Validation of N95 respirator models for pressure drop and particle capture efficiency.

Despite efforts to apply administrative and engineering controls to minimize worker exposure to aerosols, filtering facepiece respirators (FFRs) continue to be an important form of personal protective equipment in hard-to-control settings such as healthcare, agriculture, and construction. Optimizing the performance of FFRs can be advanced with the use of mathematical models that incorporate the forces that act on particles during filtration as well as those filter characteristics that influence filter pressure drop. However, a thorough investigation of these forces and characteristics using measurements of currently available FFRs has not been undertaken. Filter characteristics such as fiber diameter and filter depth were measured from samples taken from six currently-available N95 FFRs from three manufacturers. A filtration model was developed that included diffusion, inertial and electrostatic forces to estimate the filtration of an aerosol with a Boltzmann charge distribution. The diameter of the filter fibers was modeled as either a single "effective" diameter or as a lognormal distribution of diameters. Both modeling schemes produced an efficiency curve that simulated efficiency measurements made over a range of particle diameters (0.01 - 0.3 µm) with the use of a scanning mobility particle sizer in the region where efficiency is at a minimum. However, the method using a distribution of fiber diameters produced a better fit for particles > 0.1 µm. The coefficients associated with a simple form of the diffusion equation constituting a power law incorporating the Peclet number were adjusted to enhance model accuracy. Likewise, the fiber charge of the electret fibers was also adjusted to maximize model fit but remained within levels reported by others. A filter pressure drop model was also developed. Results demonstrated the need for a pressure drop model applicable to N95s relative to existing models developed with the use of fibers with larger diameters than those used in current N95 FFRs. A set of N95 FFR characteristics are provided that can be used to develop models of typical N95 FFR filter performance and pressure drop in future studies.

Gender differences in publication in emergency medicine journals.

BACKGROUND: Some studies have suggested gender disparities in both pay and academic promotion which may adversely affect salary and career progression for female physicians. The areas of research output, funding, and authorship have not been fully and systematically examined in the emergency medicine literature. We hypothesize that gender differences may exist in research output, impact, authorship, and funding. METHODS: We conducted a cross-sectional study examining all published articles in the top three emergency medicine journals as determined by Impact Factor between February 2015 and February 2018. We compared the authorship, number of citations of each article, funding, and h-index of each author by gender. RESULTS: Of the 10,118 authors representing 4166 original articles in our sample, 7562 (74.7%) were male and 2556 (25.3%) were female, with females underrepresented relative to the known proportion of female emergency medicine faculty. Males were proportionally more likely to be last authors (OR 1.65, 95% CI, 1.47-1.86) and less likely to be first authors than females (OR 0.85, 95% CI, 0.77-0.94). No difference in proportions of males and females in terms of being named as having funding was found (OR 1.02, 95% CI, 0.78-1.35). Males had higher h-indexes than females (5 vs. 3, p < .001) as well as a higher average number of citations (OR 1.068, 95% CI, 1.018-1.119). CONCLUSIONS: Males outnumber females in terms of numbers of publications, but also in number of citations, h-index, and last authorship. Future studies on physician gender disparities in emergency medicine need to account for these population differences.

Design and coverage of high throughput genotyping arrays optimized for individuals of East Asian, African American, and Latino race/ethnicity using imputation and a novel hybrid SNP selection algorithm.

Four custom Axiom genotyping arrays were designed for a genome-wide association (GWA) study of 100,000 participants from the Kaiser Permanente Research Program on Genes, Environment and Health. The array optimized for individuals of European race/ethnicity was previously described. Here we detail the development of three additional microarrays optimized for individuals of East Asian, African American, and Latino race/ethnicity. For these arrays, we decreased redundancy of high-performing SNPs to increase SNP capacity. The East Asian array was designed using greedy pairwise SNP selection. However, removing SNPs from the target set based on imputation coverage is more efficient than pairwise tagging. Therefore, we developed a novel hybrid SNP selection method for the African American and Latino arrays utilizing rounds of greedy pairwise SNP selection, followed by removal from the target set of SNPs covered by imputation. The arrays provide excellent genome-wide coverage and are valuable additions for large-scale GWA studies.

Next generation genome-wide association tool: design and coverage of a high-throughput European-optimized SNP array.

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion.

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with muM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.

The recognition pathway for the DNA cytosine methyltransferase M.HhaI.

