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Rayleigh-scatter lidar observations were made at the Atmospheric Lidar Observatory (ALO) at Utah State University (USU) from 1993–2004 from 45–90 km. The lidar operated at 532 nm with a power-aperture-product (PAP) of ~3.1 Wm2. The sensitivity of the lidar has since been increased by a factor of 66 to 205 Wm2, extending the maximum altitude into new territory, the lower thermosphere. Observations have been extended up to 115 km, almost to the 120 km goal. Early temperatures from four ~4-week periods starting in June 2014 are presented and discussed. They are compared to each other, to the ALO climatology from the original lidar [1], and to temperatures from the NRLMSISe00 empirical model [2].

Abstract Background The joint analysis of several categorical variables is a common task in many areas of biology, and is becoming central to systems biology investigations whose goal is to identify potentially complex interaction among variables belonging to a network. Interactions of arbitrary complexity are traditionally modeled in statistics by log-linear models. It is challenging to extend these to the high dimensional and potentially sparse data arising in computational biology. An important example, which provides the motivation for this article, is the analysis of so-called full-length cDNA libraries of alternatively spliced genes, where we investigate relationships among the presence of various exons in transcript species. Results We develop methods to perform model selection and parameter estimation in log-linear models for the analysis of sparse contingency tables, to study the interaction of two or more factors. Maximum Likelihood estimation of log-linear model coefficients might not be appropriate because of the presence of zeros in the table's cells, and new methods are required. We propose a computationally efficient ℓ1-penalization approach extending the Lasso algorithm to this context, and compare it to other procedures in a simulation study. We then illustrate these algorithms on contingency tables arising from full-length cDNA libraries. Conclusion We propose regularization methods that can be used successfully to detect complex interaction patterns among categorical variables in a broad range of biological problems involving categorical variables.

Abstract Background RNA metabolism, through 'combinatorial splicing', can generate enormous structural diversity in the proteome. Alternative domains may interact, however, with unpredictable phenotypic consequences, necessitating integrated RNA-level regulation of molecular composition. Splicing correlations within transcripts of single genes provide valuable clues to functional relationships among molecular domains as well as genomic targets for higher-order splicing regulation. Results We present tools to visualize complex splicing patterns in full-length cDNA libraries. Developmental changes in pair-wise correlations are presented vectorially in 'clock plots' and linkage grids. Higher-order correlations are assessed statistically through Monte Carlo analysis of a log-linear model with an empirical-Bayes estimate of the true probabilities of observed and unobserved splice forms. Log-linear coefficients are visualized in a 'spliceprint,' a signature of splice correlations in the transcriptome. We present two novel metrics: the linkage change index, which measures the directional change in pair-wise correlation with tissue differentiation, and the accuracy index, a very simple goodness-of-fit metric that is more sensitive than the integrated squared error when applied to sparsely populated tables, and unlike chi-square, does not diverge at low variance. Considerable attention is given to sparse contingency tables, which are inherent to single-gene libraries. Conclusion Patterns of splicing correlations are revealed, which span a broad range of interaction order and change in development. The methods have a broad scope of applicability, beyond the single gene – including, for example, multiple gene interactions in the complete transcriptome.

Background: New therapies have revolutionized the treatment of Crohn's disease (CD), but in some countries, the surgery rate has not changed, the frequency of emergency surgery is underestimated, and surgical risk is poorly studied., Aims: The aim of this study was to identify risk factors and clinical indications for primary surgery in CD patients at the tertiary hospital., Methods: This was a retrospective cohort of a prospectively collected database of 107 patients with CD from 2015 to 2021. The main outcomes were the incidence of surgery treatment, types of procedures performed, surgical recurrence, surgery free time, and risk factors for surgery., Results: Surgical intervention was performed in 54.2% of the patients, and most of the procedures were emergency surgeries (68.9%). The elective procedures (31.1%) were performed over 11 years after diagnosis. The main indications for surgery were ileal stricture (34.5%) and anorectal fistulas (20.7%). The most frequent procedure was enterectomy (24.1%). Recurrence surgery was most common in emergency procedures (OR 2.1; 95%CI 1.6-6.6). Montreal phenotype L1 stricture behavior (RR 1.3; 95%CI 1.0-1.8, p=0.04) and perianal disease (RR 1.43; 95%CI 1.2-1.7) increased the risk of emergency surgeries. The multiple linear regression showed age at diagnosis as a risk factor for surgery (p=0.004). The study of surgery free time showed no difference in the Kaplan-Meier curve for Montreal classification (p=0.73)., Conclusions: The risk factors for operative intervention were strictures in ileal and jejunal diseases, age at diagnosis, perianal disease, and emergency indications.

We describe rapid spread of multidrug-resistant gram-negative bacteria among patients in dedicated coronavirus disease care units in a hospital in Maryland, USA, during May-June 2020. Critical illness, high antibiotic use, double occupancy of single rooms, and modified infection prevention practices were key contributing factors. Surveillance culturing aided in outbreak recognition and control.

