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Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air–liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

During evolution of fungi, the separate tryptophan synthetase alpha and beta polypeptides of bacteria appear to have been fused in the order alpha-beta rather than the beta-alpha order that would be predicted from the order of the corresponding structural genes in all bacteria. We have fused the tryptophan synthetase polypeptides of Escherichia coli in both orders, alpha-beta and beta-alpha, with and without a short connecting (con) sequence, to explore possible explanations for the domain arrangement in fungi. We find that proteins composed of any of the four fused polypeptides, beta-alpha, beta-con-alpha, alpha-beta, and alpha-con-beta, are highly active enzymatically. However, only the alpha-beta and alpha-con-beta proteins are as active as the wild type enzyme. All four fusion proteins appear to be less soluble in vivo than the wild type enzyme; this abnormal characteristic is minimal for the alpha-con-beta enzyme. The alpha and beta domains of the four fusion polypeptides were not appreciably more heat labile than the wild type polypeptides. Competition experiments with mutant tryptophan synthetase alpha protein, and the fusion proteins suggest that in each fusion protein the joined alpha and beta domains have a functional tunnel connecting their alpha and beta active sites. Three tryptophan synthetase beta'-alpha fusion proteins were examined in which the carboxyl-terminal segment of the wild type beta polypeptide was deleted and replaced by a shorter, unnatural sequence. The resulting deletion fusion proteins were enzymatically inactive and were found predominantly in the cell debris. Evaluation of our findings in relation to the three-dimensional structure of the tryptophan synthetase enzyme complex of Salmonella typhimurium (5) and the results of mutational analyses with E. coli suggest that tryptophan synthetase may have evolved via an alpha-beta rather than a beta-alpha fusion because in beta-alpha fusions the amino-terminal helix of the alpha chain cannot assume the conformation required for optimal enzymatic activity.

Dramatic changes in smoking behaviour occurred over the half century since cigarette smoking was identified as a cause of human disease, and these changes are a major public health accomplishment.1 Perhaps no component of this change has been more dramatic than the reduction in people’s involuntary exposure to tobacco smoke pollution. The fraction of indoor workers protected by a total ban on smoking in the workplace has risen from 3% in 19862 to nearly 70% in 1999,3 and serum cotinine levels in non-smokers declined 70% between 1988 and 1998.4 Much of this progress can be attributed to the courageous and determined actions of individuals applying our advances in the medical sciences. This issue both celebrates those individuals and details their accomplishments. Almost immediately following Surgeon General Jesse Steinfeld’s articulation of involuntary smoking as a public health issue and his declaration of a non-smokers bill of rights,5 airlines became the leading edge of a societal change that would de-normalise smoking and protect non-smokers from exposure to tobacco smoke. As presented by Holm and Davis6 in this issue, aircraft were one of the first public places to have separate smoking and non-smoking sections, and regulation of airline travel would continue to lead the rest of society toward banning smoking in the work environment for the next two decades. Efforts to regulate exposure to tobacco smoke in air travel culminated in the elimination of all smoking on domestic flights lasting less than two hours in 1988, a total ban on domestic flights in 1990, and a ban on international flights in 2000. Progress on airplanes was a highly visible …

Cigarettes with lower machine measured "tar" and nicotine yields have been marketed as "safer" than high tar products over the last four decades, but there is conflicting evidence about the impact of these products on the disease burden caused by smoking. This paper critically examines the epidemiological evidence relevant to the health consequences of "reduced yield" cigarettes. Some epidemiological studies have found attenuated risk of lung cancer but not other diseases, among people who smoke "reduced yield" cigarettes compared to smokers of unfiltered, high yield products. These studies probably overestimate the magnitude of any association with lung cancer by over adjusting for the number of cigarettes smoked per day (one aspect of compensatory smoking), and by not fully considering other differences between smokers of "high yield" and "low yield" cigarettes. Selected cohort studies in the USA and UK show that lung cancer risk continued to increase among older smokers from the 1950s to the 1980s, despite the widespread adoption of lower yield cigarettes. The change to filter tip products did not prevent a progressive increase in lung cancer risk among male smokers who began smoking during and after the second world war compared to the first world war era smokers. National trends in vital statistics data show declining lung cancer death rates in young adults, especially males, in many countries, but the extent to which this is attributable to "reduced yield" cigarettes remains unclear. No studies have adequately assessed whether health claims used to market "reduced yield" cigarettes delay cessation among smokers who might otherwise quit, or increase initiation among non-smokers. There is no convincing evidence that past changes in cigarette design have resulted in an important health benefit to either smokers or the whole population. Tobacco control policies should not allow changes in cigarette design to subvert or distract from interventions proven to reduce the prevalence, intensity, and duration of smoking. Keywords: tar and nicotine; lung cancer; changing cigarette

