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The rapid detection of the spore form of Clostridioides difficile has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from C. difficile spores via a bespoke microwave lysis platform. C. difficile spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen’s two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 μg/mL to 250 μg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within tcdA and tcdB toxin genes of C. difficile and was able to detect 800 spores of C. difficile within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with C. difficile spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for C. difficile.

In order to implement the fifth generation (5G) communication system for a large number of users, the governments of many countries nominated the low 5G frequency band between 3.3 and 4.3 GHz. This paper proposes a wideband RFPA by designing the input matching network (MN) and output MN of the device using the simplified real frequency technique (SRFT) and the harmonic tuning network. The load-pull and source-pull is applied at multiple points for 100 MHz intervals over the bandwidth to obtain the optimum impedances at the output and input of the 10W Gallium Nitride (GaN) Cree CGH40010F device. To verify the design, the RFPA is simulated, and the performance is measured between 3.3 and 4.3 GHz. According to experimental findings, the measured drain efficiency (DE) throughout the whole bandwidth ranged from 57.5 to 67.5% at the output power of 40 dBm. Moreover, at the 1 dB compression point between 39.2 and 42.2 dBm output power, the drain efficiency (DE) achieves a high value of 81.2% with an output power of 42.2 dBm at a frequency of 3.3 GHz. The RFPA can obtain a maximum gain of 12.4 dB at 3.5 GHz. The linearity of the RFPA with a two-tone signal is measured and the value is less than -22 dBc all over the band.

Geochemical proxies of sea surface temperature (SST) and seawater pH (pHsw) in scleractinian coral skeletons are valuable tools for reconstructing tropical climate variability. However, most coral skeletal SST and pHsw proxies are univariate methods that are limited in their capacity to circumvent non-climate-related variability. Here we present a novel multivariate method for reconstructing SST and pHsw from the geochemistry of coral skeletons. Our Scleractinian Multivariate Isotope and Trace Element (SMITE) method optimizes reconstruction skill by leveraging the covariance across an array of coral elemental and isotopic data with SST and pHsw. First, using a synthetic proxy experiment, we find that SMITE SST reconstruction statistics (correlation, accuracy, and precision) are insensitive to noise and variable calibration period lengths relative to Sr/Ca. While SMITE pHsw reconstruction statistics remain relative to δ11B throughout the same synthetic experiment, the magnitude of the long-term trend in pHsw is progressively lost under conditions of moderate-to-high analytical uncertainty. Next, we apply the SMITE method to an array of seven coral-based geochemical variables (B/Ca, δ11B, Li/Ca, Mg/Ca, Sr/Ca, U/Ca & Li/Mg) measured from two Bermudan Porites astreoides corals. Despite a

