The impact of solvents on the toxicity of the banned parathion insecticide.

The aerial crop dusting and spraying of fields with the phosphorothioate insecticide parathion in the late 1900s, significantly improved crop yields but resulted in high levels of occupational toxicity in handlers and agricultural workers, as well as cases of intentional self-harm poisoning, culminating in its banning in many western countries by early 2000s. However because of the low solubility and volatility of parathion, most available products were formulated using organic solvents e.g. xylene, to increase the efficacy of the aerosols and dusts. In the present study, the toxicity of parathion was assessed when formulated in an aqueous solvents (ethanol/PBS (1:9)), and delivered to macaques as an aerosol. Doses of 780 µg/kg and 1.56 mg/kg were delivered one day apart, using a modified nebulizer calculated to result in lung deposition of ∼480 µg/kg with a similar or larger amount being swallowed; these doses being similar to the estimated lethal oral dose 286ug/kg - 1.43 mg/kg of formulated parathion in humans. Surprisingly, this dose (a combined amount of ∼14 mg) caused only low AChE inhibition and moderate BChE inhibition with no clinical symptoms, indicating that the use of organic solvents may have previously played a critical role in the severity of parathion toxicity following inhalation exposure. In addition, unlike constitutively toxic OPs, which are highly toxic when inhaled, these results are consistent with the idea that phosphorothioate insecticides appear to be more intoxicating following oral than inhalation exposure. However, this still remains uncertain because the presence of organic solvents in the ingested parathion studies was not always known.

Compositional shifts within the denture-associated bacteriome in pneumonia - an analytical cross-sectional study.

Introduction. Bacterial pneumonia is a common cause of morbidity and mortality in elderly individuals. While the incidence of edentulism is falling, approximately 19 % of the UK population wear a full or partial removable denture. Despite advances in denture biomaterials, the majority of dentures are fabricated using polymethyl-methacrylate. Growing evidence suggests that colonization of the oral cavity by putative respiratory pathogens predisposes individuals to respiratory infection, by translocation of these microorganisms along the respiratory tract.Hypothesis/Gap Statement. We hypothesized that denture surfaces provide a susceptible colonization site for putative respiratory pathogens, and thus could increase pneumonia risk in susceptible individuals.Aim. This study aimed to characterize the bacterial community composition of denture-wearers in respiratory health compared with individuals with a confirmed diagnosis of pneumonia.Methodology. This was an analytical cross-sectional study, comparing frail elderly individuals without respiratory infection (n=35) to hospitalized patients with pneumonia (n=26). The primary outcome was the relative abundance of putative respiratory pathogens identified by 16S rRNA metataxonomic sequencing, with quantitative PCR used to identified Streptococcus pneumoniae.Results. There was a statistically significant increase in the overall relative abundance of putative respiratory pathogens (P<0.0001), with a greater than 20-fold increase in the bioburden of these microorganisms. In keeping with these findings, there were significant shifts in bacterial community diversity (Chao index, P=0.0003) and richness (Inverse Simpson index P<0.0001) in the denture-associated microbiota of pneumonia patients compared with control subjects.Conclusion. Within the limitations of this study, our evidence supports the role of denture acrylic biomaterials as a potential colonization site for putative respiratory pathogens, which may lead to an increased risk of pneumonia in susceptible individuals. These findings support prior observational studies which have found denture-wearers to be at increased risk of respiratory infection. Further research is needed to confirm the sequence of colonization and translocation to examine potential causal relationships.

Evidence for the Role of a Second Fc-Binding Receptor in Placental IgG Transfer in Nonhuman Primates.

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.

A single post-exposure oxime RS194B treatment rapidly reactivates acetylcholinesterase and reverses acute symptoms in macaques exposed to diethylphosphorothioate parathion and chlorpyrifos insecticides.

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticides, significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins that target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with an oxime to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently used oximes either reactivate poorly, do not readily cross the blood-brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure, for example RS194B, have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticides or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms, and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these findings and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos. These OPs require conversion by P450 in the liver of the inactive thions to the active toxic oxon forms, and once again demonstrated RS194B efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1-2 h was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.

Structural and energetic analyses of SARS-CoV-2 N-terminal domain characterise sugar binding pockets and suggest putative impacts of variants on COVID-19 transmission.

