policy legislation and compliance: direct advertising ρ = -0.003, p = 0.970; promotion and sponsorship ρ = 0.140, p = 0.107; smoke-free environments ρ = 0.158, p = 0.070. Conclusion: There is a clear need to understand the barriers in achieving tobacco control policy compliance and to routinely collect and incorporate data on compliance in research in order to generate a more reliable basis for further improvements in tobacco control.

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season.

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.

A SAGE approach to discovery of genes involved in autophagic cell death.

Programmed cell death (PCD), important in normal animal physiology and disease, can be divided into at least two morphological subtypes, including type I, or apoptosis, and type II, or autophagic cell death. While many molecules involved in apoptosis have been discovered and studied intensively during the past decade, autophagic cell death is not well characterized molecularly. Here we report the first comprehensive identification of molecules associated with autophagic cell death during normal metazoan development in vivo. During Drosophila metamorphosis, the larval salivary glands undergo autophagic cell death regulated by a hormonally induced transcriptional cascade. To identify and analyze the genes expressed, we examined wild-type patterns of gene expression in three predeath stages of Drosophila salivary glands using serial analysis of gene expression (SAGE) [7]. 1244 transcripts, including genes involved in autophagy, defense response, cytoskeleton remodeling, noncaspase proteolysis, and apoptosis, were expressed differentially prior to salivary gland death. Mutant expression analysis indicated that several of these genes were regulated by E93, a gene required for salivary gland cell death. Our analyses strongly support both the emerging notion that there is overlap with respect to the molecules involved in autophagic cell death and apoptosis, and that there are important differences.

Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis.

As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation.

Tobacco Control Progress in Low and Middle Income Countries in Comparison to High Income Countries.

The study aimed to describe worldwide levels and trends of tobacco control policy by comparing low and middle income countries with other income categories from 2007 to 2014 and to analyze the corresponding relation to recent changes in smoking prevalence. Policy measure data representing years 2007 to 2014 were collected from all available World Health Organization (WHO) reports on the global tobacco epidemic. Corresponding policy percentage scores (PS) were calculated based on MPOWER measures. Age-standardized smoking prevalence data for years 2010 and 2015 were collected from the WHO Global Health Observatory Data Repository. Trends of PS were analysed with respect to WHO region and OECD country income category. Scatter plots and regression analysis were used to depict the relationship between tobacco control policy of 2010 and change in smoking prevalence between 2015 and 2010 by sex and income category. Combined PS for all countries increased significantly from 47% in 2007 to 61% by 2014 (p < 0.001). When grouped by income category and region, policies were strengthened in all categories, albeit with varying progression. By 2014, tobacco control policy legislation had reached 45% in the Least Developed Countries (LDCs), 59% in Low Middle Income Countries (LMICs), 66% in Upper Middle Income Countries (UMICs) and 70% in High Income Countries (HICs). Overall, there was a negative relationship between higher policy scores and change in smoking prevalence. Although policy strengthening had been conducted between 2007 and 2014, room for considerable global improvement remains, particularly in LDCs.

Hepatitis B immune status in adolescents vaccinated during infancy: A retrospective cohort study from a pediatric practice in Germany.

In Germany, vaccination of infants against hepatitis B is recommended since 1995. However, data on long-term immunity is sparse and the necessity of a booster dose remains uncertain. Aims of this study were to assess the long-term persistence of antibodies to the hepatitis B surface antigen (anti-HBs) after immunization during infancy and the effect of a subsequent hepatitis B booster vaccination during adolescence on anti-HBs levels. Patients from a private pediatric practice who had received a full vaccination course of hepatitis B as infants and who were quantitatively tested for anti-HBs during adolescence (pre-booster levels) were included. In those participants who received a hepatitis B booster, post-booster anti-HBs levels were measured. Univariate analyses were conducted to determine factors associated with pre- and post-booster anti-HBs levels, respectively. 106 participants (53% male) were included in the study. At an average of 13.7 y after primary vaccination, 14% of participants had an anti-HBs level of ≥100 IU/l, while 46% were at 10-99 IU/l and 40% had anti-HBs levels of <10 IU/l. In total, 34 received a booster vaccination. Of those, 97% (33/34) had post-booster anti-HBs levels ≥ 100 IU/l, which were independent from pre-booster levels. No other patient characteristics were associated with pre-booster or post-booster anti-HBs≥ 100 IU/l. Although almost half of study participants showed low anti-HBs levels at follow-up, robust responses to booster vaccination suggest that adolescents who received the full vaccination course during infancy are still protected against hepatitis B infection.