Characterization of Ag-Ion Releasing Zeolite Filled 3D Printed Resins.

There has been profound growth in the use of 3D printed materials in dentistry in general, including orthodontics. The opportunity to impart antimicrobial properties to 3D printed parts from existing resins requires the capability of forming a stable colloid incorporating antimicrobial fillers. The objective of this research was to characterize a colloid consisting of a 3D printable resin mixed with Ag-ion releasing zeolites and fumed silica to create 3D printed parts with antiviral properties. The final composite was tested for antiviral properties against SARS-CoV-2 and HIV-1. Antiviral activity was measured in terms of the half-life of SARS-CoV-2 and HIV-1 on the composite surface. The inclusion of the zeolite did not interfere with the kinetics measured on the surface of the ATR crystal. While the depth of cure, measured following ISO4049 guidelines, was reduced from 3.8 mm to 1.4 mm in 5 s, this greatly exceeded the resolution required for 3D printing. The colloid was stable for at least 6 months and the rheological behavior was dependent upon the fumed silica loading. The inclusion of zeolites and fumed silica significantly increased the flexural strength of the composite as measured by a 3 point bend test. The composite released approximately 2500 µg/L of silver ion per gram of composite as determined by potentiometry. There was a significant reduction of the average half-life of SARS-CoV-2 (1.9 fold) and HIV-1 (2.7 fold) on the surface of the composite. The inclusion of Ag-ion releasing zeolites into 3D-printable resin can result in stable colloids that generate composites with improved mechanical properties and antiviral properties.

The RNA Atlas expands the catalog of human non-coding RNAs.

Existing compendia of non-coding RNA (ncRNA) are incomplete, in part because they are derived almost exclusively from small and polyadenylated RNAs. Here we present a more comprehensive atlas of the human transcriptome, which includes small and polyA RNA as well as total RNA from 300 human tissues and cell lines. We report thousands of previously uncharacterized RNAs, increasing the number of documented ncRNAs by approximately 8%. To infer functional regulation by known and newly characterized ncRNAs, we exploited pre-mRNA abundance estimates from total RNA sequencing, revealing 316 microRNAs and 3,310 long non-coding RNAs with multiple lines of evidence for roles in regulating protein-coding genes and pathways. Our study both refines and expands the current catalog of human ncRNAs and their regulatory interactions. All data, analyses and results are available for download and interrogation in the R2 web portal, serving as a basis for future exploration of RNA biology and function.

Messenger RNA capture sequencing of extracellular RNA from human biofluids using a comprehensive set of spike-in controls.

Comprehensive transcriptome analysis of extracellular RNA (exRNA) purified from human biofluids is challenging because of the low RNA concentration and compromised RNA integrity. Here, we describe an optimized workflow to (1) isolate exRNA from different types of biofluids and (2) to prepare messenger RNA (mRNA)-enriched sequencing libraries using complementary hybridization probes. Importantly, the workflow includes 2 sets of synthetic spike-in RNA molecules as processing controls for RNA purification and sequencing library preparation and as an alternative data normalization strategy. For complete details on the use and execution of this protocol, please refer to Hulstaert et al. (2020).

Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking.

Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this critical process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development using both in vitro imaging and biochemistry as well as in vivo models. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 results in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrate that Rab10 is Notch responsive, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune basement membrane secretion during blood vessel development.

Medical countermeasures during the 2018 Ebola virus disease outbreak in the North Kivu and Ituri Provinces of the Democratic Republic of the Congo: a rapid genomic assessment.

BACKGROUND: The real-time generation of information about pathogen genomes has become a vital goal for transmission analysis and characterisation in rapid outbreak responses. In response to the recently established genomic capacity in the Democratic Republic of the Congo, we explored the real-time generation of genomic information at the start of the 2018 Ebola virus disease (EVD) outbreak in North Kivu Province. METHODS: We used targeted-enrichment sequencing to produce two coding-complete Ebola virus genomes 5 days after declaration of the EVD outbreak in North Kivu. Subsequent sequencing efforts yielded an additional 46 genomes. Genomic information was used to assess early transmission, medical countermeasures, and evolution of Ebola virus. FINDINGS: The genomic information demonstrated that the EVD outbreak in the North Kivu and Ituri Provinces was distinct from the 2018 EVD outbreak in Équateur Province of the Democratic Republic of the Congo. Primer and probe mismatches to Ebola virus were identified in silico for all deployed diagnostic PCR assays, with the exception of the Cepheid GeneXpert GP assay. INTERPRETATION: The first two coding-complete genomes provided actionable information in real-time for the deployment of the rVSVΔG-ZEBOV-GP Ebola virus envelope glycoprotein vaccine, available therapeutics, and sequence-based diagnostic assays. Based on the mutations identified in the Ebola virus surface glycoprotein (GP12) observed in all 48 genomes, deployed monoclonal antibody therapeutics (mAb114 and ZMapp) should be efficacious against the circulating Ebola virus variant. Rapid Ebola virus genomic characterisation should be included in routine EVD outbreak response procedures to ascertain efficacy of medical countermeasures. FUNDING: Defense Biological Product Assurance Office.

2018 Ebola virus disease outbreak in Équateur Province, Democratic Republic of the Congo: a retrospective genomic characterisation.