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.

Enhanced HIV SOSIP Envelope yields in plants through transient co-expression of peptidyl-prolyl isomerase B and calreticulin chaperones and ER targeting.

High yield production of recombinant HIV SOSIP envelope (Env) trimers has proven elusive as numerous disulfide bonds, proteolytic cleavage and extensive glycosylation pose high demands on the host cell machinery and stress imposed by accumulation of misfolded proteins may ultimately lead to cellular toxicity. The present study utilized the Nicotiana benthamiana/p19 (N.b./p19) transient plant system to assess co-expression of two ER master regulators and 5 chaperones, crucial in the folding process, to enhance yields of three Env SOSIPs, single chain BG505 SOSIP.664 gp140, CH505TF.6R.SOSIP.664.v4.1 and CH848-10.17-DT9. Phenotypic changes in leaves induced by SOSIP expression were employed to rapidly identify chaperone-assisted improvement in health and expression. Up to 15-fold increases were obtained by co-infiltration of peptidylprolvl isomerase (PPI) and calreticulin (CRT) which were further enhanced by addition of the ER-retrieval KDEL tags to the SOSIP genes; levels depending on individual SOSIP type, day of harvest and chaperone gene dosage. Results are consistent with reducing SOSIP misfolding and cellular stress due to increased exposure to the plant host cell's calnexin/calreticulin network and accelerating the rate-limiting cis-trans isomerization of Xaa-Pro peptide bonds respectively. Plant transient co-expression facilitates rapid identification of host cell factors and will be translatable to other complex glycoproteins and mammalian expression systems.

Audible acoustics from low-magnitude fluid-induced earthquakes in Finland.

Earthquakes are frequently accompanied by public reports of audible low-frequency noises. In 2018, public reports of booms or thunder-like noises were linked to induced earthquakes during an Engineered Geothermal System project in the Helsinki Metropolitan area. In response, two microphone arrays were deployed to record and study these acoustic signals while stimulation at the drill site continued. During the 11 day deployment, we find 39 earthquakes accompanied by possible atmospheric acoustic signals. Moment magnitudes of these events ranged from [Formula: see text] to 1.87 with located depths of 4.8-6.5 km. Analysis of the largest event revealed a broadband frequency content, including in the audible range, and high apparent velocities across the arrays. We conclude that the audible noises were generated by local ground reverberation during the arrival of seismic body waves. The inclusion of acoustic monitoring at future geothermal development projects will be beneficial for studying seismic-to-acoustic coupling during sequences of induced earthquakes.

Protection of Newborn Macaques by Plant-Derived HIV Broadly Neutralizing Antibodies: a Model for Passive Immunotherapy during Breastfeeding.

Preventing human immunodeficiency virus (HIV) infection in newborns by vertical transmission remains an important unmet medical need in resource-poor areas where antiretroviral therapy (ART) is not available and mothers and infants cannot be treated prepartum or during the breastfeeding period. In the present study, the protective efficacy of the potent HIV-neutralizing antibodies PGT121 and VRC07-523, both produced in plants, were assessed in a multiple-SHIV (simian-human immunodeficiency virus)-challenge breastfeeding macaque model. Newborn macaques received either six weekly subcutaneous injections with PGT121 alone or as a cocktail of PGT121-LS plus VRC07-523-LS injected three times every 2 weeks. Viral challenge with SHIVSF162P3 was twice weekly over 5.5 weeks using 11 exposures. Despite the transient presence of plasma viral RNA either immediately after the first challenge or as single-point blips, the antibodies prevented a productive infection in all babies with no sustained plasma viremia, compared to viral loads ranging from 103 to 5 × 108 virions/ml in four untreated controls. No virus was detected in peripheral blood mononuclear cells (PBMCs), and only 3 of 159 tissue samples were weakly positive in the treated babies. Newborn macaques proved to be immunocompetent, producing transient anti-Env antibodies and anti-drug antibody (ADA), which were maintained in the circulation after passive broadly neutralizing antibody clearance. ADA responses were directed to the IgG1 Fc CH2-CH3 domains, which has not been observed to date in adult monkeys passively treated with PGT121 or VRC01. In addition, high levels of VRC07-523 anti-idiotypic antibodies in the circulation of one newborn was concomitant with the rapid elimination of VRC07. Plant-expressed antibodies show promise as passive immunoprophylaxis in a breastfeeding model in newborns. IMPORTANCE Plant-produced human neutralizing antibody prophylaxis is highly effective in preventing infection in newborn monkeys during repeated oral exposure, modeling virus in breastmilk, and offers advantages in cost of production and safety. These findings raise the possibility that anti-Env antibodies may contribute to the control of viral replication in this newborn model and that the observed immune responsiveness may be driven by the long-lived presence of immune complexes.

