High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro.

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.

High-throughput organ-on-chip platform with integrated programmable fluid flow and real-time sensing for complex tissue models in drug development workflows.

Drug development suffers from a lack of predictive and human-relevant in vitro models. Organ-on-chip (OOC) technology provides advanced culture capabilities to generate physiologically appropriate, human-based tissue in vitro, therefore providing a route to a predictive in vitro model. However, OOC technologies are often created at the expense of throughput, industry-standard form factors, and compatibility with state-of-the-art data collection tools. Here we present an OOC platform with advanced culture capabilities supporting a variety of human tissue models including liver, vascular, gastrointestinal, and kidney. The platform has 96 devices per industry standard plate and compatibility with contemporary high-throughput data collection tools. Specifically, we demonstrate programmable flow control over two physiologically relevant flow regimes: perfusion flow that enhances hepatic tissue function and high-shear stress flow that aligns endothelial monolayers. In addition, we integrate electrical sensors, demonstrating quantification of barrier function of primary gut colon tissue in real-time. We utilize optical access to the tissues to directly quantify renal active transport and oxygen consumption via integrated oxygen sensors. Finally, we leverage the compatibility and throughput of the platform to screen all 96 devices using high content screening (HCS) and evaluate gene expression using RNA sequencing (RNA-seq). By combining these capabilities in one platform, physiologically-relevant tissues can be generated and measured, accelerating optimization of an in vitro model, and ultimately increasing predictive accuracy of in vitro drug screening.

Effects of breeding history and crop management on the root architecture of wheat.

AIMS: Selection for optimal root system architecture (RSA) is important to ensure genetic gains in the sustainable production of wheat (Triticum aestivum L.). Here we examine the hypothesis that past wheat breeding has led to changes in RSA and that future breeding efforts can focus directly on RSA to improve adaptation to target environments. METHODS: We conducted field trials using diverse wheat varieties, including modern and historic UK varieties and non-UK landraces, tested under contrasting tillage regimes (non-inversion tillage versus conventional ploughing) for two trial years or different seeding rates (standard versus high rate) for one trial year. We used field excavation, washing and measurement of root crowns ('shovelomics') to characterise RSA traits, including: numbers of seminal, crown and nodal roots per plant, and crown root growth angle. RESULTS: We found differences among genotypes for all root traits. Modern varieties generally had fewer roots per plant than historic varieties. On average, there were fewer crown roots and root angles were wider under shallow non-inversion tillage compared with conventional ploughing. Crown root numbers per plant also tended to be smaller at a high seeding rate compared with the standard. There were significant genotype-by-year, genotype-by-tillage and genotype-by-seeding-rate interactions for many root traits. CONCLUSIONS: Smaller root systems are likely to be a result of past selection that facilitated historical yield increases by reducing below-ground competition within the crop. The effects of crop management practices on RSA depend on genotype, suggesting that future breeding could select for improved RSA traits in resource-efficient farming systems.

RNA interference for the identification of ectoparasite vaccine candidates.

Ectoparasites present a major challenge for disease management globally. With drug resistance increasingly observed in many disease-causing species, the need for novel control measures is pressing. Ever-expanding genomic resources from 'next generation' sequencing are now available for a number of arthropod ectoparasites, necessitating an effective means of screening these data for novel candidates for vaccine antigens or targets for chemotherapeutics. Such in vitro screening methods must be developed if we are to make discoveries in a timely and cost-effective manner. This review will discuss the potential that RNA interference (RNAi) has demonstrated thus far in the context of arthropod ectoparasites and the potential roles for this technology in the development of novel methods for parasite control.

Spontaneous alpha-N-6-phosphogluconoylation of a "His tag" in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins.

