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Our ability to fight pathogens relies on major histocompatibility complex class I (MHC-I) molecules presenting diverse antigens on the surface of diseased cells. The transporter associated with antigen processing (TAP) transports nearly the entire repertoire of antigenic peptides into the endoplasmic reticulum for MHC-I loading. How TAP transports peptides specific for MHC-I is unclear. In this study, we used cryo-EM to determine a series of structures of human TAP, both in the absence and presence of peptides with various sequences and lengths. The structures revealed that peptides of eight or nine residues in length bind in a similarly extended conformation, despite having little sequence overlap. We also identified two peptide-anchoring pockets on either side of the transmembrane cavity, each engaging one end of a peptide with primarily main chain atoms. Occupation of both pockets results in a global conformational change in TAP, bringing the two halves of the transporter closer together to prime it for isomerization and ATP hydrolysis. Shorter peptides are able to bind to each pocket separately but are not long enough to bridge the cavity to bind to both simultaneously. Mutations that disrupt hydrogen bonds with the N and C termini of peptides almost abolish MHC-I surface expression. Our findings reveal that TAP functions as a molecular caliper that selects peptides according to length rather than sequence, providing antigen diversity for MHC-I presentation. [ABSTRACT FROM AUTHOR]

Introduction: For in vivo inhalation studies, aerosol dosimetry links aerosol exposure and deposited dose within the respiratory tract. Aerosol dosimetry programs like the Multiple Path Particle Deposition model utilize respiratory tract geometry, respiratory physiology, and aerosol properties to predict particle deposition within the respiratory tract. A challenge to wider use of these dosimetry programs for in vivo inhalation studies is the lack of species-specific or strain specific respiratory tract anatomy, and in some cases, strain specific respiratory physiology data. The objective of this work was to develop aerosol dosimetry predictions for the in vivo human disease models of chronic obstructive pulmonary disease (COPD; C57BL/6 mice) cardiovascular disease (CVD; ApoE-/- mice), and lung cancer (AJ mice) using the Multiple Path Particle Deposition dosimetry model. Methods: Microcomputed tomography derived tracheobronchial geometry data were combined with available pulmonary geometry data for C57BL/6, ApoE-/-, and AJ mice and incorporated into the Multiple Path Particle Deposition dosimetry model. Mouse strain specific respiratory physiology literature values were used as input into the Multiple Path Particle Deposition dosimetry model. Results: The resulting particle deposition predictions compared well with the very limited strain specific experimental particle deposition data. Since multiple tracheobronchial geometries and thus pulmonary anatomies were used for C57BL/6 and ApoE-/- mice, an estimate of intrastrain variability in predicted particle deposition was obtained. Discussion and Conclusions: The intrastrain variability in predicted particle deposition for C57BL/6 and ApoE-/- mice was always smaller in the tracheobronchial compared to pulmonary airways. The differences in reported respiratory physiology values for C57BL/6 mice resulted in greater intrastrain variability in predicted particle deposition than observed with the different tracheobronchial geometries and pulmonary anatomies. These mouse strain specific aerosol dosimetry predictions can provide insight into the delivered dose to specific respiratory tract regions that can be correlated with in vivo endpoints. [ABSTRACT FROM AUTHOR]

Introduction: WS-5 is a flavor ingredient that provides a cooling sensation similar to that of menthol but without the characteristic menthol flavor. In the available toxicological evaluations of WS-5 there was no data specific to inhalation exposure. Methods: A 90-days nose-only inhalation study was conducted using Sprague Dawley rats exposed to Filtered air, propylene glycol/glycerin (40/60 by weight; vehicle control), or one of three WS-5 concentrations (0.2, 0.4, or 0.8% by weight) to assess the inhalation toxicity of WS-5. The study design was consistent with the Organization for Economic Development and Cooperation test guideline 413 and included a 28-day and 6-week recovery sacrifices, in addition to the 13-week assessment. Results: Food consumption and body weights were unaffected by WS-5 exposure compared to vehicle control or Filtered air. No WS-5 exposure related alterations were observed in serum chemistry, hematology, coagulation, urinalysis or bronchoalveolar lavage fluid cytology and clinical chemistry. Macroscopic examinations and terminal organ weights revealed no observations associated with exposure to WS-5. Discussion: Histopathology findings of alveolar hemorrhage and mixed cell infiltration at the 28-day necropsy were either not present in the WS-5 exposed groups or were present at similar frequencies in the vehicle control and/or Filtered air groups at the 13-weeks necropsy and thus, were not considered to be vehicle control or WS-5 related. Conclusion: Based on the absence of adverse effects, the no-observed-adverse effect concentration was considered to be 2.5 mg/L of aerosolized 0.8 w% WS-5 that achieved a mean measured exposure concentration of approximately 18 μg WS-5/L [ABSTRACT FROM AUTHOR]

Introduction: For in vivo inhalation studies, aerosol dosimetry links aerosol exposure and deposited dose within the respiratory tract. Aerosol dosimetry programs like the Multiple Path Particle Deposition model utilize respiratory tract geometry, respiratory physiology, and aerosol properties to predict particle deposition within the respiratory tract. A challenge to wider use of these dosimetry programs for in vivo inhalation studies is the lack of species-specific or strain specific respiratory tract anatomy, and in some cases, strain specific respiratory physiology data. The objective of this work was to develop aerosol dosimetry predictions for the in vivo human disease models of chronic obstructive pulmonary disease (COPD; C57BL/6 mice) cardiovascular disease (CVD; ApoE-/- mice), and lung cancer (AJ mice) using the Multiple Path Particle Deposition dosimetry model. Methods: Microcomputed tomography derived tracheobronchial geometry data were combined with available pulmonary geometry data for C57BL/6, ApoE-/-, and AJ mice and incorporated into the Multiple Path Particle Deposition dosimetry model. Mouse strain specific respiratory physiology literature values were used as input into the Multiple Path Particle Deposition dosimetry model. Results: The resulting particle deposition predictions compared well with the very limited strain specific experimental particle deposition data. Since multiple tracheobronchial geometries and thus pulmonary anatomies were used for C57BL/6 and ApoE-/- mice, an estimate of intrastrain variability in predicted particle deposition was obtained. Discussion and Conclusions: The intrastrain variability in predicted particle deposition for C57BL/6 and ApoE-/- mice was always smaller in the tracheobronchial compared to pulmonary airways. The differences in reported respiratory physiology values for C57BL/6 mice resulted in greater intrastrain variability in predicted particle deposition than observed with the different tracheobronchial geometries and pulmonary anatomies. These mouse strain specific aerosol dosimetry predictions can provide insight into the delivered dose to specific respiratory tract regions that can be correlated with in vivo endpoints. [ABSTRACT FROM AUTHOR]