ABSTRACT Transplacental transfer of maternal antibodies provides the fetus and newborn with passive protection against infectious diseases. While the role of the highly conserved neonatal Fc receptor (FcRn) in transfer of IgG in mammals is undisputed, recent reports have suggested that a second receptor may contribute to transport in humans. We report poor transfer efficiency of plant-expressed recombinant HIV-specific antibodies, including engineered variants with high FcRn affinity, following subcutaneous infusion into rhesus macaques close to parturition. Unexpectedly, unlike those derived from mammalian tissue culture, plant-derived antibodies were essentially unable to cross macaque placentas. This defect was associated with poor Fcγ receptor binding and altered Fc glycans and was not recapitulated in mice. These results suggest that maternal-fetal transfer of IgG across the three-layer primate placenta may require a second receptor and suggest a means of providing maternal antibody treatments during pregnancy while avoiding fetal harm. IMPORTANCE This study compared the ability of several human HIV envelope-directed monoclonal antibodies produced in plants with the same antibodies produced in mammalian cells for their ability to cross monkey and mouse placentas. We found that the two types of antibodies have comparable transfer efficiencies in mice, but they are differentially transferred across macaque placentas, consistent with a two-receptor IgG transport model in primates. Importantly, plant-produced monoclonal antibodies have excellent binding characteristics for human FcRn receptors, permitting desirable pharmacokinetics in humans. The lack of efficient transfer across the primate placenta suggests that therapeutic plant-based antibody treatments against autoimmune diseases and cancer could be provided to the mother while avoiding transfer and preventing harm to the fetus.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is an ongoing pandemic that causes significant health/socioeconomic burden. Variants of concern (VOCs) have emerged affecting transmissibility, disease severity and re-infection risk. Studies suggest that the - N-terminal domain (NTD) of the spike protein may have a role in facilitating virus entry via sialic-acid receptor binding. Furthermore, most VOCs include novel NTD variants. Despite global sequence and structure similarity, most sialic-acid binding pockets in NTD vary across coronaviruses. Our work suggests ongoing evolutionary tuning of the sugar-binding pockets and recent analyses have shown that NTD insertions in VOCs tend to lie close to loops.We extended the structural characterisation of these sugar-binding pockets and explored whether variants could enhance sialic acid-binding. We found that recent NTD insertions in VOCs (i.e., Gamma, Delta and Omicron variants) and emerging variants of interest (VOIs) (i.e., Iota, Lambda and Theta variants) frequently lie close to sugar-binding pockets. For some variants, including the recent Omicron VOC, we find increases in predicted sialic acid-binding energy, compared to the original SARS-CoV-2, which may contribute to increased transmission. These binding observations are supported by molecular dynamics simulations (MD).We examined the similarity of NTD across Betacoronaviruses to determine whether the sugar-binding pockets are sufficiently similar to be exploited in drug design. Whilst most pockets are too structurally variable, we detected a previously unknown highly structurally conserved pocket which can be investigated in pursuit of a generic pan-Betacoronavirus drug. Our structure-based analyses help rationalise the effects of VOCs and provide hypotheses for experiments. Our findings suggest a strong need for experimental monitoring of changes in NTD of VOCs.

Many countries have allocated new frequency bands for fifth generation (5G) communication systems. In this paper, a wideband continuous class-F (CCF) radio frequency power amplifier (RFPA) is presented for the new 5G frequency band, from 3.3 GHz to 4.3 GHz using a 10 W Cree CGH40100F device. A unique wideband RFPA design approach for the output matching network (OMN) is also presented by applying a harmonic tuning network (HTN) for the harmonics and a low pass matching technique (LPMT) for the fundamental frequency. The RFPA is fabricated, and promising measurement results show a drain efficiency of 55.9% to 65.3% is achieved at an output power of 40 dBm (± 0.3 dBm) over a frequency range of 3.3 GHz to 4.3 GHz. A two-tone signal with a 10 MHz spacing was also applied to investigate the linearity of the RFPA.

Subglacial hydrological systems require innovative technological solutions to access and observe. Wireless sensor platforms can be used to collect and return data, but their performance in deep and fast-moving ice requires quantification. We report experimental results from Cryoegg: a spherical probe that can be deployed into a borehole or moulin and transit through the subglacial hydrological system. The probe measures temperature, pressure and electrical conductivity in situ and returns all data wirelessly via a radio link. We demonstrate Cryoegg's utility in studying englacial channels and moulins, including in situ salt dilution gauging. Cryoegg uses VHF radio to transmit data to a surface receiving array. We demonstrate transmission through up to 1.3 km of cold ice – a significant improvement on the previous design. The wireless transmission uses Wireless M-Bus on 169 MHz; we present a simple radio link budget model for its performance in cold ice and experimentally confirm its validity. Cryoegg has also been tested successfully in temperate ice. The battery capacity should allow measurements to be made every 2 h for more than a year. Future iterations of the radio system will enable Cryoegg to transmit data through up to 2.5 km of ice.

Modeling harmonic tremor recorded at volcanoes is an essential practice in improving eruption forecasting methods and warning systems. We model the conduit dynamics of quasi-periodic tremor (chugging) recorded at Karymsky, Tungurahua, and Fuego volcanoes to estimate its source characteristics. Chugging mechanisms are estimated using two theoretical models originally derived in Garcés [1997, doi: https://doi.org/10.1029/1999JB900096] and Girona et al. [2019, doi: https://doi.org/10.1029/2019JB017482], respectively. Comparisons of the conduit and fluid output parameters suggests that chugging is primarily limited to near-surface oscillations and outgassing due to an accumulation of gas between eruptive episodes. The modeled results indicate clustered release of volatiles exsolved from a deeper magma conduit region, triggered by an initial explosion. This interpretation is consistent with both infrasonic and seismic observations at each volcano.