In previous studies, we have shown that calcitonin gene-related peptide (CGRP) acutely inhibits 45Ca2+ uptake in osteoblastic UMR106 cells, and we have proposed that ATP-sensitive potassium (K(ATP)) channels are involved in mediating this action of CGRP. To directly test this proposed mechanism, we have now examined the effects of CGRP on both membrane potential (Em) and K+ mobilization in UMR106 cells, using specific fluorometric dye assays. CGRP (0.01-100 nM) induced membrane hyperpolarization in a dose-dependent manner, with a half maximal effect (ED50) at approximately 0.2 nM and a maximal effect at 100 nM. Both pinacidil (Pina; a K(ATP) channel opener) and forskolin (FSK) induced similar membrane hyperpolarization, but the actions of these three agents could be easily distinguished: both CGRP and Pina actions were well antagonized by glibenclamide (Glib; a selective K(ATP) channel blocker), whereas FSK action was strongly attenuated only by tetraethylammonium (a K(Ca) channel blocker) or compound H-89 (an inhibitor of cAMP-dependent protein kinases). Cells pretreated with Pina no longer responded to CGRP, but they could still respond to FSK; furthermore, pretreatment with FSK failed to block successive treatment with either CGRP or Pina. In parallel with observed changes in Em, CGRP (0.01-100 nM) decreased intracellular K+ concentrations ([K+]i) in a dose-dependent manner, with an ED50 identical to that obtained for alterations in Em. This action of CGRP was sensitive to Glib and had only slight sensitivity to tetraethylammonium; this CGRP effect was mimicked by Pina but not by FSK. Interestingly, CGRP significantly elicited changes in cell shape by a Glib-sensitive mechanism that included notable decreases in cross-sectional cytoplasmic area. These observations strongly support our proposal that CGRP primarily stimulates K+ efflux via activation of K(ATP) channels and thereby induces membrane hyperpolarization in UMR106 cells. Furthermore, our data also suggest that this cascade of initial cellular events may result in rapid changes in cell morphology and decreases in cellular area of the type that are thought to act as triggers for proliferation and/or differentiation in many cellular phenotypes.

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The cpdB gene encodes a periplasmic 2',3'-cyclic phosphodiesterase (3'-nucleotidase). This enzyme has been purified previously and the gene is located at 96 min on the Escherichia coli chromosome. In this study the cpdB gene was cloned from ClaI-cleaved DNA, and the gene product was identified. DNA blotting experiments showed that the recombinant plasmid contains a deletion with respect to the expected genomic fragment of approximately 4 kilobases, which extends into the vector. Furthermore, the gene was absent from three other recombinant libraries. Together, these findings suggest the presence in the genome of an adjacent gene whose product is lethal when it is present on a multicopy plasmid. The nucleotide sequence of the cpdB gene was also determined. The 5' and 3' untranslated sequences contain characteristic sequences that are involved in the initiation and termination of transcription, including two possible promoters, one of which may contain two overlapping -10 sequences. A strong Shine-Dalgarno sequence is followed by an open reading frame which corresponds to a protein having a molecular weight of 70,954. The first 19 amino acid residues have the characteristics of a signal peptide. The 3' untranslated sequence contains two putative rho-independent transcription terminators having low thermodynamic stability.

Rat liver glucosidase II, an endoplasmic reticulum hydrolase involved in the biosynthesis of the N-linked class of glycoproteins, has been purified in good yield to a state approaching homogeneity. The purified enzyme hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, 4-methylumbelliferyl-alpha-D-glucopyranoside, maltose, and the precursor oligosaccharides glucose1-2mannose9N-acetylglucosamine, but it does not act on glucose3mannose9N-acetylglucosamine or p-nitrophenyl-beta-D-glucopyranoside. The ratio of the rate at which glucose is released from p-nitrophenyl-alpha-D-glucopyranoside to that from glucose2mannose9N-acetylglucosamine or glucose1mannose9N-acetylglucosamine remains constant throughout the 8-step purification procedure; thus it appears that a single enzyme is responsible for the activities toward both the artificial and oligosaccharide substrates. The fact that the enzyme cleaves both of the inner 1,3-linked glucosyl residues from the precursor oligosaccharides supports the view that they are linked in the alpha-configuration. The pH dependence of enzymatic activity is quite similar for different substrates, showing a broad optimum between pH 6 and 7.5. Activity toward p-nitrophenyl-alpha-D-glucopyranoside is enhanced by 12 mM 2-deoxy-D-glucose (260-300% activation) and 25 mM mannose (150% activation), but these two compounds inhibit the action of the enzyme toward the precursor oligosaccharides. By isoelectrofocusing the purified enzyme exhibits one form, which has a pI of 3.5-3.8. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicates that glucosidase II has a subunit molecular weight of 65,000. Ferguson plot analysis of the behavior of native enzyme in polyacrylamide gels indicates that it is a 262,000-dalton tetramer. Gel filtration gives a molecular weight of 288,000. Several lines of evidence indicate that the enzyme is a glycoprotein.