Introduction: WS-5 is a flavor ingredient that provides a cooling sensation similar to that of menthol but without the characteristic menthol flavor. In the available toxicological evaluations of WS-5 there was no data specific to inhalation exposure. Methods: A 90-days nose-only inhalation study was conducted using Sprague Dawley rats exposed to Filtered air, propylene glycol/glycerin (40/60 by weight; vehicle control), or one of three WS-5 concentrations (0.2, 0.4, or 0.8% by weight) to assess the inhalation toxicity of WS-5. The study design was consistent with the Organization for Economic Development and Cooperation test guideline 413 and included a 28-day and 6-week recovery sacrifices, in addition to the 13-week assessment. Results: Food consumption and body weights were unaffected by WS-5 exposure compared to vehicle control or Filtered air. No WS-5 exposure related alterations were observed in serum chemistry, hematology, coagulation, urinalysis or bronchoalveolar lavage fluid cytology and clinical chemistry. Macroscopic examinations and terminal organ weights revealed no observations associated with exposure to WS-5. Discussion: Histopathology findings of alveolar hemorrhage and mixed cell infiltration at the 28-day necropsy were either not present in the WS-5 exposed groups or were present at similar frequencies in the vehicle control and/or Filtered air groups at the 13-weeks necropsy and thus, were not considered to be vehicle control or WS-5 related. Conclusion: Based on the absence of adverse effects, the no-observed-adverse effect concentration was considered to be 2.5 mg/L of aerosolized 0.8 w%WS-5 that achieved a mean measured exposure concentration of approximately 18 μg WS-5/L [ABSTRACT FROM AUTHOR]

[Display omitted] • OhyA carboxy terminus is a membrane binding domain used for interfacial activation. • Cryo-EM dimer of dimers structure matches cryo-EM dimer and crystal structure packing. • OhyA interaction with liposomal membrane drives dimer to high order oligomer switch. Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyA•membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20–30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme. [ABSTRACT FROM AUTHOR]

To assess potential exposure of non-users to exhaled constituents from pod and cartridge electronic nicotine delivery systems (ENDS) products, an environmental clinical study was conducted with (n = 43) healthy adult smokers. Room air concentrations of 34 selected constituents (nicotine, propylene glycol, glycerin, 15 carbonyls, 12 volatile organic compounds, and 4 trace metals) and particle number concentration (0.3 to 25 µm) were compared from use of two ENDS products and conventional cigarettes using room ventilations representative of a residential, an office or a hospitality setting over a 4-h. exposure period. Products used were JUUL ENDS, Virginia Tobacco flavor (Group I), VUSE Solo, Original flavor (Group II) (5.0 and 4.8% nicotine by weight, respectively) and subjects' own conventional cigarettes (Group III). Cumulative 4-h room air sampling and particle counting were performed during prescribed (Groups I and II) and ad libitum product use (all Groups). Conventional cigarette use resulted in significantly more constituents detected and higher 4-h cumulative constituent concentrations compared to use of the ENDS products tested, except for the predominant ENDS ingredients, propylene glycol and glycerin. Use of conventional cigarettes also resulted in greater total particle number concentration than either prescribed or ad libitum use of either of the ENDS used in this study. [ABSTRACT FROM AUTHOR]

A key to understanding biological response due to cell exposure to chemical constituents in aerosols is to accurately be able to determine the delivered dose. Deposition efficiency and uniformity of deposition was measured experimentally in the Vitrocell® 24/48 air–liquid-interface (ALI) in vitro exposure system using monodisperse solid fluorescent particles with mass median aerodynamic diameters (MMAD) of 0.51, 1.1, 2.2 and 3.3 µm. Experimental results were compared with computational fluid dynamics (CFD; using both Lagrangian and Eulerian approaches) predicted deposition efficiency and uniformity for a single row (N = 6) of cell culture inserts in the Vitrocell® 24/48 system. Deposited fluorescent monodisperse particles were quantified using fluorescent microscopy and Image J software. Experiments were conducted using a suspension of two particle MMADs with each experiment being conducted a total of three times on different days. The average experimentally measured deposition efficiency ranged from a low of 0.013% for 0.51 µm MMAD particles to a maximum 0.86% for 3.3 µm MMAD particles. There was good agreement between the average experimentally measured and the CFD predicted particle deposition efficiency (regardless of approach) with agreement being slightly better at the smaller MMADs. Experimentally measured and CFD predicted average uniformity of deposition was >45% of the mean and within 15% of the mean for 0.51 µm and 2.2 MMAD µm particles, respectively. Experimentally measured average uniformity of deposition was between 15 and 45% of the mean while CFD predictions were within 15% of the mean for 1.1 and 3.3 µm MMAD particles. The deposition efficiency and uniformity across the cell culture inserts for solid particles should be considered when designing exposure regimens using the Vitrocell® 24/48 ALI in vitro exposure system. Copyright © 2019 American Association for Aerosol Research [ABSTRACT FROM AUTHOR]

Summary: Objective: To evaluate the clinical efficacy and safety of quinidine in patients with KCNT1‐related epilepsy of infancy with migrating focal seizures (EIMFS) in the infantile period and to compare with the effect of quinidine on mutant channels in vitro. Methods: We identified 4 patients with EIMFS with onset in the neonatal period, pathogenic variants in the KCNT1 gene, and lack of response to AEDs. Patients were prospectively enrolled, treated with quinidine, and monitored according to a predefined protocol. Electroclinical, neuroimaging, and genetic data were reviewed. Two patients had novel variants in the KCNT1 gene that were modeled in Xenopus oocytes with channel properties characterized using electrophysiology recordings. Results: Three of four patients were treated with quinidine early in their disease course, prior to 6 months of age. No significant side effects were noted with quinidine therapy. In addition, there were no significant changes in electroencephalography (EEG)–confirmed seizure burden during therapy, and patients had near hourly seizures before, during, and after treatment. Two patients had previously reported gain‐of‐function mutations, which demonstrated sensitivity to high levels of quinidine in the oocyte assay. Two patients with novel variants, showed characteristic gain‐of‐function and were thus predicted to be pathogenic. Of interest, these variants were essentially insensitive to high levels of quinidine. Significance: Patients had no reported benefit to quinidine therapy despite age at treatment initiation. Pharmacogenetic results in oocytes were consistent with clinical treatment failure in 2 patients, suggesting that single‐dose pharmacologic assessment may be helpful in predicting which patients are exceedingly unlikely to achieve benefit with quinidine. In the 2 patients who had a lack of therapeutic benefit despite sensitivity to high concentrations of quinidine with in vitro oocyte assay, it is likely that the achievable exposure levels in the brain were too low to cause significant in vivo channel blockade. [ABSTRACT FROM AUTHOR]

Dosimetry models can be used to predict the dose of inhaled material, but they require several parameters including particle size distribution. The reported particle size distributions for aerosols from electronic nicotine delivery system (ENDS) products vary widely and don't always identify a specific product. A low-flow cascade impactor was used to determine the particle size distribution [mass median aerodynamic diameter (MMAD); geometric standard deviation (GSD)] from 20 different cartridge based ENDS products. To assess losses and vapor phase amount, collection efficiency of the system was measured by comparing the collected mass in the impactor to the difference in ENDS product mass. The levels of nicotine, glycerin, propylene glycol, water, and menthol in the formulations of each product were also measured. Regardless of the ENDS product formulation, the MMAD of all tested products was similar and ranged from 0.9 to 1.2 μm with a GSD ranging from 1.7 to 2.2. There was no consistent pattern of change in the MMAD and GSD as a function of number of puffs (cartridge life). The collection efficiency indicated that 9%–26% of the generated mass was deposited in the collection system or was in the vapor phase. The particle size distribution data are suitable for use in aerosol dosimetry programs. [ABSTRACT FROM AUTHOR]