Enzymatic sequence-specific DNA modification involves multiple poorly understood intermediates. DNA methyltransferases like M.HhaI initially bind nonspecific DNA and then selectively bind and modify a unique sequence. High-resolution NMR was used to map conformational changes occurring in M.HhaI upon binding nonspecific DNA, a one base pair altered noncognate DNA sequence, and both hemimethylated and unmethylated cognate DNA sequences. Comparisons with previous NMR studies of the apoenzyme and enzyme-cofactor complex provide snapshots of the pathway to sequence-specific complex formation. Dramatic chemical shift perturbations reaching many distal sites within the protein are detected with cognate DNA, while much smaller changes are observed upon nonspecific and noncognate DNA binding. A cooperative rather than stepwise transition from a nonspecific to a cognate complex is revealed. Furthermore, switching from unmethylated to hemimethylated cognate DNA involves detectable protein conformational changes 20-30 A away from the methyl group, indicating high protein sensitivity and plasticity to DNA modification.

Long-range structural and dynamical changes induced by cofactor binding in DNA methyltransferase M.HhaI.

The bacterial DNA cytosine methyltransferase M.HhaI sequence-specifically modifies DNA in an S-adenosylmethionine dependent reaction. The enzyme stabilizes the target cytosine (GCGC) into an extrahelical position, with a concomitant large movement of an active site loop involving residues 80-99. We used multidimensional, transverse relaxation-optimized NMR experiments to assign nearly 80% of all residues in the cofactor-bound enzyme form, providing a basis for detailed structural and dynamical characterization. We examined details of the previously unknown effects of the cofactor binding with M.HhaI in solution. Addition of the cofactor results in numerous structural changes throughout the protein, including those decorating the cofactor binding site, and distal residues more than 30 A away. The active site loop is involved in motions both on a picosecond to nanosecond time scale and on a microsecond to millisecond time scale and is not significantly affected by cofactor binding except for a few N-terminal residues. The cofactor also affects residues near the DNA binding cleft, suggesting a role for the cofactor in regulating DNA interactions. The allosteric properties we observed appear to be closely related to the significant amount of dynamics and dynamical changes in response to ligand binding detected in the protein.

Mechanism of O2 activation by cytochrome P450cam studied by isotope effects and transient state kinetics.

The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.

Characterization of 3'hExo, a 3' exonuclease specifically interacting with the 3' end of histone mRNA.

The 3' end of mammalian histone mRNAs consisting of a conserved stem-loop and a terminal ACCCA interacts with a recently identified human 3' exonuclease designated 3'hExo. The sequence-specific interaction suggests that 3'hExo may participate in the degradation of histone mRNAs. ERI-1, a Caenorhabditis elegans homologue of 3'hExo, has been implicated in degradation of small interfering RNAs. We introduced a number of mutations to 3'hExo to identify residues required for RNA binding and catalysis. To assure that the introduced mutations specifically target one of these two activities of 3'hExo rather than cause global structural defects, the mutant proteins were tested in parallel for the ability both to bind the stem-loop RNA and to degrade RNA substrates. Our analysis confirms that 3'hExo is a member of the DEDDh family of 3' exonucleases. Specific binding to the RNA requires the SAP domain and two lysines located immediately to its C terminus. 3'hExo binds with the highest affinity to the wild-type 3' end of histone mRNA, and any changes to this sequence reduce efficiency of binding. 3'hExo has only residual, if any, 3' exonuclease activity on DNA substrates and localizes mostly to the cytoplasm, suggesting that in vivo it performs exclusively RNA-specific functions. Efficient degradation of RNA substrates by 3'hExo requires 2' and 3' hydroxyl groups at the last nucleotide. 3'hExo removes 3' overhangs of small interfering RNAs, whereas the double-stranded region is resistant to the enzymatic activity.

A CPSF-73 homologue is required for cell cycle progression but not cell growth and interacts with a protein having features of CPSF-100.