In this 2-phase real-world evaluation of chlorhexidine gluconate (CHG) skin concentrations in intensive care unit patients, we found lower skin CHG concentrations when rinsing with water after CHG solution bath (compared with no rinse), but no significant difference in concentrations between the use of CHG solution without rinse and preimpregnated CHG wipes. CHG concentration audits could be useful in assessing the quality of bathing practice, and CHG solution without rinsing may be an alternative to preimpregnated CHG wipes., (Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Objective: To identify factors associated with the development of surgical site infection (SSI) among adult patients undergoing renal transplantation, Design: A retrospective cohort study., Setting: An urban tertiary care center in Baltimore, Maryland, with a well-established renal transplantation program that performs ~200-250 renal transplant procedures annually., Results: At total of 441 adult patients underwent renal transplantation between January 1, 2010, and December 31, 2011. Of these 441 patients, 66 (15%) developed an SSI; of these 66, 31 (47%) were superficial incisional infections and 35 (53%) were deep-incisional or organ-space infections. The average body mass index (BMI) among this patient cohort was 29.7; 84 (42%) were obese (BMI >30). Patients who developed an SSI had a greater mean BMI (31.7 vs 29.4; P=.004) and were more likely to have a history of peripheral vascular disease, rheumatologic disease, and narcotic abuse. History of cerebral vascular disease was protective. Multivariate analysis showed BMI (odds ratio [OR] 1.06; 95% confidence interval [CI], 1.02-1.11) and past history of narcotic use/abuse (OR, 4.86; 95% CI, 1.24-19.12) to be significantly associated with development of SSI after controlling for National Healthcare Surveillance Network (NHSN) score and presence of cerebrovascular, peripheral vascular, and rheumatologic disease., Conclusions: We identified higher BMI as a risk factor for the development of SSI following renal transplantation. Notably, neither aggregate comorbidity scores nor NHSN risk index were associated with SSI in this population. Additional risk adjustment measures and research in this area are needed to compare SSIs across transplant centers.

To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.

Alternative splicing of pre-mRNA may generate many distinct proteins from a single gene: regulation of alternative exon selection constitutes control of molecular structure downstream of transcription. Identifying natural splice variants among hundreds or thousands of theoretical alternatives, and examining the regulation of exon selection at multiple sites, may require screening many full-length cDNAs. We describe methods for preparing full-length cDNA libraries comprising the splice variants from single genes. The methods employ robust long distance reverse transcription, gene-specific second strand synthesis, long PCR, and cloning: with these methods cDNAs coding full-length open reading frames were prepared for 21 ion channels (1.2-15 kb). Exon combinations in isolated clones are determined by multiplex PCR. Approximately 85% of the clones contain full-length inserts. Screening can detect even rare variants (0.1%) in linear proportion to their abundance in initial mRNA pools. Tissue-specific expression patterns are reproducible. We describe methods for quantifying and minimizing artifactual exon recombination by template switching. These methods can be used to generate thousands of full-length clones of even large transcripts (>8 kb) for the systematic identification of splice variants and the analysis of regulation of alternative exon selection., (Copyright 2000 Academic Press.)

The structure of CACNA1I, the gene encoding alpha1I, a human brain T Ca2+ channel alpha1 subunit, was determined by comparison of polymerase chain reaction-amplified brain cDNA and genomic sequences. The gene consists of at least 36 exons spanning at least 115,168 basepairs of chromosome 22q12.3-13.2. The predicted protein has 2016 amino acids and 28 potential phosphorylation sites. Alternative splicing of the gene occurs at two sites: cassette exon 9 and an alternative acceptor in exon 33. Molecular diversity generated by alternative splicing and post-translational modification of this and other members of the T alpha1 subunit gene family may account for the observed heterogeneity of T currents in central neurons.

Experiments designed to identify Na pump structural features which tag the molecule for asymmetric cell-surface localization are inherently complex because either subunit, or both, may contain targeting information and because the cells which recognize those targeting signals and maintain asymmetric plasma membrane domains also express their own Na pumps, the subunits of which can assemble into hybrid pump molecules with pump subunits expressed from transfected cDNA clones. Cotransfecting cDNA for both subunits only complicates matters further by resulting in expression of four distinct dimeric molecular species. To eliminate the potential for cross-assembly in these and other experiments we have constructed cDNA encoding a "single-subunit" Na pump (called "alpha-beta") in which the alpha and beta subunits are joined by a linker of 17 amino acids. By all criteria tested alpha-beta functioned as a normal heterodimeric Na pump. It was expressed in a variety of mammalian cell lines as a single, high molecular weight polypeptide located primarily on the surface membrane, with the beta subunit exposed to the extracellular medium. Binding of the conformation-sensitive monoclonal antibody 24 to the beta subunit indicated that the fusion protein was folded as a properly "assembled" sodium pump. Expression of alpha-beta in ouabain-resistant mouse L cells resulted in high affinity ouabain binding and ouabain-sensitive, sodium-dependent rubidium transport. The enzyme was properly targeted to the basolateral plasma membrane in polarized epithelial cells. The functional integrity of the fusion protein renders it suitable for site-directed mutagenesis studies of targeting and enzymology where control of subunit assembly is desired. These results also support topological models in which the carboxyl terminus of the alpha subunit is cytoplasmic.