Concerns have been raised about the potential health effects of potential bystander exposure to exhaled aerosols from e-vapor products (EVPs). An exhaled breath collection system (EBS) was developed and analytical methods were verified for collection and analysis of exhaled breath from users of EVPs. Analytical methods were adapted and verified for collection of environmental air samples during EVP use in an exposure chamber. Analysis of constituents in exhaled breath focused on nicotine, propylene glycol, and glycerin (because these are reported as the major constituents in EVPs) and selected carbonyl compounds (acetaldehyde, acrolein, and formaldehyde). Analysis of environmental samples included nicotine, propylene glycol, glycerin, 12 volatile organic compounds (VOCs), 15 carbonyl compounds and 4 metals. The EBS and analytical methods used were found to be suitable for collection and analysis of the target constituents in exhaled breath. Environmental sampling for background levels of VOCs and carbonyl compounds found only acetone, acetaldehyde, benzene, ethylbenzene, formaldehyde, isoprene, methyl ethyl ketone, hexaldehyde, propionaldehyde, and toluene above the limit of quantification in some samples. None of the targeted metals were detected. Background levels of VOCs and carbonyl compounds were consistent with levels previously reported for ambient air. [ABSTRACT FROM AUTHOR]

The transporter associated with antigen processing (TAP) is an ATP-binding cassette (ABC) transporter essential to cellular immunity against viral infection. Some persistent viruses have evolved strategies to inhibit TAP so that they may go undetected by the immune system. The herpes simplex virus for example evades immune surveillance by blocking peptide transport with a small viral protein ICP47. In this study, we determined the structure of human TAP bound to ICP47 by electron cryo-microscopy (cryo-EM) to 4.0 Å. The structure shows that ICP47 traps TAP in an inactive conformation distinct from the normal transport cycle. The specificity and potency of ICP47 inhibition result from contacts between the tip of the helical hairpin and the apex of the transmembrane cavity. This work provides a clear molecular description of immune evasion by a persistent virus. It also establishes the molecular structure of TAP to facilitate mechanistic studies of the antigen presentation process. [ABSTRACT FROM AUTHOR]

Site-specific dosimetry was used to address the hypothesis that, given the airway dimensions of one subject, the airway dimensions of a second subject can be obtained by comparing responses to a modified bronchoconstrictor inhalation-protocol. Fourteen healthy, nonsmoking subjects received a high-resolution computed tomography (HRCT) scan prior to performing a methacholine challenge and measurement of change in forced-expiratory-volume-in-one-second, ( ∆FEV 1 ). Scale factors of average length ( L ) and diameter ( d ) of the first six tracheobronchial airway generations were used to construct typical path lungs. The Multiple Path Particle Dosimetry (MPPD) model calculated surface density of methacholine. Airway circumference change, ∆C / C 0 , was used to calculate ∆FEV 1 in terms of airway smooth muscle sensitivity, K (cm/µg/cm 2 ). Virtual protocols were modeled such that fractional changes in ∆FEV 1, “∆Y” , were independent of K . Eight subjects responded to methacholine with ∆FEV 1 >3% and K s from 0.011 to 35.8. For one virtual protocol, the linear relations between ∆Y and length and diameter scale factors had R 2 s respectively of 0.49 and 0.57. Within the limits of these measurements, the hypothesis is demonstrated; — any continuously increasing function of ∆Y vs. scale factor being sufficient to allow (without HRCT scans) classification of subjects according to airway size. [ABSTRACT FROM AUTHOR]

From 2009-2014, incidental collections of terrestrial molluscs were made from the Far North of Ontario, Canada. Thirty-four species of terrestrial molluscs were identified from these collections, including one exotic species, Deroceras reticulatum, and three newly reported species from the Far North, Vallonia pulchella, Vertigo cf. genesii, and Gastrocopta similis. Vertigo cf. genesii is newly reported from Ontario. Some species have not been collected in Ontario for many years and some of these only once before. Subnational conservation ranks are discussed for higher-ranked species. [ABSTRACT FROM AUTHOR]

Purpose Approximately 20% of children with epilepsy are drug-resistant, incurring considerable costs. Epilepsy surgery has been shown to be an effective intervention in this population. This study provides an initial look at the costs associated with surgical management of children with drug-resistant epilepsy as compared with medical management alone. Procedures In a retrospective cohort study of children with drug-resistant epilepsy referred for possible surgical intervention, we compared direct costs of those treated surgically versus those offered surgery but managed medically instead. We also assessed the difference in seizure frequency between the two groups. Findings There were 94 total patients, 78 (83%) in the surgical group and 16 (17%) in the medical group. The median (25th-75th percentile) cost of the epilepsy surgery hospitalization was $118,400 ($101,900-$143,800). Total median annual follow-up costs, not including the cost of surgical hospitalization, were not significantly different between the two groups at 1- or 2-year follow-up. However, the surgical patients who were seizure-free at 1-year follow-up, and those that remained seizure-free at 2-year follow-up, had significantly lower costs compared with the medical group ($8000 versus $16,200, P = 0.04 and $4300 versus $7600, P = 0.05, respectively). The surgical group had significantly fewer seizures compared with the medical group at 1-year follow-up. Conclusions Although epilepsy surgery is expensive and the overall costs of surgical and medical management are similar in the first 2 years, patients who achieved seizure freedom after surgery had lower costs compared with those treated medically. [ABSTRACT FROM AUTHOR]

Xerolenta obvia is a Central European land snail that has been introduced to Ontario, Canada, where it was first recorded in the literature in 1975 from a single population at Bethany (City of Kawartha Lakes). Over the four decades since that publication, additional records have been found clustered near Bethany, within the City of Kawartha Lakes, neighbouring Peterborough and Northumberland counties, as well as the Regional Municipality of Durham. Two distant sites are also now known, one in rural Ottawa (southeastern Ontario), and one at Windsor, Essex County (southwestern Ontario). [ABSTRACT FROM AUTHOR]

The geographic distribution and habitat of Patera appressa (Say, 1821) in Canada are described. This is a North American land snail native to the southern Appalachians but it has been known, since 1994, from a cluster of sites in Trenton, City of Quinte West, Ontario, where it is presumably introduced. It may no longer be extant at any of the known Ontario sites. [ABSTRACT FROM AUTHOR]