Tracing Evolution Through Protein Structures: Nature Captured in a Few Thousand Folds.

This article is dedicated to the memory of Cyrus Chothia, who was a leading light in the world of protein structure evolution. His elegant analyses of protein families and their mechanisms of structural and functional evolution provided important evolutionary and biological insights and firmly established the value of structural perspectives. He was a mentor and supervisor to many other leading scientists who continued his quest to characterise structure and function space. He was also a generous and supportive colleague to those applying different approaches. In this article we review some of his accomplishments and the history of protein structure classifications, particularly SCOP and CATH. We also highlight some of the evolutionary insights these two classifications have brought. Finally, we discuss how the expansion and integration of protein sequence data into these structural families helps reveal the dark matter of function space and can inform the emergence of novel functions in Metazoa. Since we cover 25 years of structural classification, it has not been feasible to review all structure based evolutionary studies and hence we focus mainly on those undertaken by the SCOP and CATH groups and their collaborators.

Monitoring changes in human activity during the COVID-19 shutdown in Las Vegas using infrasound microbarometers.

While studies of urban acoustics are typically restricted to the audio range, anthropogenic activity also generates infrasound (<20 Hz, roughly at the lower end of the range of human hearing). Shutdowns related to the COVID-19 pandemic unintentionally created ideal conditions for the study of urban infrasound and low frequency audio (20-500 Hz), as closures reduced human-generated ambient noise, while natural signals remained relatively unaffected. An array of infrasound sensors deployed in Las Vegas, NV, provides data for a case study in monitoring human activity during the pandemic through urban acoustics. The array records a sharp decline in acoustic power following the temporary shutdown of businesses deemed nonessential by the state of Nevada. This decline varies spatially across the array, with stations close to McCarran International Airport generally recording the greatest declines in acoustic power. Further, declines in acoustic power fluctuate with the time of day. As only signals associated with anthropogenic activity are expected to decline, this gives a rough indication of periodicities in urban acoustics throughout Las Vegas. The results of this study reflect the city's response to the pandemic and suggest spatiotemporal trends in acoustics outside of shutdowns.

Biological impact of mutually exclusive exon switching.

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.

Dynamic changes in the brain protein interaction network correlates with progression of Aß42 pathology in Drosophila.

Alzheimer's disease (AD), the most prevalent form of dementia, is a progressive and devastating neurodegenerative condition for which there are no effective treatments. Understanding the molecular pathology of AD during disease progression may identify new ways to reduce neuronal damage. Here, we present a longitudinal study tracking dynamic proteomic alterations in the brains of an inducible Drosophila melanogaster model of AD expressing the Arctic mutant Aß42 gene. We identified 3093 proteins from flies that were induced to express Aß42 and age-matched healthy controls using label-free quantitative ion-mobility data independent analysis mass spectrometry. Of these, 228 proteins were significantly altered by Aß42 accumulation and were enriched for AD-associated processes. Network analyses further revealed that these proteins have distinct hub and bottleneck properties in the brain protein interaction network, suggesting that several may have significant effects on brain function. Our unbiased analysis provides useful insights into the key processes governing the progression of amyloid toxicity and forms a basis for further functional analyses in model organisms and translation to mammalian systems.

Characterisation of protein isoforms encoded by the Drosophila Glycogen Synthase Kinase 3 gene shaggy.

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-3ß proteins encoded by independent vertebrate genes. Our analysis suggests that different proteoforms generated by alternative splicing are likely to perform distinct functions.

Aerosolized recombinant human butyrylcholinesterase delivered by a nebulizer provides long term protection against inhaled paraoxon in macaques.