Formation of the mature 3' ends of the vast majority of cellular mRNAs occurs through cleavage and polyadenylation and requires a cleavage and polyadenylation specificity factor (CPSF) containing, among other proteins, CPSF-73 and CPSF-100. These two proteins belong to a superfamily of zinc-dependent beta-lactamase fold proteins with catalytic specificity for a wide range of substrates including nucleic acids. CPSF-73 contains a zinc-binding histidine motif involved in catalysis in other members of the beta-lactamase superfamily, whereas CPSF-100 has substitutions within the histidine motif and thus is unlikely to be catalytically active. Here we describe two previously unknown human proteins, designated RC-68 and RC-74, which are related to CPSF-73 and CPSF-100 and which form a complex in HeLa and mouse cells. RC-68 contains the intact histidine motif, and hence it might be a functional counterpart of CPSF-73, whereas RC-74 lacks this motif, thus resembling CPSF-100. In HeLa cells RC-68 is present in both the cytoplasm and the nucleus whereas RC-74 is exclusively nuclear. RC-74 does not interact with CPSF-73, and neither RC-68 nor RC-74 is found in a complex with CPSF-160, indicating that these two proteins form a separate entity independent of the CPSF complex and are likely involved in a pre-mRNA processing event other than cleavage and polyadenylation of the vast majority of cellular pre-mRNAs. RNA interference-mediated depletion of RC-68 arrests HeLa cells early in G(1) phase, but surprisingly the arrested cells continue growing and reach the size typical of G(2) cells. RC-68 is highly conserved from plants to humans and may function in conjunction with RC-74 in the 3' end processing of a distinct subset of cellular pre-mRNAs encoding proteins required for G(1) progression and entry into S phase.

Statistical coevolution analysis and molecular dynamics: identification of amino acid pairs essential for catalysis.

Molecular dynamics (MD) simulations of HhaI DNA methyltransferase and statistical coupling analysis (SCA) data on the DNA cytosine methyltransferase family were combined to identify residues that are coupled by coevolution and motion. The highest ranking correlated pairs from the data matrix product (SCA.MD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 A and form a clear focal point centered near the active site. We suggest that these distal anti-correlated pairs are involved in mediating active-site compressions that may be important for catalysis. Mutants that disrupt the implicated interactions support the validity of our combined SCA.MD approach.

Steady-state kinetic investigation of cytochrome P450cam: interaction with redox partners and reaction with molecular oxygen.

Cytochrome P450cam (CYP101) is a prokaryotic monooxygenase that requires two proteins, putidaredoxin reductase (PdR) and putidaredoxin (Pdx), to supply electrons from NADH. This study addresses the mechanism by which electrons are transported from PdR to P450cam through Pdx and used to activate O(2) at the heme of P450cam. It is shown that k(cat)/Km(O2) is independent of the PdR concentration and hyperbolically dependent on Pdx. The phenomenon of saturation of reaction rates with either P450cam or PdR at high ratios of one enzyme to the other is investigated and shown to be consistent with a change in the rate limiting step. Either the reduction of Pdx by PdR (high P450) or the reduction of P450 by Pdx (high PdR) determines the rate. These data support a mechanism where Pdx acts as a shuttle for transport of electrons from PdR to P450cam, effectively ruling out the formation of a kinetically significant PdR/Pdx/P450cam complex.

Cloning and characterization of the Drosophila U7 small nuclear RNA.

Base pairing between the 5' end of U7 small nuclear RNA (snRNA) and the histone downstream element (HDE) in replication-dependent histone pre-mRNAs is the key event in 3'-end processing that leads to generation of mature histone mRNAs. We have cloned the Drosophila U7 snRNA and demonstrated that it is required for histone pre-mRNA 3'-end processing in a Drosophila nuclear extract. The 71-nt Drosophila U7 snRNA is encoded by a single gene that is embedded in the direct orientation in an intron of the Eip63E gene. The U7 snRNA gene contains conserved promoter elements typical of other Drosophila snRNA genes, and the coding sequence is followed by a 3' box indicating that the Drosophila U7 snRNA gene is an independent transcription unit. Drosophila U7 snRNA contains a trimethyl-guanosine cap at the 5' end and a putative Sm-binding site similar to the unique Sm-binding site found in other U7 snRNAs. Drosophila U7 snRNA is approximately 10 nt longer than mammalian U7 snRNAs because of an extended 5' sequence and has only a limited potential to form a stem-loop structure near the 3' end. The extended 5' end of Drosophila U7 snRNA can base pair with the HDE in all five Drosophila histone pre-mRNAs. Blocking the 5' end of the U7 snRNA with a complementary oligonucleotide specifically blocks processing of a Drosophila histone pre-mRNA. Changes in the HDE that abolish or decrease processing efficiency result in a reduced ability to recruit U7 snRNA to the pre-mRNA.