Several proteins expressed in Escherichia coli with the N-terminus Gly-Ser-Ser-[His]6- consisted partly (up to 20%) of material with 178 Da of excess mass, sometimes accompanied by a smaller fraction with an excess 258 Da. The preponderance of unmodified material excluded mutation, and the extra masses were attributed to posttranslational modifications. As both types of modified protein were N-terminally blocked, the alpha-amino group was modified in each case. Phosphatase treatment converted +258-Da protein into +178-Da protein. The modified His tags were isolated, and the mass of the +178-Da modification estimated as 178.06 +/- 0.02 Da by tandem mass spectrometry. As the main modification remained at +178 Da in 15N-substituted protein, it was deemed nitrogen-free and possibly carbohydrate-like. Limited periodate oxidations suggested that the +258-Da modification was acylation with a 6-phosphohexonic acid, and that the +178-Da modification resulted from its dephosphorylation. NMR spectra of cell-derived +178-Da His tag and synthetic alpha-N-d-gluconoyl-His tag were identical. Together, these results suggested that the +258-Da modification was addition of a 6-phosphogluconoyl group. A plausible mechanism was acylation by 6-phosphoglucono-1,5-lactone, produced from glucose 6-phosphate by glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Supporting this, treating a His-tagged protein with excess d-glucono-1,5-lactone gave only N-terminal gluconoylation.

Sound localization in subjects with impaired hearing. Spatial-discrimination and interaural-discrimination tests.

In order to get a systematic picture of how various hearing impairments and neurologic disorders may affect sound localization, psychophysical spatial- and lateralization-discrimination measurements were performed on 140 subjects, including 69 with different types of hearing impairments, 32 with neurological diseases, and 39 with normal hearing. The quantities measured were: in the freefield, the horizontal minimum audible angle (MAA) at eight reference azimuths around the head and the vertical MAA straight ahead; with headphones, the just-noticeable difference (JND) in interaural time delay and the JND in interaural intensity difference. The standard stimulus was broadband (0.25-10 kHz), pulsed (1-sec), noise presented at a suprathreshold level for both ears (65-100 dB SPL). The results show that there exist characteristic impairments of sound localization in the different types of hearing impairments tested. On a general level, the results are consistent with the concept that the localization of sound relies on a decision made by the central auditory system based on a number of cues present in the acoustic signal at the two ears. The cues tested in our study are: 1) the interaural time difference, 2) the interaural intensity difference, and 3) the spectrum of the received signal at each ear. At a more specific level, the sound localization impairments found in conductive hearing losses are interpreted as bone-conduction effects, the results found in sensorineural hearing losses are interpreted as consequences of impaired or preserved spectral processing, the results in neurinomas are interpreted as impaired signal transmission in the auditory nerves, and the results of subjects with central involvements suggest that separate processors exist at some level in the central auditory system for the different localization cues. Finally, comments are made about the practical clinical significance of sound localization tests in the audiological and neurological evaluation of patients.

Auditory-nerve potentials from ear canals of patients with otologic problems.

Summary--The feasibility of making rountine measurements of human auditory-nerve potentials in an office environment is demonstrated. Using a portable device for stimulus generating and response recording, auditory-nerve potentials are recorded from subjects with normal and abnormal hearing by means of an electrode placed on the skin of the ear canal. The results can be compared with those of others obtained under laboratory conditions. Preliminary results indicate that in many instances, the nature of the hearing deficit is related to the latency and size of the click-evoked auditory-nerve potentials. The precise relationships between nerve responses and specific disease conditions are still difficult to formulate.

cis-terpin hydrate metabolism by a Brevibacterium: patterns of enzyme induction, and accumulation of -terpineol in growth.

A brevibacterium, strain TH-4, previously isolated by aerobic enrichment on the monocyclic monoterpenoid cis-terpin hydrate as a sole carbon and energy source, was found to grow on alpha-terpineol and on a number of common sugars and organic acids. Oxidation of these terpenoids was shown to occur via an induced enzyme system, as measured manometrically by oxygen uptake and prevention of protein synthesis with chloramphenicol or puromycin. Oxidation of terpin hydrate by cell suspensions appeared to be coincidentally induced by growth on alpha-terpineol, and oxidation of alpha-terpineol similarly appeared to be induced by growth on terpin hydrate. Culture fluids in which the TH-4 organism was grown at the expense of cis-terpin hydrate were found to contain (-)-alpha-terpineol in combined butanol-ether extracts. The isolated compound was shown to be chromatographically and spectrophotometrically identical to an authentic sample of alpha-terpineol. The stereospecificity of an enzymatic dehydration of terpin hydrate to alpha-terpineol is considered.