A total of 20 commercial cigarette and 16 commercial smokeless tobacco products were assayed for 96 compounds listed as harmful and potentially harmful constituents (HPHCs) by the US Food and Drug Administration. For each product, a single lot was used for all testing. Both International Organization for Standardization and Health Canada smoking regimens were used for cigarette testing. For those HPHCs detected, measured levels were consistent with levels reported in the literature, however substantial assay variability (measured as average relative standard deviation) was found for most results. Using an abbreviated list of HPHCs, statistically significant differences for most of these HPHCs occurred when results were obtained 4–6 months apart (i.e., temporal variability). The assay variability and temporal variability demonstrate the need for standardized analytical methods with defined repeatability and reproducibility for each HPHC using certified reference standards. Temporal variability also means that simple conventional comparisons, such as two-sample t -tests, are inappropriate for comparing products tested at different points in time from the same laboratory or from different laboratories. Until capable laboratories use standardized assays with established repeatability, reproducibility, and certified reference standards, the resulting HPHC data will be unreliable for product comparisons or other decision making in regulatory science. [ABSTRACT FROM AUTHOR]

The geographic distribution of Strobilops aeneus Pilsbry, 1926, a rare species in Canada, is reviewed and all known records are mapped. Two recent records, the only ones since 1941, are reported from the province of Ontario. One of these records represents a small range extension ca. 85 km north of the closest previous site. Specimens identified as S. aeneus from Ontario, New Brunswick, and Nova Scotia were re-examined and found to be another species. [ABSTRACT FROM AUTHOR]

Context: In addition to tobacco and cigarette ingredients, there are many non-tobacco components and processes used to manufacture commercial cigarettes. Proposed cigarette components and manufacturing process changes were evaluated using a tiered toxicological testing program. Objective: The toxicological testing and evaluation of proposed changes to selected non-tobacco cigarette components and manufacturing processes are described in this special report. Materials and methods: Selected non-tobacco cigarette components and manufacturing processes were evaluated using experimental and control cigarettes. These experimental cigarettes were evaluated using mainstream smoke chemistry, bacterial mutagenicity and cytotoxicity assays. In some evaluations, 90-day nose-only mainstream smoke inhalation studies using rats were performed. Results: Some proposed design changes were not implemented, or limitations on their use were established. Most study results, however, were similar to those previously reported in the scientific literature for design changes in cigarette construction. Conclusion: The studies reported in the series of publication demonstrate that our testing approach is feasible for evaluation of cigarette component and manufacturing process changes. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters are molecular pumps that harness the chemical energy of ATP hydrolysis to translocate solutes across the membrane. The substrates transported by different ABC transporters are diverse, ranging from small ions to large proteins. Although crystal structures of several ABC transporters are available, a structural basis for substrate recognition is still lacking. For the Escherichia coli maltose transport system, the selectivity of sugar binding to maltose-binding protein (MBP), the periplasmic binding protein, does not fully account for the selectivity of sugar transport. To obtain a molecular understanding of this observation, we determined the crystal structures of the transporter complex MBP-MalFGK2 bound with large malto-oligosaccharide in two different conformational states. In the pretranslocation structure, we found that the transmembrane subunit MalG forms two hydrogen bonds with malto-oligosaccharide at the reducing end. In the outward-facing conformation, the transmembrane subunit MalF binds three glucosyl units from the nonreducing end of the sugar. These structural features explain why modified malto-oligosaccharides are not transported by MalFGK2 despite their high binding affinity to MBP. They also show that in the transport cycle, substrate is channeled from MBP into the transmembrane pathway with a polarity such that both MBP and MalFGK2 contribute to the overall substrate selectivity of the system. [ABSTRACT FROM AUTHOR]

Efficient carbon utilization is critical to the survival of microorganisms in competitive environments. To optimize energy usage, bacteria have developed an integrated control system to preferentially uptake carbohydrates that support rapid growth. The availability of a preferred carbon source, such as glucose, represses the synthesis and activities of proteins necessary for the transport and metabolism of secondary carbon sources. This regulatory phenomenon is defined as carbon catabolite repression. In enteric bacteria, the key player of carbon catabolite repression is a component of the glucose-specific phosphotransferase system, enzyme IIA (EIIAGlc). It is known that unphosphorylated EIIAGlc binds to and inhibits a variety of transporters when glucose is available. However, understanding the underlying molecular mechanism has been hindered by the complete absence of structures for any EIIAGlc-transporter complexes. Here we present the 3.9 Å crystal structure of Escherichia coli EIIAGlc in complex with the maltose transporter, an ATP-binding cassette (ABC) transporter. The structure shows that two EIIAGlc molecules bind to the cytoplasmic ATPase subunits, stabilizing the transporter in an inward-facing conformation and preventing the structural rearrangements necessary for ATP hydrolysis. We also show that the half-maximal inhibitory concentrations of the full-length EIIAGlc and an amino-terminal truncation mutant differ by 60-fold, consistent with the hypothesis that the amino-terminal region, disordered in the crystal structure, functions as a membrane anchor to increase the effective EIIAGlc concentration at the membrane. Together these data suggest a model of how the central regulatory protein EIIAGlc allosterically inhibits maltose uptake in E. coli. [ABSTRACT FROM AUTHOR]

For some individuals, the degree of response to inhaled allergens or the efficacy of inhaled pharmaceuticals may be influenced by their lung morphometry. Fourteen healthy, nonsmoking subjects received a high-resolution computed tomography (HRCT) scan just prior to performance of a methacholine challenge. Following the methacholine challenge, subjects were given a bronchodilator and received a second HRCT scan. HRCT scans were reconstructed and measurements of airway length, diameter, branch angle, and inclination to gravity were made for the first six tracheobronchial airway generations. The average length (L) and diameter (d) of each airway generation were used to calculate the average airway generation circumference (πd) and surface area (πLd). The product of airway circumference and surface area was incorporated into an airway volume factor (Ld2), inversely proportional to the fractional decrease in airway circumference due to deposited methacholine.Ld2was used to compare methacholine challenge responders with nonresponders. Six subjects did not respond to the methacholine challenge, while eight subjects responded with a greater than 3% decrease in their forced expiratory volume in 1 s. Compared with the nonresponders, these responders had smaller airway volume factors (from about the same in airway generation 1 to 36% smaller in airway generation 5). They also entered the study with less airway patency (the largest difference was in airway generation 5; 28% compared with 13% in the nonresponsive). Comparison of first and second (after bronchodilation) CT scans indicated that all subjects started the study with each airway generation having a unique degree of airway patency (2–33%). Copyright 2013 American Association for Aerosol Research [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp) is an ATP-binding cassette transporter that confers multidrug resistance in cancer cells. It also affects the absorption, distribution and clearance of cancer-unrelated drugs and xenobiotics. For these reasons, the structure and function of P-gp have been studied extensively for decades. Here we present biochemical characterization of P-gp from Caenorhabditis elegans and its crystal structure at a resolution of 3.4?ångströms. We find that the apparent affinities of P-gp for anticancer drugs actinomycin?D and paclitaxel are approximately 4,000 and 100 times higher, respectively, in the membrane bilayer than in detergent. This affinity enhancement highlights the importance of membrane partitioning when a drug accesses the transporter in the membrane. Furthermore, the transporter in the crystal structure opens its drug pathway at the level of the membrane's inner leaflet. In the helices flanking the opening to the membrane, we observe extended loops that may mediate drug binding, function as hinges to gate the pathway or both. We also find that the interface between the transmembrane and nucleotide-binding domains, which couples ATP hydrolysis to transport, contains a ball-and-socket joint and salt bridges similar to the ATP-binding cassette importers, suggesting that ATP-binding cassette exporters and importers may use similar mechanisms to achieve alternating access for transport. Finally, a model of human P-gp derived from the structure of C. elegans P-gp not only is compatible with decades of biochemical analysis, but also helps to explain perplexing functional data regarding the Phe335Ala mutant. These results increase our understanding of the structure and function of this important molecule. [ABSTRACT FROM AUTHOR]

