SHORT PALATE LUNG AND NASAL EPITHELIUM 1 AND AIRWAY DISEASE

Airway disease such as asthma and infection is the cause substantial morbidity and mortality in the world today. Although modern medicine has developed many drugs for these conditions, these diseases remain highly prevalent and are often difficult to treat. Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is an abundant multi-functional protein in the airway. It has been reported to have immune-modulatory, surfactant and anti-microbial functions, and it regulates the airway surface liquid (ASL) height through the epithelial sodium channel (ENaC). This study focuses on utilizing SPLUNC1’s protective properties in combatting airway disease. Airway hyperresponsiveness (AHR) is a characteristic feature of asthma, yet its pathophysiology is still poorly understood. SPLUNC1 is dysregulated in allergic rhinitis and chronic rhinosinusitis with nasal polyps. However, SPLUNC1 regulation in asthmatics has not been investigated. Here, we show that in allergic asthmatic humans and house dust mite (HDM)-allergic mice, SPLUNC1 in the bronchoalveolar lavage (BAL) is reduced. We demonstrate that administration of SPLUNC1 to mice decreases their AHR and show that the molecular basis for this effect involves the coordination of the N-terminus with an electrostatic patch on the protein’s body. We propose that SPLUNC1 be further investigated for use in reducing AHR. Pseudomonas aeruginosa, a primary lung pathogen in nosocomial pneumonia and in lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease (COPD), causes considerable morbidity and mortality. SPLUNC1 has been shown to neutralize and combat P. aeruginosa in vivo and in vitro. Here, we sought to establish a model for evaluating delivery of exogenous SPLUNC1 in acute lung infection and provide evidence that preemptive administration of SPLUNC1 may decrease bacterial burden. Lastly, we suggest that the administration of SPLUNC1 which we propose for asthma and lung infection results in SPLUNC1 mediated SPLUNC1 release in the lungs, effectively increasing the local protein concentration. This effect may utilize SPLUNC1’s natural protective properties to combat airway disease. We conclude that SPLUNC1 should be investigated further for use in asthma and bacterial pneumonia.Doctor of Philosoph

2-Selenouridine Triphosphate Synthesis and Se-RNA Transcription

2-Selenouridine (SeU) is one of the naturally occurring modifications of Se-tRNAs (SeU-RNA) at the wobble position of the anticodon loop. Its role in the RNA-RNA interaction, especially during the mRNA decoding, is elusive. To assist the research exploration, herein we report the enzymatic synthesis of the SeU-RNA via 2-selenouridine triphosphate (SeUTP) synthesis and RNA transcription. Moreover, we have demonstrated that the synthesized SeUTP is stable and recognizable by T7 RNA polymerase. Under the optimized conditions, the transcription yield of SeU-RNA can reach up to 85% of the corresponding native RNA. Furthermore, the transcribed SeU-hammerhead ribozyme has the similar activity as the corresponding native, which suggests usefulness of SeU-RNAs in function and structure studies of noncoding RNAs, including the Se-tRNAs

A General Model for Binary Cell Fate Decision Gene Circuits with Degeneracy: Indeterminacy and Switch Behavior in the Absence of Cooperativity

BACKGROUND: The gene regulatory circuit motif in which two opposing fate-determining transcription factors inhibit each other but activate themselves has been used in mathematical models of binary cell fate decisions in multipotent stem or progenitor cells. This simple circuit can generate multistability and explains the symmetric "poised" precursor state in which both factors are present in the cell at equal amounts as well as the resolution of this indeterminate state as the cell commits to either cell fate characterized by an asymmetric expression pattern of the two factors. This establishes the two alternative stable attractors that represent the two fate options. It has been debated whether cooperativity of molecular interactions is necessary to produce such multistability. PRINCIPAL FINDINGS: Here we take a general modeling approach and argue that this question is not relevant. We show that non-linearity can arise in two distinct models in which no explicit interaction between the two factors is assumed and that distinct chemical reaction kinetic formalisms can lead to the same (generic) dynamical system form. Moreover, we describe a novel type of bifurcation that produces a degenerate steady state that can explain the metastable state of indeterminacy prior to cell fate decision-making and is consistent with biological observations. CONCLUSION: The general model presented here thus offers a novel principle for linking regulatory circuits with the state of indeterminacy characteristic of multipotent (stem) cells