The recent intentional use of nerve agents and pesticides in Europe and Afghanistan highlights the need for an effective countermeasure against organophosphates (OP) toxins. The most developed pretreatment candidate to date is plasma (native) human butyrylcholinesterase (HuBChE), which is limited in availability and because of its 1:1 stoichiometry with OPs, a large dose will present challenges when delivered parenterally both in terms of pharmacokinetics and manageability in the field. A tetrameric recombinant (r) form of human BChE produced in CHO-K1 cells with similar structure, in vivo stability and antidotal efficacy as the native form, has been developed to deliver rHuBChE as an aerosol (aer) to form a pulmonary bioshield capable of neutralizing inhaled OPs in situ and prevent AChE inhibition in the blood and in the brain; the latter associated with the symptoms of OP toxicity. Previous proof-of-concept macaque studies demonstrated that delivery of 9 mg/kg using a microsprayer inserted down the trachea, resulted in protection against an inhaled dose of 15ug/kg of aer-paraoxon (aer-Px) given 72 h later. In the present studies, pulmonary delivery of rHuBChE in macaques was achieved using Aerogen vibrating mesh nebulizers, similar to that used for human self-administration. The promising findings indicate that despite the poor lung deposition observed in macaques using nebulizers (13-20%), protective levels of RBC-AChE were still present in the blood even when exposure aer-Px (55 µg/kg) was delayed for five days. This long term retention of 5 mg/kg rHuBChE deposited in the lung bodes well for the use of an aer-rHuBChE pretreatment in humans where a user-friendly customized nebulizer with increased lung deposition up to 50% will provide even longer protection at a lower dose.

Comparison of Drosophila melanogaster Embryo and Adult Proteome by SWATH-MS Reveals Differential Regulation of Protein Synthesis, Degradation Machinery, and Metabolism Modules.

An important area of modern biology consists of understanding the relationship between genotype and phenotype. However, to understand this relationship it is essential to investigate one of the principal links between them: the proteome. With the development of recent mass-spectrometry approaches, it is now possible to quantify entire proteomes and thus relate them to different phenotypes. Here, we present a comparison of the proteome of two extreme developmental states in the well-established model organism Drosophila melanogaster: adult and embryo. Protein modules such as ribosome, proteasome, tricarboxylic acid cycle, glycolysis, or oxidative phosphorylation were found differentially expressed between the two developmental stages. Analysis of post-translation modifications of the proteins identified in this study indicates that they generally follow the same trend as their corresponding protein. Comparison between changes in the proteome and the transcriptome highlighted patterns of post-transcriptional regulation for the subunits of protein complexes such as the ribosome and the proteasome, whereas protein from modules such as TCA cycle, glycolysis, and oxidative phosphorylation seem to be coregulated at the transcriptional level. Finally, the impact of the endosymbiont Wolbachia pipientis on the proteome of both developmental states was also investigated.

Bioluminescence of Vibrio fischeri: bacteria respond quickly and sensitively to pulsed microwave electric (but not magnetic) fields.

Biological systems with intrinsic luminescent properties serve as powerful and noninvasive bioreporters for real-time and label-free monitoring of cell physiology. This study employs the bioluminescent marine bacterium Vibrio fischeri to investigate the effects of separated microwave electric (E) and magnetic (H) fields. Using a cylindrical TM010 mode aluminum resonant cavity, designed to spatially separate E and H fields of a pulsed microwave (2.45 GHz) input, we sampled at 100-ms intervals the 490-nm emission of bioluminescence from suspensions of the V. fischeri. E-field exposure (at 4.24 and 13.4 kV/m) results in rapid and sensitive responses to 100-ms pulses. H-field excitation elicits no measurable responses, even at 100-fold higher power input levels (equivalent to 183 A/m). The observed effects on bacterial light output partially correlate with measured E-field-induced temperature increases. In conclusion, the endogenous bioluminescence of V. fischeri provides a sensitive and noninvasive method to assess the biological effects of microwave fields.

Introduction of the YTE mutation into the non-immunogenic HIV bnAb PGT121 induces anti-drug antibodies in macaques.