Abstract: A tiered approach for testing ingredients in a cigarette matrix was developed and includes chemical–analytical testing and a standard battery of biological toxicity assays. These assays were adapted for comparative evaluation of mainstream smoke from experimental cigarettes with or without ingredients at various inclusion levels. This adaptation to test cigarette mainstream smoke may impact assay response. Since it is difficult to a priori determine discriminatory power, it was evaluated using a large experimental dataset from a multi-year program of cigarette ingredient testing performed at two separate laboratories. A statistical method, minimum detectable difference (MDD), was used as a measure of assay discriminatory power. MDD of cigarette smoke constituents ranged from 6% to 29% of the average. Salmonella mutagenicity and cytotoxicity test MDDs ranged from 20% to 81% and 18% to 49%, respectively. Body weight gain in 90-day nose-only inhalation studies yielded an MDD of 30–40%. Histopathological findings with severity scores between 0.5 and 1.5 had the lowest MDDs of 23% and higher. In general, discriminatory power decreased with increasing biological complexity and toxicological relevance of the assay. Beyond statistical analysis, however, a weight-of-the-evidence analysis by experienced researchers is required for toxicological assessment of a cigarette ingredient. [Copyright &y& Elsevier]

Background: Genomic datasets generated by new technologies are increasingly prevalent in disparate areas of biological research. While many studies have sought to characterize relationships among genomic features, commensurate efforts to characterize relationships among biological samples have been less common. Consequently, the full extent of sample variation in genomic studies is often under-appreciated, complicating downstream analytical tasks such as gene co-expression network analysis. Results: Here we demonstrate the use of network methods for characterizing sample relationships in microarray data generated from human brain tissue. We describe an approach for identifying outlying samples that does not depend on the choice or use of clustering algorithms. We introduce a battery of measures for quantifying the consistency and integrity of sample relationships, which can be compared across disparate studies, technology platforms, and biological systems. Among these measures, we provide evidence that the correlation between the connectivity and the clustering coefficient (two important network concepts) is a sensitive indicator of homogeneity among biological samples. We also show that this measure, which we refer to as cor(K,C), can distinguish biologically meaningful relationships among subgroups of samples. Specifically, we find that cor(K,C) reveals the profound effect of Huntington's disease on samples from the caudate nucleus relative to other brain regions. Furthermore, we find that this effect is concentrated in specific modules of genes that are naturally co-expressed in human caudate nucleus, highlighting a new strategy for exploring the effects of disease on sets of genes. Conclusions: These results underscore the importance of systematically exploring sample relationships in large genomic datasets before seeking to analyze genomic feature activity. We introduce a standardized platform for this purpose using freely available R software that has been designed to enable iterative and interactive exploration of sample networks. [ABSTRACT FROM AUTHOR]

The purpose of this paper is to document the spread of Dyssodia papposa (Asteraceae; Fetid Dogweed) along roadsides in eastern Canada. Fetid Dogweed is reported new to the provinces of Manitoba and Québec, Canada, and has greatly expanded its range along southern Ontario highways in the past 15 years. It is expected to continue to expand in northeastern North America. The distinctive appearance of this plant combined with earlier botanical fieldwork along Ontario highways makes it highly unlikely that this species was previously overlooked. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters convert chemical energy from ATP hydrolysis to mechanical work for substrate translocation. They function by alternating between two states, exposing the substrate-binding site to either side of the membrane. A key question that remains to be addressed is how substrates initiate the transport cycle. Using x-ray crystallography, we have captured the maltose transporter in an intermediate step between the inward- and outward-facing states. We show that interactions with substrate-loaded maltose-binding protein in the periplasm induce a partial closure of the MalK dimer in the cytoplasm. ATP binding to this conformation then promotes progression to the outward-facing state. These results, interpreted in light of biochemical and functional studies, provide a structural basis to understand allosteric communication in ABC transporters. [ABSTRACT FROM AUTHOR]

Context: Ingredients have been used in modern cigarette manufacturing to facilitate tobacco processing, provide flavor, and preserve tobacco. Concern has been raised regarding the use of ingredients in cigarette manufacturing due to the possible generation of toxic chemicals resulting from their combustion when added to tobacco. Objective: Investigate the impact of individual ingredients on cigarette smoke toxicity. Materials and methods: A total of 95 ingredients were tested individually through addition at different concentrations to the tobacco of experimental cigarettes. Mainstream cigarette smoke chemistry analysis, bacterial mutagenicity testing, and cytotoxicity testing were conducted. Additionally, 31 of the ingredients were tested in 90-day nose-only rat inhalation studies using mainstream cigarette smoke. Studies were designed following conventional toxicity testing methods employed for food additives and other consumer products. Results: The studies reported here demonstrate that high levels of some ingredients can change the quantity of some smoke constituents, altering the smoke chemistry profile. From the panel of biological endpoints monitored, these added ingredients produced minimal changes in the overall toxicity profile of mainstream cigarette smoke. In some cases, the addition of high levels of an ingredient caused a small reduction in toxicity findings, probably due to displacement of burning tobacco. Conclusion: The battery of testing results presented here is a useful addition to the available scientific information for cigarette ingredients and extends the dataset which can be used for evaluating their appropriate use. [ABSTRACT FROM AUTHOR]

Background: Menstrual cups have been available for decades, but their use is limited by bulky design and the need for multiple sizes. The Softcup® (Instead, Inc., San Diego, CA) is a simple single-size disposable over-the-counter (OTC) menstrual cup that compresses to tampon shape to facilitate insertion and can be worn during coitus. This report describes preclinical evaluation, clinical testing, and postmarketing monitoring of the Softcup. Methods: Preclinical testing complied with U.S. Food and Drug Administration (FDA) guidelines and used standard United States Pharmacopoeia methodologies for assessment of potential toxicity. Clinical testing enrolled 406 women in seven U.S. centers. A detailed written questionnaire assessed safety, acceptability, and effectiveness for menstrual collection. Study safety parameters included pelvic examinations, Pap smears, colposcopy, urinalysis, vaginal pH, wet mounts, gram stain, and vaginal microflora cultures. Postmarketing surveillance of over 100 million Softcups has been conducted by the manufacturer and by the FDA Medwatch system. Results: No toxicity or mutagenicity was observed in preclinical evaluations. In clinical testing, after three cycles of cup use, 37% of subjects rated the cup as better than, 29% as worse than, and 34% as equal to pads or tampons. The cup was preferred for comfort, dryness, and less odor. Cups received lower ratings for disposal and convenience. Eighty-one percent of enrolled women were able to insert and remove their first cup using only written instructions. Use difficulties resulting in study discontinuations included cramping (1%), leakage (1%), and improper fit (3%). No safety parameters were adversely affected. No significant health risks were reported during postmarketing surveillance. Conclusions: These results demonstrate that a single-size vaginal device has no significant health risks and is acceptable to many women without the need for fitting or other medical services. [ABSTRACT FROM AUTHOR]