Glacially- and magmatically-derived seismic events have been noted to heavily overlap in characteristics, thus there exists the potential for false-alarms or missed warnings at ice-covered volcanoes. Here we present the first study to specifically target icequakes at an ice-covered volcano in Southern Chile. Two months of broadband seismic data collected at Llaima volcano in 2015 were analyzed in order to quantify, characterize, and locate glacially-derived seismic events at one of the most active ice-covered volcanoes in the region. We find over 1,000 repeating seismic events across 11 families, the largest of which contains 397 events. Approximate locations and characteristics of the largest families lead us to conclude that these events were derived from persistent stick-slip motion along the ice-rock interface at the base of a glacier near the volcano summit. These results have implications for future seismic monitoring at Llaima volcano and other ice-covered active volcanoes in the region.

Alternative splicing can expand the diversity of proteomes. Homologous mutually exclusive exons (MXEs) originate from the same ancestral exon and result in polypeptides with similar structural properties but altered sequence. Why would some genes switch homologous exons and what are their biological impact? Here, we analyse the extent of sequence, structural and functional variability in MXEs and report the first large scale, structure-based analysis of the biological impact of MXE events from different genomes. MXE-specific residues tend to map to single domains, are highly enriched in surface exposed residues and cluster at or near protein functional sites. Thus, MXE events are likely to maintain the protein fold, but alter specificity and selectivity of protein function. This comprehensive resource of MXE events and their annotations is available at: http://gene3d.biochem.ucl.ac.uk/mxemod/. These findings highlight how small, but significant changes at critical positions on a protein surface are exploited in evolution to alter function.

The Drosophila shaggy gene (sgg, GSK-3) encodes multiple protein isoforms with serine/threonine kinase activity and is a key player in diverse developmental signalling pathways. Currently it is unclear whether different Sgg proteoforms are similarly involved in signalling or if different proteoforms have distinct functions. We used CRISPR/Cas9 genome engineering to tag eight different Sgg proteoform classes and determined their localization during embryonic development. We performed proteomic analysis of the two major proteoform classes and generated mutant lines for both of these for transcriptomic and phenotypic analysis. We uncovered distinct tissue-specific localization patterns for all of the tagged proteoforms we examined, most of which have not previously been characterised directly at the protein level, including one proteoform initiating with a non-standard codon. Collectively, this suggests complex developmentally regulated splicing of the sgg primary transcript. Further, affinity purification followed by mass spectrometric analyses indicate a different repertoire of interacting proteins for the two major proteoforms we examined, one with ubiquitous expression (Sgg-PB) and one with nervous system specific expression (Sgg-PA). Specific mutation of these proteoforms shows that Sgg-PB performs the well characterised maternal and zygotic segmentations functions of the sgg locus, while Sgg-PA mutants show adult lifespan and locomotor defects consistent with its nervous system localisation. Our findings provide new insights into the role of GSK-3 proteoforms and intriguing links with the GSK-3α and GSK-3β proteins encoded by independent vertebrate genes. Our analysis suggests that different proteoforms generated by alternative splicing are likely to perform distinct functions.

Abstract Fibrosis-related events play a part in most blinding diseases worldwide. However, little is known about the mechanisms driving this complex multifactorial disease. Here we have carried out the first genome-wide RNA-Sequencing study in human conjunctival fibrosis. We isolated 10 primary fibrotic and 7 non-fibrotic conjunctival fibroblast cell lines from patients with and without previous glaucoma surgery, respectively. The patients were matched for ethnicity and age. We identified 246 genes that were differentially expressed by over two-fold and p