Design and synthesis of boronic-acid-labeled thymidine triphosphate for incorporation into DNA

The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection

Identification of BPIFA1/SPLUNC1 as an epithelium-derived smooth muscle relaxing factor

Asthma is a chronic airway disease characterized by inflammation, mucus hypersecretion and abnormal airway smooth muscle (ASM) contraction. Bacterial permeability family member A1, BPIFA1, is a secreted innate defence protein. Here we show that BPIFA1 levels are reduced in sputum samples from asthmatic patients and that BPIFA1 is secreted basolaterally from healthy, but not asthmatic human bronchial epithelial cultures (HBECs), where it suppresses ASM contractility by binding to and inhibiting the Ca2+ influx channel Orai1. We have localized this effect to a specific, C-terminal α-helical region of BPIFA1. Furthermore, tracheas from Bpifa1−/− mice are hypercontractile, and this phenotype is reversed by the addition of recombinant BPIFA1. Our data suggest that BPIFA1 deficiency in asthmatic airways promotes Orai1 hyperactivity, increased ASM contraction and airway hyperresponsiveness. Strategies that target Orai1 or the BPIFA1 deficiency in asthma may lead to novel therapies to treat this disease

The Need for Laboratory Measurements and Ab Initio Studies to Aid Understanding of Exoplanetary Atmospheres

We are now on a clear trajectory for improvements in exoplanet observations that will revolutionize our ability to characterize their atmospheric structure, composition, and circulation, from gas giants to rocky planets. However, exoplanet atmospheric models capable of interpreting the upcoming observations are often limited by insufficiencies in the laboratory and theoretical data that serve as critical inputs to atmospheric physical and chemical tools. Here we provide an up-to-date and condensed description of areas where laboratory and/or ab initio investigations could fill critical gaps in our ability to model exoplanet atmospheric opacities, clouds, and chemistry, building off a larger 2016 white paper, and endorsed by the NAS Exoplanet Science Strategy report. Now is the ideal time for progress in these areas, but this progress requires better access to, understanding of, and training in the production of spectroscopic data as well as a better insight into chemical reaction kinetics both thermal and radiation-induced at a broad range of temperatures. Given that most published efforts have emphasized relatively Earth-like conditions, we can expect significant and enlightening discoveries as emphasis moves to the exotic atmospheres of exoplanets.Comment: Submitted as an Astro2020 Science White Pape

In‐person interventions to reduce social isolation and loneliness: An evidence and gap map