Abstract: Propylene glycol (PG) is a humectant commonly used in cigarettes. Previous toxicological examinations of the effects on the addition of PG to tobacco used mixtures with several other flavoring agents. In the present work, toxicological comparisons were made of experimental cigarettes containing no added PG against otherwise similar cigarettes with three different amounts of PG added to the tobacco. The main toxicological comparison was a sub-chronic inhalation study with mainstream smoke in Sprague–Dawley rats (exposures of 150mg/m3 of total particulate matter, 6h exposure per day, for 90 consecutive days). The target PG concentrations in the tobacco of the four cigarette types were 0, 4, 7 and 10%. Additional studies with mainstream smoke were bacterial mutagenicity (5 Salmonella strains, both with and without metabolic activation, particulate phase only), cytotoxicity of both particulate and gas/vapor phases (using the neutral red uptake assay), and analytical chemistry (41 analytes). The graded inclusion of PG into experimental cigarettes resulted in increases in the smoke concentrations of propylene oxide, at very low concentrations. Broadly similar responses were seen across the four cigarette types, and the responses were similar to those previously described in the scientific literature. The addition of PG to experimental cigarettes reduced concentrations of some smoke components (e.g. nicotine), but had minimal effects on the biological responses. Most of the changes produced in the 90-days of exposure were resolved in a 42-day post-inhalation period. [Copyright &y& Elsevier]

Individuals with a proximal urea cycle disorder, such as carbamoyl phosphate synthetase deficiency 1 or ornithine transcarbamylase deficiency, may present with encephalopathy resulting from hyperammonemia. The clinical presentation of arginase deficiency is considerably different, characterized by progressive spasticity involving the lower extremities and usually dementia. Diagnosis may be delayed, and patients are often thought to have cerebral palsy. The true etiology of brain injury in arginase deficiency is unknown, but is not thought to be due to hyperammonemia and brain swelling, the mechanism of injury recognized in ornithine transcarbamylase deficiency. Elevated arginine could augment nitric oxide synthesis, leading to oxidative damage. The hypothesis for the present study was that specific brain vulnerability in arginase deficiency would involve microstructural alterations in corticospinal tracts and that this finding, as measured by diffusion tensor imaging, would differ from age-matched control subjects and those with ornithine transcarbamylase deficiency. Diffusion tensor imaging data were compared for a 17-year-old male patient with arginase deficiency, age-matched normal control subjects, and age-matched individuals with ornithine transcarbamylase deficiency. Significant differences were found in suspected areas of interest, specifically in the corticospinal tracts. This finding confirms the hypothesis that the mechanism of injury in arginase deficiency, although still unknown, is unlikely to be similar to that causing ornithine transcarbamylase deficiency. [ABSTRACT FROM AUTHOR]

Cigarette mainstream smoke (MS) is a dynamic aerosol consisting of a gas–vapor phase and a particulate phase. In recent years, novel in vitro whole smoke exposure systems have been developed to expose cells directly to whole MS. One such system is the Burghart Mimic Smoker-01 (MSB-01). Our previous data using the MSB-01 indicated that a 50 ± 10% loss of particulate matter occurred prior to MS delivery into the exposure chamber. Additionally, a change in aerosol particle diameter was also measured, suggesting that the chemical composition of MS might be changing within the system. In this study, we have expanded on our previous work and compared the particulate phase chemical composition of undiluted and diluted MS generated by the instrument and that of the MS delivered into the exposure chamber. The average percent delivery of cigarette smoke condensate (CSC) detected for all the measured chemical constituents was 35 ± 13% for undiluted MS and 23 ± 8% for 1:1 diluted MS. The data also indicate that under our experimental conditions, incomplete mixing of the freshly generated MS occurs during its dilution by the system. Taken together, the data presented here show that significant chemical changes occur between the generation of MS by the system and its delivery into the exposure chamber. This indicates that due to the dynamic nature of cigarette smoke, it is important to characterize the exposure conditions in order to gain the best insight and accurately correlate exposure with biological endpoints. [ABSTRACT FROM AUTHOR]

The use of respiratory tract dosimetry predictions to estimate the desired aerosol exposure concentration required for a specific target deposited dose in animal inhalation studies has been hindered by: (1) a lack of species/strain specific quantitative respiratory tract anatomy; and (2) verification by comparison of calculated and experimentally measured deposited doses. Using recent literature on tracheobronchial and pulmonary respiratory tract anatomy, dosimetry predictions for the Balb/c mouse were compared with deposited doses for 0.5, 1.0, and 2.0 micrometer diameter aerosols in a newly available nose-only exposure system. Spatial and temporal aerosol port to port uniformity of this nose-only exposure system was within ± 10% of the mean. Dosimetry predictions were in agreement with the measured mean deposited doses for the aerosols tested. [ABSTRACT FROM AUTHOR]

Nicotine’s dose and rate of delivery to the brain play an important role in its addiction and cardiovascular effects. Nicotine is mainly present in the particulate phase of cigarette smoke, and since particle size distribution controls the deposition behavior of particles in the respiratory tract, changes in the particle size distribution can produce variations in its regional and total dose to the lung. These variations can change its absorption rate and delivery to the brain. The particle size distribution of mainstream smoke (MS) varies with changes in puffing regimen and cigarette design and composition. This study examined nicotine in different particle size fractions of MS generated from 2R4F, Marlboro Medium, and Quest1 cigarettes using 3 puffing regimens: (1) FTC-like puff, 35 ml over 2 s; (2) short puff, 50 ml over 2 s; and (3) long puff, 100 ml over 10 s. MS was generated in a chamber at 37°C and>95% relative humidity (RH), and size-segregated particles were collected using RJR cascade impactors. Particle size distribution was determined by spectrophotometry. Nicotine was anayzed using gas chromatography and mass spectrometry. Results showed that nicotine speciates in larger particles (1.1–1.9 μm diameter) under the long puffing regimen and in smaller particles (0.4–1.1 μm diameter) under the short puffing regimen, while mass median aerodynamic diameter of mainstream smoke particles was found to be approximately constant (0.9–1.0 μm) for the three puffing regimens. Overall, changes in puffing regimen have a significant effect on particle size distribution of nicotine and its deposited dose. [ABSTRACT FROM AUTHOR]

Summary: ATP-binding cassette transporters couple ATP hydrolysis to substrate translocation through an alternating access mechanism, but the nature of the conformational changes in a transport cycle remains elusive. Previously we reported the structure of the maltose transporter MalFGK2 in an outward-facing conformation in which the transmembrane (TM) helices outline a substrate-binding pocket open toward the periplasmic surface and ATP is poised for hydrolysis along the closed nucleotide-binding dimer interface. Here we report the structure of the nucleotide-free maltose transporter in which the substrate binding pocket is only accessible from the cytoplasm and the nucleotide-binding interface is open. Comparison of the same transporter crystallized in two different conformations reveals that alternating access involves rigid-body rotations of the TM subdomains that are coupled to the closure and opening of the nucleotide-binding domain interface. The comparison also reveals that point mutations enabling binding protein-independent transport line dynamic interfaces in the TM region. [Copyright &y& Elsevier]