Recombinant antibodies play increasingly important roles as immunotherapeutic treatments for human cancers as well as inflammatory and infectious diseases and have revolutionized their management. In addition, their therapeutic potential may be enhanced by the introduction of defined mutations in the crystallizable fragment (Fc) domains eg YTE (M252Y/S254T/T256E) and LS (M428L/N434S), as a consequence of increased half-lives and prolonged duration of protection. However, the functional properties of any biologic may be compromised by unanticipated immunogenicity in humans, rendering them ineffective. Several potent broadly neutralizing HIV monoclonal antibodies (bnAbs) have been identified that protect against SHIV challenge in macaque models and reduce HIV viremia in HIV-infected individuals. In the present study, the pharmacokinetics and immunogenicity of one or more 5mg/kg subcutaneous (SC) injections in naïve macaques of the HIV bnAb PGT121 and its PGT121-YTE mutant, both produced in plants, have been compared towards prolonging efficacy. Induction of anti-drug/anti-idiotypic antibodies (ADA, anti-id) has been monitored using both binding ELISAs and more functional inhibition of virus neutralization (ID50) assays. Timing of the anti-Id responses and their impact on pharmacokinetic profiles (clearance) and efficacy (protection) have also been assessed. The results indicate that ADA induction in naïve macaques may result both from injection of the previously non-immunogenic PGT121 into pre-primed animals and also by the introduction of the YTE mutation. Binding ADA antibody levels, induced in 7/10 macaques within two weeks of a first or second PGT121-YTE injection, were closely associated with both reduced pharmacokinetic profiles and loss of protection. However no correlation was observed with inhibitory ADA activity. These studies provide insights into both the structural features of bnAb and the immune status of the host which may contribute to the development of ADA in macaques and describe possible YTE-mediated changes in structure/orientation of HIV bnAbs that trigger such responses.

Requirement for PRC1 subunit BMI1 in host gene activation by Epstein-Barr virus protein EBNA3C.

Epstein-Barr virus proteins EBNA3A, EBNA3B and EBNA3C control hundreds of host genes after infection. Changes in epigenetic marks around EBNA3-regulated genes suggest that they exert transcriptional control in collaboration with epigenetic factors. The roles of polycomb repressive complex (PRC)2 subunit SUZ12 and of PRC1 subunit BMI1 were assessed for their importance in EBNA3-mediated repression and activation. ChIP-seq experiments for SUZ12 and BMI1 were performed to determine their global localization on chromatin and analysis offered further insight into polycomb protein distribution in differentiated cells. Their localization was compared to that of each EBNA3 to resolve longstanding questions about the EBNA3-polycomb relationship. SUZ12 did not co-localize with any EBNA3, whereas EBNA3C co-localized significantly and co-immunoprecipitated with BMI1. In cells expressing a conditional EBNA3C, BMI1 was sequestered to EBNA3C-binding sites after EBNA3C activation. When SUZ12 or BMI1 was knocked down in the same cells, SUZ12 did not contribute to EBNA3C-mediated regulation. Surprisingly, after BMI1 knockdown, EBNA3C repressed equally efficiently but host gene activation by EBNA3C was impaired. This overturns previous assumptions about BMI1/PRC1 functions during EBNA3C-mediated regulation, for the first time identifies directly a host factor involved in EBNA3-mediated activation and provides a new insight into how PRC1 can be involved in gene activation.

CATH: expanding the horizons of structure-based functional annotations for genome sequences.

This article provides an update of the latest data and developments within the CATH protein structure classification database (http://www.cathdb.info). The resource provides two levels of release: CATH-B, a daily snapshot of the latest structural domain boundaries and superfamily assignments, and CATH+, which adds layers of derived data, such as predicted sequence domains, functional annotations and functional clustering (known as Functional Families or FunFams). The most recent CATH+ release (version 4.2) provides a huge update in the coverage of structural data. This release increases the number of fully- classified domains by over 40% (from 308 999 to 434 857 structural domains), corresponding to an almost two- fold increase in sequence data (from 53 million to over 95 million predicted domains) organised into 6119 superfamilies. The coverage of high-resolution, protein PDB chains that contain at least one assigned CATH domain is now 90.2% (increased from 82.3% in the previous release). A number of highly requested features have also been implemented in our web pages: allowing the user to view an alignment between their query sequence and a representative FunFam structure and providing tools that make it easier to view the full structural context (multi-domain architecture) of domains and chains.

A Compact Microwave Microfluidic Sensor Using a Re-Entrant Cavity.

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.