By testing serum samples taken between 1980 and 1984 from men attending a department of sexually transmitted diseases, it was shown that antibodies to human immunodeficiency virus (HIV) first appeared in 1981. Homosexual men were significantly more likely to have antibodies to HIV and to cytomegalovirus (CMV) than were heterosexual men attending the same clinic. This shows that homosexuals are exposed to both HIV, the cause of the acquired immune deficiency syndrome (AIDS), and to CMV, which can reactivate to cause life threatening disease once immunosuppression has developed. All homosexuals, not just those with antibodies against HIV, had raised levels of CMV antibodies. This suggests that they experience frequent antigenic stimulation after reinfections with CMV or reactivation of endogenous virus.

In [23], Schiffer and Spencer prove that all small deformations of complex structure on a compact Riemann surface may be realized by altering the complex structure only within an arbitrarily small neighborhood of a point on the surface. It seems interesting in general to consider whether it is possible to construct deformations of an algebraic variety or complex manifold from deformations of neighborhoods of certain subvarieties. Further motivation for trying to understand the role of subvarieties in deformations is suggested by the instability under deformation of the Neron-Severi group, i.e., the group of divisors modulo numerical equivalence. As an example, one may consider the family of affine surfaces Vt: x 2 + y 2 + z 2 = t z (t is a parameter; V o has a nodal singularity at the origin). This family admits a resolution {Xt} --, {V~}, with {X,} a smooth family of non-singular surfaces, and each X~ is a minimal resolution of V t ([4] or [5]). The exceptional curve E in X o is a IP 1 with self-intersection 2 which does not appear in any Xt, for t#:0. One may ask whether every smooth surface X with such a curve in it admits a one-parameter family of deformations arising from this local model. Furthermore, if X contains several disjoint such curves, does each one independently contribute one dimension to the moduli of X? The Hartogs ' theorem of [14] says that one cannot simply plumb in the local deformation, leaving the structure of X unchanged outside a small neighborhood of E. Moreover, old examples of Segre [26] show that the nodes on certain hypersurfaces V in IP 3 are not "independent", i.e., there aren't enough deformations of the resolution X of V to allow for a one-dimensional contribution from each node. Theorem (3.7) of this paper says that the regularity of the Kuranishi variety of X is sufficient for the deformations of X to realize independently

As a first step in studying the biosynthesis of the peptide hormone calcitonin, we have identified procalcitonin species in CA-77 cells, a newly developed rat medullary thyroid carcinoma cell line. mRNA extracted from the cells directed the synthesis of a putative procalcitonin in a reticulocyte lysate translation system containing microsomal membranes. Both this species and a radiolabeled form of immunoreactive calcitonin from intact cells had the same retention time during reverse phase high performance liquid chromatography. The putative cellular procalcitonin was also immunoprecipitated by antiserum to a synthetic peptide whose sequence constitutes the COOH-terminal 16 residues of preprocalcitonin. The polypeptide had a Mr = 13,400, as estimated by gel filtration chromatography under denaturing conditions. Microsequencing of the [35S]methionine-labeled polypeptide indicated that residues 13, 32, and 34 of procalcitonin were methionine. Similar analysis of the peptide labeled with [3H]proline indicated that residues 2 and 11 of the precursor were proline. The positions of methionine and proline could be aligned in a unique manner with the NH2-terminal half of the preprocalcitonin sequence inferred from cDNA analyses. These results indicate that procalcitonin consists of 111 amino acids and suggest that a 25-residue signal sequence is cotranslationally cleaved from preprocalcitonin. From the procalcitonin sequence we can now predict the sequence of likely biosynthetic intermediates and mature secretory products derived from the NH2-terminal as well as COOH-terminal regions of the precursor.

The complete nucleotide sequence of the Neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure. A comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae (encoded by the TRP5 gene) and Escherichia coli (encoded by the trpA and trpB genes) shows that the A and B domains (amino acid segments homologous to the trpA and trpB polypeptides, respectively) of the N. crassa and yeast polypeptides are in the same order (NH2-A-B-COOH). This arrangement is the reverse of the gene order characteristic of all prokaryotes that have been examined. N. crassa tryptophan synthetase has strong homology to the yeast TRP5 polypeptide (A domains have 54% identity; B domains have 75% identity), and somewhat weaker homology to the E. coli trpA and trpB polypeptides (A domains have 31% identity; B domains have 50% identity). The two domains of the N. crassa polypeptide are linked by a connector of 54-amino acid residues that has less than 25% identity to the 45-residue connector of the yeast polypeptide, although secondary structure analysis predicts both connectors would be alpha-helical. In contrast to the yeast TRP5 gene, which has no introns, the trp-3 coding region is interrupted by two introns 77 and 71 nucleotides in length. Both introns are located near the 5'-end of the gene and therefore not near the segment encoding the connector.