In vitro systems are frequently used to study mechanisms of mainstream cigarette smoke (MS)-induced lung injury. Traditional methods of exposure involve the capture of MS particulate phase with filter pads or bubbling MS through phosphate buffered saline (PBS) or cell culture medium. Although useful for in vitro experiments, these exposure methods may fail to capture potential interactions between the gas and particulate phases. To better understand the effect of MS on the human airway, in vitro whole smoke exposure systems that utilize freshly generated whole smoke are needed. Here we report the characterization of a new in vitro whole smoke exposure system (Burghart Mimic Smoker-01 (MSB-01)). This system uses a smoke distribution manifold to simultaneously deliver MS to each well of a 96-well plate. Intraday and interday variations for particulate matter deposition were less than 5% and 13% respectively. Cytotoxicity measurements using lung epithelial BEAS-2B cells indicate variations in calculated EC50 (half maximal effective concentration) values of 13% intraday and 20% interday. Smoke particulate losses and changes in particle size distribution were also analyzed. The data indicate that 45-50% of the MS generated at the smoking ports is lost within the system prior to delivery into the exposure chamber; however, no changes in particle size distribution were detected throughout the system. Overall, the MSB-01 reproducibly delivered mainstream cigarette smoke in a dose dependent manner across the multiwell plate. The MSB-01 is a high throughput system capable of exposing cells to both the MS particulate and gas/vapor phases simultaneously. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation. [Copyright &y& Elsevier]

The enormous complexity of the human brain ultimately derives from a finite set of molecular instructions encoded in the human genome. These instructions can be directly studied by exploring the organization of the brain's transcriptome through systematic analysis of gene coexpression relationships. We analyzed gene coexpression relationships in microarray data generated from specific human brain regions and identified modules of coexpressed genes that correspond to neurons, oligodendrocytes, astrocytes and microglia. These modules provide an initial description of the transcriptional programs that distinguish the major cell classes of the human brain and indicate that cell type–specific information can be obtained from whole brain tissue without isolating homogeneous populations of cells. Other modules corresponded to additional cell types, organelles, synaptic function, gender differences and the subventricular neurogenic niche. We found that subventricular zone astrocytes, which are thought to function as neural stem cells in adults, have a distinct gene expression pattern relative to protoplasmic astrocytes. Our findings provide a new foundation for neurogenetic inquiries by revealing a robust and previously unrecognized organization to the human brain transcriptome. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder affecting millions of elderly individuals worldwide. Advances in the genetics of AD have led to new levels of understanding and treatment opportunities. Here, we used a systems biology approach based on weighted gene coexpression network analysis to determine transcriptional networks in AD. This method permits a higher order depiction of gene expression relationships and identifies modules of coexpressed genes that are functionally related, rather than producing massive gene lists. Using this framework, we characterized the transcriptional network in AD, identifying 12 distinct modules related to synaptic and metabolic processes, immune response, and white matter, nine of which were related to disease progression. We further examined the association of gene expression changes with progression of AD and normal aging, and were able to compare functional modules of genes defined in both conditions. Two biologically relevant modules were conserved betweenADand aging, one related to mitochondrial processes such as energy metabolism, and the other related to synaptic plasticity. We also identified several genes that were central, or hub, genes in both aging and AD, including the highly abundant signaling molecule 14.3.3 ζ(YWHAZ), whose role in AD and aging is uncharacterized. Finally, we found that presenilin 1 (PSEN1) is highly coexpressed with canonical myelin proteins, suggesting a role for PSEN1 in aspects of glial-neuronal interactions related to neurodegenerative processes. [ABSTRACT FROM AUTHOR]

Abstract: Simulations of fine aerosols (100nm ) in the respiratory tract are challenging due to low particle deposition rates and the combined effects of diffusional and inertial deposition mechanisms. Furthermore, validations of fine respiratory aerosol deposition are difficult due to a lack of localized experimental data. The objective of this study is to develop and test a continuous two-phase model for simulating the regional and local deposition of dilute fine aerosols in an idealized double bifurcation segment of the respiratory tract. To evaluate the developed transport model, novel in vitro deposition results for 400nm particles have been determined in a double bifurcation geometry of respiratory generations G3–G5. In addition, previously reported local deposition characteristics for aerosols have also been considered. Computational two-phase models that have been evaluated include a standard chemical species (CS) mass fraction approximation, the drift flux (DF) approach to account for finite particle inertia, and two novel extensions of the DF model to correct for near-wall particle velocity. The first velocity correction model (DF-VC1) applies a continuous field solution for particle slip at the wall surface. As an alternative, a sub-grid near-wall Lagrangian solution has been proposed as the DF-VC2 model. Localized experimental results for the deposition of 400nm particles indicated elevated deposition contours ranging from 1% to 5% of total deposition at the first bifurcation and 0.1–1% at the second. Of the computational models tested, the DF-VC2 method provided the best match to experimental deposition values on a regional and highly localized basis. Specifically, the DF-VC2 model matched total experimental deposition results to within 10% for both 400nm and particles. Considering the local deposition of fine aerosols, the DF-VC2 model matched the experimentally determined elevated contours at the first and second bifurcations for both 400nm and particles. In conclusion, a DF particle transport model with near-wall velocity corrections appears to provide a highly effective solution for the deposition of fine respiratory aerosols. [Copyright &y& Elsevier]

The maltose uptake system of Escherichia coli is a well-characterized member of the ATP-binding cassette transporter superfamily. Here we present the 2.8-Å crystal structure of the intact maltose transporter in complex with the maltose-binding protein, maltose and ATP. This structure, stabilized by a mutation that prevents ATP hydrolysis, captures the ATP-binding cassette dimer in a closed, ATP-bound conformation. Maltose is occluded within a solvent-filled cavity at the interface of the two transmembrane subunits, about halfway into the lipid bilayer. The binding protein docks onto the entrance of the cavity in an open conformation and serves as a cap to ensure unidirectional translocation of the sugar molecule. These results provide direct evidence for a concerted mechanism of transport in which solute is transferred from the binding protein to the transmembrane subunits when the cassette dimer closes to hydrolyse ATP. [ABSTRACT FROM AUTHOR]

Comparisons of gene expression between human and non-human primate brains have identified hundreds of differentially expressed genes, yet translating these lists into key functional distinctions between species has proved difficult. Here we provide a more integrated view of human brain evolution by examining the large-scale organization of gene coexpression networks in human and chimpanzee brains. We identify modules of coexpressed genes that correspond to discrete brain regions and quantify their conservation between the species. Module conservation in cerebral cortex is significantly weaker than module conservation in sub-cortical brain regions, revealing a striking gradient that parallels known evolutionary hierarchies. We introduce a method for identifying species-specific network connections and demonstrate how differential network connectivity can be used to identify key drivers of evolutionary change. By integrating our results with comparative genomic sequence data and estimates of protein sequence divergence rates, we confirm a number of network predictions and validate these findings. Our results provide insights into the molecular bases of primate brain organization and demonstrate the general utility of weighted gene coexpression network analysis. [ABSTRACT FROM AUTHOR]