BackgroundSocial isolation and loneliness can occur in all age groups, and they are linked to increased mortality and poorer health outcomes. There is a growing body of research indicating inconsistent findings on the effectiveness of interventions aiming to alleviate social isolation and loneliness. Hence the need to facilitate the discoverability of research on these interventions.ObjectivesTo map available evidence on the effects of in-person interventions aimed at mitigating social isolation and/or loneliness across all age groups and settings.Search MethodsThe following databases were searched from inception up to 17 February 2022 with no language restrictions: Ovid MEDLINE, Embase, EBM Reviews—Cochrane Central Register of Controlled Trials, APA PsycInfo via Ovid, CINAHL via EBSCO, EBSCO (all databases except CINAHL), Global Index Medicus, ProQuest (all databases), ProQuest ERIC, Web of Science, Korean Citation Index, Russian Science Citation Index, and SciELO Citation Index via Clarivate, and Elsevier Scopus.Selection CriteriaTitles, abstracts, and full texts of potentially eligible articles identified were screened independently by two reviewers for inclusion following the outlined eligibility criteria.Data Collection and AnalysisWe developed and pilot tested a data extraction code set in Eppi-Reviewer. Data was individually extracted and coded. We used the AMSTAR2 tool to assess the quality of reviews. However, the quality of the primary studies was not assessed.Main ResultsA total of 513 articles (421 primary studies and 92 systematic reviews) were included in this evidence and gap map which assessed the effectiveness of in-person interventions to reduce social isolation and loneliness. Most (68%) of the reviews were classified as critically low quality, while less than 5% were classified as high or moderate quality. Most reviews looked at interpersonal delivery and community-based delivery interventions, especially interventions for changing cognition led by a health professional and group activities, respectively. Loneliness, wellbeing, and depression/anxiety were the most assessed outcomes. Most research was conducted in high-income countries, concentrated in the United States, United Kingdom, and Australia, with none from low-income countries. Major gaps were identified in societal level and community-based delivery interventions that address policies and community structures, respectively. Less than 5% of included reviews assessed process indicators or implementation outcomes. Similar patterns of evidence and gaps were found in primary studies. All age groups were represented but more reviews and primary studies focused on older adults (≥60 years, 63%) compared to young people (≤24 years, 34%). Two thirds described how at-risk populations were identified and even fewer assessed differences in effect across equity factors for populations experiencing inequities

Molecular basis of antibiotic multiresistance transfer in Staphylococcus aureus

Multidrug-resistant Staphylococcus aureus infections pose a significant threat to human health. Antibiotic resistance is most commonly propagated by conjugative plasmids like pLW1043, the first vancomycin-resistant S. aureus vector identified in humans. We present the molecular basis for resistance transmission by the nicking enzyme in S. aureus (NES), which is essential for conjugative transfer. NES initiates and terminates the transfer of plasmids that variously confer resistance to a range of drugs, including vancomycin, gentamicin, and mupirocin. The NES N-terminal relaxase–DNA complex crystal structure reveals unique protein–DNA contacts essential in vitro and for conjugation in S. aureus. Using this structural information, we designed a DNA minor groove-targeted polyamide that inhibits NES with low micromolar efficacy. The crystal structure of the 341-residue C-terminal region outlines a unique architecture; in vitro and cell-based studies further establish that it is essential for conjugation and regulates the activity of the N-terminal relaxase. This conclusion is supported by a small-angle X-ray scattering structure of a full-length, 665-residue NES–DNA complex. Together, these data reveal the structural basis for antibiotic multiresistance acquisition by S. aureus and suggest novel strategies for therapeutic intervention

Pax6 Represses Androgen Receptor-Mediated Transactivation by Inhibiting Recruitment of the Coactivator SPBP

The androgen receptor (AR) has a central role in development and maintenance of the male reproductive system and in the etiology of prostate cancer. The transcription factor Pax6 has recently been reported to act as a repressor of AR and to be hypermethylated in prostate cancer cells. SPBP is a transcriptional regulator that previously has been shown to enhance the activity of Pax6. In this study we have identified SPBP to act as a transcriptional coactivator of AR. We also show that Pax6 inhibits SPBP-mediated enhancement of AR activity on the AR target gene probasin promoter, a repression that was partly reversed by increased expression of SPBP. Enhanced expression of Pax6 reduced the amount of SPBP associated with the probasin promoter when assayed by ChIP in HeLa cells. We mapped the interaction between both AR and SPBP, and AR and Pax6 to the DNA-binding domains of the involved proteins. Further binding studies revealed that Pax6 and SPBP compete for binding to AR. These results suggest that Pax6 represses AR activity by displacing and/or inhibiting recruitment of coactivators to AR target promoters. Understanding the mechanism for inhibition of AR coactivators can give rise to molecular targeted drugs for treatment of prostate cancer

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data