Fluorene, C 13 H 10 , crystallizes in the orthorhombic system, space group Pnam with four molecules per unit cell. The molecule possesses a plane of symmetry which is parallel to the (001) plane of the crystal. The structure has been determined by trial-and-error methods, followed (i) by a least-squares refinement and (ii) by two-dimensional Fourier syntheses. The results from the two methods of refinement are compared and for (ii) a method is given for systematically correcting for overlapping of atoms in projections. The accuracy of the co-ordinates given by these methods is discussed in detail, and it is concluded that the co-ordinates adopted are correct to ±0.017 Å. The carbon skeleton is planar to within the experimental error and the equation of the plane is y = 1.4375 x + 11.0947.

The co-ordinates of the carbon and hydrogen atoms and the anisotropic thermal vibrations of the carbon atoms of chrysene have been determined by three-dimensional least-squares refinement and by three-dimensional Fourier, and difference Fourier, syntheses. The value of R = Ʃ || F o | - | F c ||/Ʃ| F o for over 1000 planes observed with Cu Kα radiation is 0·076. The standard deviations of the carbon atom co-ordinates are less than 0·0033 Å. One C–C bond is significantly longer than the mean and longer than would be expected by any theoretical consideration. The value 1·465 Å can be explained by overcrowding of the hydrogen atoms attached to the carbon atoms concerned. The ‘K’ bond of the phenanthrene nucleus has a length of 1·365 Å and this is in the same benzene ring and diametrically opposite to the very long bond mentioned above. It is shown that the thermal vibrations obtained for the individual carbon atoms can be well represented by considering the molecule to vibrate as a rigid body.

As a first step in studying the biosynthesis of the peptide hormone calcitonin, we have identified procalcitonin species in CA-77 cells, a newly developed rat medullary thyroid carcinoma cell line. mRNA extracted from the cells directed the synthesis of a putative procalcitonin in a reticulocyte lysate translation system containing microsomal membranes. Both this species and a radiolabeled form of immunoreactive calcitonin from intact cells had the same retention time during reverse phase high performance liquid chromatography. The putative cellular procalcitonin was also immunoprecipitated by antiserum to a synthetic peptide whose sequence constitutes the COOH-terminal 16 residues of preprocalcitonin. The polypeptide had a Mr = 13,400, as estimated by gel filtration chromatography under denaturing conditions. Microsequencing of the [35S]methionine-labeled polypeptide indicated that residues 13, 32, and 34 of procalcitonin were methionine. Similar analysis of the peptide labeled with [3H]proline indicated that residues 2 and 11 of the precursor were proline. The positions of methionine and proline could be aligned in a unique manner with the NH2-terminal half of the preprocalcitonin sequence inferred from cDNA analyses. These results indicate that procalcitonin consists of 111 amino acids and suggest that a 25-residue signal sequence is cotranslationally cleaved from preprocalcitonin. From the procalcitonin sequence we can now predict the sequence of likely biosynthetic intermediates and mature secretory products derived from the NH2-terminal as well as COOH-terminal regions of the precursor.

The complete nucleotide sequence of the Neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure. A comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae (encoded by the TRP5 gene) and Escherichia coli (encoded by the trpA and trpB genes) shows that the A and B domains (amino acid segments homologous to the trpA and trpB polypeptides, respectively) of the N. crassa and yeast polypeptides are in the same order (NH2-A-B-COOH). This arrangement is the reverse of the gene order characteristic of all prokaryotes that have been examined. N. crassa tryptophan synthetase has strong homology to the yeast TRP5 polypeptide (A domains have 54% identity; B domains have 75% identity), and somewhat weaker homology to the E. coli trpA and trpB polypeptides (A domains have 31% identity; B domains have 50% identity). The two domains of the N. crassa polypeptide are linked by a connector of 54-amino acid residues that has less than 25% identity to the 45-residue connector of the yeast polypeptide, although secondary structure analysis predicts both connectors would be alpha-helical. In contrast to the yeast TRP5 gene, which has no introns, the trp-3 coding region is interrupted by two introns 77 and 71 nucleotides in length. Both introns are located near the 5'-end of the gene and therefore not near the segment encoding the connector.