In order to better understand aerosol dynamics and deposition in the complex flow field of the respiratory tract, both in vitro experiments and numerical modeling techniques have widely been employed. Computational fluid dynamics (CFD) modeling offers the flexibility of easily modifying system parameters such as flow rates, particle sizes, system geometry, and heterogeneous outlet conditions. However, a number of numerical errors and artifacts can lead to nonphysical CFD results. Experimental methods offer the advantage of physical realism; however, parameter variation is often difficult. The objective of this study is to illustrate the use of CFD to enhance the understanding of experimental results. In parallel, the selected experimental results have been used to partially validate the CFD predictions. A specific case study has been considered focusing on 1-μm particle depositions in a physiologically realistic bifurcation (PRB) model of respiratory generations 3–5. Previous experiments in this system report a deposition rate of approximately 0.01%. An in-depth CFD analysis has been employed to evaluate two cases of the empirical model. The first case consists of only the PRB double bifurcation geometry. The second case includes a portion of the experimental particle delivery system, which may influence the entering velocity and particle profiles. To assess the influence of upstream transition and turbulence, each of the two cases considered has been evaluated using laminar and low Reynolds number k –ω approximations. Results indicate that both upstream flow effects and turbulent or transitional flow play a significant role in determining the deposition of 1-μm particles in the model considered. Simulating upstream flow effects and laminar flow was required to match the empirically reported deposition fraction and provided a two orders of magnitude improvement over initial CFD estimates. This study highlights the need to consider the effects of experimental particle generation systems on velocity and particle profiles entering respiratory models. Future work is necessary to investigate the mechanisms responsible for the experimentally observed local deposition patterns. [ABSTRACT FROM AUTHOR]

Computational fluid dynamic (CFD) techniques have provided unprecedented opportunity for investigating inhaled particle deposition in realistic human airway geometries. Several recent articles describing local aerosol deposition predictions based upon “validated” CFD models have highlighted the challenges in validating local aerosol deposition predictions. These challenges include: (1) defining what is meant by validation; (2) defining appropriate experimental data for validation; and (3) determining when the agreement is not fortuitous. The term validation has numerous meanings, depending on the field and context in which it is used. For example, in computer programming it means the code executes as intended, to the experimentalist it means predicted results agree with matched experimental measurements, and to the risk assessor it implies that predictions using new parameters can be trusted. Based on the current literature it is not clear that a consensus exists for what constitutes a validated CFD model. It is also not clear what types of experimental data are needed or how closely the CFD input values and experimental conditions should be matched (similar or identical airway geometries, entrance airflow, or aerosol profiles) to validate CFD derived predictions. Due to the complexity of CFD computer codes and the multiplicity of deposition mechanisms, it is possible that total aerosol deposition may be accurately predicted and the resulting local particle deposition patterns are incorrect, or vice versa. Specific examples and suggestions for several challenges to experimentalists and modelers are presented. [ABSTRACT FROM AUTHOR]

Most current deterministic computer dosimetry models use idealized dichotomous symmetrical infant, child, and adolescent tracheobronchial geometries for dosimetry predictions. These tracheobronchial geometries were derived from either the morphometric measurements of J. D. Mortensen and colleagues or those of R. F. Phalen and colleagues. The airway growth curves used to create these geometries are typically means from all airways within a single generation in the tracheobronchial tree. This idealized approach implies that the growth rate of all airways in the same generation is identical (symmetric growth, SG), which is unlikely. In this study, existing morphometric measurements of airway dimensions were used to determine growth equations for airway length, diameter, branch angle, and inclination to gravity as a function of body height for each airway in the first four tracheobronchial generations. Tracheobronchial geometries (asymmetrical growth, AG) were created from these growth curves for the first 4 tracheobronchial generations representing a 4- and 11-yr-old child and an adult. Particle deposition predictions for steady inspiratory flow using 0.1- to 20-μm diameter monodisperse particles in these AG geometries were compared with predictions using the SG geometries using the uniform growth rate approach. Each airway in the first four generations had a unique airway length and diameter growth rate. Airway branch angle and inclination to gravity did not change as a function of body height. The unique airway growth rates resulted in significant differences in predicted deposition efficiency between the AG and SG geometry. Use of AG geometry may provide improved estimates of regional particle deposition in infants, children, and adolescents. [ABSTRACT FROM AUTHOR]

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The nucleocapsid (N) protein plays an essential role in SARS-CoV genome packaging and virion assembly. We have previously shown that SARS-CoV N protein forms a dimer in solution through its C-terminal domain. In this study, the crystal structure of the dimerization domain, consisting of residues 270-370, is determined to 1.75 Å resolution. The structure shows a dimer with extensive interactions between the two subunits, suggesting that the dimeric form of the N protein is the functional unit in vivo. Although lacking significant sequence similarity, the dimerization domain of SARS-CoV N protein has a fold similar to that of the nucleocapsid protein of the porcine reproductive and respiratory syndrome virus. This finding provides structural evidence of the evolutionary link between Coronaviridae and Arteriviridae, suggesting that the N proteins of both viruses have a common origin. [ABSTRACT FROM AUTHOR]

Lipoxygenases (LOXs) catalyze the regio- and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. We report here the 3.2 Å resolution structure of 8R-LOX from the Caribbean sea whip coral Plexaura homomalla, a LOX isozyme with calcium dependence and the uncommon R chiral stereospecificity. Structural and spectroscopic analyses demonstrated calcium binding in a C2-like membrane-binding domain, illuminating the function of similar amino acids in calcium-activated mammalian 5-LOX, the key enzyme in the pathway to the pro-inflammatory leukotrienes. Mutation of Ca2+-ligating amino acids in 8R-LOX resulted not only in a diminished capacity to bind membranes, as monitored by fluorescence resonance energy transfer, but also in an associated loss of Ca2+-regulated enzyme activity. Moreover, a structural basis for R chiral specificity is also revealed; creation of a small oxygen pocket next to Gly428 (Ala in all S-LOX isozymes) promoted C-8 oxygenation with R chirality on the activated fatty acid substrate. [ABSTRACT FROM AUTHOR]

8R-Lipoxygenase and allene oxide synthase (AOS) are parts of a naturally occurring fusion protein from the coral Plexaura homomalla. AOS catalyses the production of an unstable epoxide (an allene oxide) from the fatty acid hydroperoxide generated by the lipoxygenase activity. Here, we report the structure of the AOS domain and its striking structural homology to catalase. Whereas nominal sequence identity between the enzymes had been previously described, the extent of structural homology observed was not anticipated, given that this enzyme activity had been exclusively associated with the P450 superfamily, and conservation of a catalase fold without catalase activity is unprecedented. Whereas the heme environment is largely conserved, the AOS heme is planar and the distal histidine is flanked by two hydrogen-bonding residues. These critical differences likely facilitate the switch from a catalatic activity to that of a fatty acid hydroperoxidase. [ABSTRACT FROM AUTHOR]