Previous studies have shown that the short-motif electroplax Na channel is phosphorylated in vitro by cyclic AMP-dependent protein kinase (PKA) at serines 6 or 7 and 1776 and threonine 17 (Emerick & Agnew, 1989). We here show that phosphatase treatment of solubilized, purified Na channels enhanced subsequent PKA labeling of four of five tryptic phosphopeptides, indicating that these sites are phosphorylated in vivo. Microsequencing and analysis of PTH-amino acid products revealed endogenous labeling of serines 6, 444, 1680, and 1776. Serines 1680 and 1776 lie in the carboxyl-terminal cytoplasmic domain, while serine 6 lies in the amino terminus and serine 444 is in the cytoplasmic loop between domains I and II. Endogenous phosphorylation of serine 6 establishes experimentally that the Na channel amino terminus is cytoplasmic. In electrophysiological experiments, brief exposure of inside-out membrane patches excised from Sachs-organ cells to MgATP and purified PKA catalytic subunit produced rapid, sustained reduction of Na current amplitude by approximately 80% and a hyperpolarizing shift in the conductance/voltage relation by 10-12 mV. The effect was absent in controls omitting either PKA or MgATP. Serines 6 and 1776 and threonine 17 are labeled rapidly and extensively in vitro, and only threonine 17 appears to be unphosphorylated in vivo. We suggest that phosphorylation of the amino and carboxyl domains, perhaps especially at threonine 17, underlies the demonstrated downregulation of the electroplax Na channel.

A fluorescence assay for measuring Na channel activation in liposomes containing voltage-sensitive Na channels isolated from Electrophorus electricus is described. The assay is based on transport of a heavy-metal cation, T1+, through the activated channel to quench fluorescence of an internalized, water-soluble chromophore. The channel is "locked" in a chronically opened configuration with alkaloid neurotoxins such as veratridine or batrachotoxin. Diffusion potentials are used to amplify the signal, and enlarged liposomes (greater than 8000 A) result in time courses extended to the range of seconds. Analysis of the kinetics of quenching yields parameters that behave as linear functions of channel activation and reflect vesicle size and channel abundance. The k1/2's for activation by veratridine and batrachotoxin were 5 microM and 169 nM, respectively, and that for tetrodotoxin blockade was 4 nM. Externally applied QX-222 and tetrodotoxin each acted to partially block the stimulated signal, as expected for compounds that act on oppositely oriented channels in the membrane. Single-channel conductances estimated with either veratridine or batrachotoxin ranged between 0.6 and 40.7 pS, corresponding to transport numbers of (1.2 X 10(5)) to (8.1 X 10(6)) ions s-1 channel-1 under the conditions of assay. The assay is approximately 100-fold more sensitive than radiotracer influx assays, requiring 1 fmol of protein per time course.

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including alpha-(2----8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis alpha-(2----8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3200 pmol of [3H]TTX-binding sites/mg of protein and a single polypeptide of approximately 285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

The voltage-sensitive sodium channel from the electroplax of Electrophorus electricus is selectively phosphorylated by the catalytic subunit of cyclic-AMP-dependent protein kinase (protein kinase A) but not by protein kinase C. Under identical limiting conditions, the protein was phosphorylated 20% as rapidly as the synthetic model substrate kemptamide. A maximum of 1.7 +/- 0.6 equiv of phosphate is incorporated per mole. Phosphoamino acid analysis revealed labeled phosphoserine and phosphothreonine at a constant ratio of 3.3:1. Seven distinct phosphopeptides were identified among tryptic fragments prepared from radiolabeled, affinity-purified protein and resolved by HPLC. The three most rapidly labeled fragments were further purified and sequenced. Four phosphorylated amino acids were identified deriving from three consensus phosphorylation sites. These were serine 6, serine 7, and threonine 17 from the amino terminus and a residue within 47 amino acids of the carboxyl terminus, apparently serine 1776. The alpha-subunits of brain sodium channels, like the electroplax protein, are readily phosphorylated by protein kinase A. However, these are also phosphorylated by protein kinase C and exhibit a markedly different pattern of incorporation. Each of three brain alpha-subunits displays an approximately 200 amino acid segment between homologous repeat domains I and II, which is missing from the electroplax and skeletal muscle proteins [Noda et al. (1986) Nature (London) 320, 188; Kayano et al. (1988) FEBS Lett. 228, 1878; Trimmer et al. (1989) Neuron 3, 33]. Most of the phosphorylation of the brain proteins occurs on a cluster of consensus phosphorylation sites located in this segment. This contrasts with the pattern of highly active sites on the amino and carboxyl termini of the electroplax protein. The detection of seven labeled tryptic phosphopeptides compared to the maximal labeling stoichiometry of approximately 2 suggests that many of the acceptor sites on the protein may be blocked by endogenous phosphorylation.