Pseudokinases are a member of the kinase superfamily that lack one or more of the canonical residues required for catalysis. Protein pseudokinases are widely distributed across species and are present in proteins that perform a great diversity of roles in the cell. They represent approximately 10% to 40% of the kinome of a multicellular organism. In the human, the pseudokinase subfamily consists of approximately 60 unique proteins. Despite their lack of one or more of the amino acid residues typically required for the productive interaction with ATP and metal ions, which is essential for the phosphorylation of specific substrates, pseudokinases are important functional molecules that can act as dynamic scaffolds, competitors, or modulators of protein−protein interactions. Indeed, pseudokinase misfunctions occur in diverse diseases and represent a new therapeutic window for the development of innovative therapeutic approaches. In this contribution, we describe the structural features of pseudokinases that are used as the basis of their classification; analyse the interactome space of human pseudokinases and discuss their potential as suitable drug targets for the treatment of various diseases, including metabolic, neurological, autoimmune, and cell proliferation disorders.

A miniaturized 2.4 GHz re-entrant cavity has been designed, manufactured and tested as a sensor for microfluidic compositional analysis. It has been fully evaluated experimentally with water and common solvents, namely methanol, ethanol, and chloroform, with excellent agreement with the expected behaviour predicted by the Debye model. The sensor’s performance has also been assessed for analysis of segmented flow using water and oil. The samples’ interaction with the electric field in the gap region has been maximized by aligning the sample tube parallel to the electric field in this region, and the small width of the gap (typically 1 mm) result in a highly localised complex permittivity measurement. The re-entrant cavity has simple mechanical geometry, small size, high quality factor, and due to the high concentration of electric field in the gap region, a very small mode volume. These factors combine to result in a highly sensitive, compact sensor for both pure liquids and liquid mixtures in capillary or microfluidic environments.

The mitotic spindle is an essential molecular machine involved in cell division, whose composition has been studied extensively by detailed cellular biology, high-throughput proteomics, and RNA interference experiments. However, because of its dynamic organization and complex regulation it is difficult to obtain a complete description of its molecular composition. We have implemented an integrated computational approach to characterize novel human spindle components and have analysed in detail the individual candidates predicted to be spindle proteins, as well as the network of predicted relations connecting known and putative spindle proteins. The subsequent experimental validation of a number of predicted novel proteins confirmed not only their association with the spindle apparatus but also their role in mitosis. We found that 75% of our tested proteins are localizing to the spindle apparatus compared to a success rate of 35% when expert knowledge alone was used. We compare our results to the previously published MitoCheck study and see that our approach does validate some findings by this consortium. Further, we predict so-called "hidden spindle hub", proteins whose network of interactions is still poorly characterised by experimental means and which are thought to influence the functionality of the mitotic spindle on a large scale. Our analyses suggest that we are still far from knowing the complete repertoire of functionally important components of the human spindle network. Combining integrated bio-computational approaches and single gene experimental follow-ups could be key to exploring the still hidden regions of the human spindle system.

Abstract??To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were ?153 and ?162??, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl2-containing Ringer?s solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl2than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr andmbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave ?20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout?intermediate protein 1 (24?kDa) and intermediate protein 2 (28?kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of ?7?kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability. [ABSTRACT FROM AUTHOR]

The most recent eruptive cycle of Tungurahua volcano began in May 2004, and reached its highest level of activity in July 2004. This activity cycle is the last one of a series of four cycles that followed the reawakening and major eruption of Tungurahua in 1999. Between June 30 and August 12, 2004, three temporary seismic and infrasonic stations were installed on the flanks of the volcano and recorded over 2,000 degassing events. The events are classified by waveform character and include: explosion events (the vast majority, spanning three orders of pressure amplitudes at 3.5 km from the vent, 0.1–180 Pa), jetting events, and sequences of repetitive infrasonic pulses, called chugging events. Travel-time analysis of seismic first arrivals and infrasonic waves indicates that explosions start with a seismic event at a shallow depth (<200 m), followed ∼1 s later by an out-flux of gas, ash and solid material through the vent. Cluster analysis of infrasonic signals from explosion events was used to isolate four groups of similar waveforms without apparent correlation to event size, location, or time. The clustering is thus associated with source mechanism and probably spatial distribution. Explosion clusters do not exhibit temporal dependence. [ABSTRACT FROM AUTHOR]