BACKGROUND: The 2018 Ebola virus disease (EVD) outbreak in Équateur Province, Democratic Republic of the Congo, began on May 8, and was declared over on July 24; it resulted in 54 documented cases and 33 deaths. We did a retrospective genomic characterisation of the outbreak and assessed potential therapeutic agents and vaccine (medical countermeasures). METHODS: We used target-enrichment sequencing to produce Ebola virus genomes from samples obtained in the 2018 Équateur Province outbreak. Combining these genomes with genomes associated with known outbreaks from GenBank, we constructed a maximum-likelihood phylogenetic tree. In-silico analyses were used to assess potential mismatches between the outbreak strain and the probes and primers of diagnostic assays and the antigenic sites of the experimental rVSVΔG-ZEBOV-GP vaccine and therapeutics. An in-vitro flow cytometry assay was used to assess the binding capability of the individual components of the monoclonal antibody cocktail ZMapp. FINDINGS: A targeted sequencing approach produced 16 near-complete genomes. Phylogenetic analysis of these genomes and 1011 genomes from GenBank revealed a distinct cluster, confirming a new Ebola virus variant, for which we propose the name "Tumba". This new variant appears to have evolved at a slower rate than other Ebola virus variants (0·69 × 10-3 substitutions per site per year with "Tumba" vs 1·06 × 10-3 substitutions per site per year without "Tumba"). We found few sequence mismatches in the assessed assay target regions and antigenic sites. We identified nine amino acid changes in the Ebola virus surface glycoprotein, of which one resulted in reduced binding of the 13C6 antibody within the ZMapp cocktail. INTERPRETATION: Retrospectively, we show the feasibility of using genomics to rapidly characterise a new Ebola virus variant within the timeframe of an outbreak. Phylogenetic analysis provides further indications that these variants are evolving at differing rates. Rapid in-silico analyses can direct in-vitro experiments to quickly assess medical countermeasures. FUNDING: Defense Biological Product Assurance Office.

Correction to: Diel rewiring and positive selection of ancient plant proteins enabled evolution of CAM photosynthesis in Agave.

Following publication of the original article.

Enrichment post-library preparation enhances the sensitivity of high-throughput sequencing-based detection and characterization of viruses from complex samples.

BACKGROUND: Sequencing-based detection and characterization of viruses in complex samples can suffer from lack of sensitivity due to a variety of factors including, but not limited to, low titer, small genome size, and contribution of host or environmental nucleic acids. Hybridization-based target enrichment is one potential method for increasing the sensitivity of viral detection via high-throughput sequencing. RESULTS: This study expands upon two previously developed panels of virus enrichment probes (for filoviruses and for respiratory viruses) to include other viruses of biodefense and/or biosurveillance concern to the U.S. Department of Defense and various international public health agencies. The newly expanded and combined panel is tested using carefully constructed synthetic metagenomic samples that contain clinically relevant amounts of viral genetic material. Target enrichment results in a dramatic increase in sensitivity for virus detection as compared to shotgun sequencing, yielding full, deeply covered viral genomes from materials with Ct values suggesting that amplicon sequencing would be likely to fail. Increased pooling to improve cost- and time-effectiveness does not negatively affect the ability to obtain full-length viral genomes, even in the case of co-infections, although as expected, it does decrease depth of coverage. CONCLUSIONS: Hybridization-based target enrichment is an effective solution to obtain full-length viral genomes for samples from which virus detection would fail via unbiased, shotgun sequencing or even via amplicon sequencing. As the development and testing of probe sets for viral target enrichment expands and continues, the application of this technique, in conjunction with deeper pooling strategies, could make high-throughput sequencing more economical for routine use in biosurveillance, biodefense and outbreak investigations.

Diel rewiring and positive selection of ancient plant proteins enabled evolution of CAM photosynthesis in Agave.

BACKGROUND: Crassulacean acid metabolism (CAM) enhances plant water-use efficiency through an inverse day/night pattern of stomatal closure/opening that facilitates nocturnal CO2 uptake. CAM has evolved independently in over 35 plant lineages, accounting for ~ 6% of all higher plants. Agave species are highly heat- and drought-tolerant, and have been domesticated as model CAM crops for beverage, fiber, and biofuel production in semi-arid and arid regions. However, the genomic basis of evolutionary innovation of CAM in genus Agave is largely unknown. RESULTS: Using an approach that integrated genomics, gene co-expression networks, comparative genomics and protein structure analyses, we investigated the molecular evolution of CAM as exemplified in Agave. Comparative genomics analyses among C3, C4 and CAM species revealed that core metabolic components required for CAM have ancient genomic origins traceable to non-vascular plants while regulatory proteins required for diel re-programming of metabolism have a more recent origin shared among C3, C4 and CAM species. We showed that accelerated evolution of key functional domains in proteins responsible for primary metabolism and signaling, together with a diel re-programming of the transcription of genes involved in carbon fixation, carbohydrate processing, redox homeostasis, and circadian control is required for the evolution of CAM in Agave. Furthermore, we highlighted the potential candidates contributing to the adaptation of CAM functional modules. CONCLUSIONS: This work provides evidence of adaptive evolution of CAM related pathways. We showed that the core metabolic components required for CAM are shared by non-vascular plants, but regulatory proteins involved in re-reprogramming of carbon fixation and metabolite transportation appeared more recently. We propose that the accelerated evolution of key proteins together with a diel re-programming of gene expression were required for CAM evolution from C3 ancestors in Agave.

Targeted Sequencing of Respiratory Viruses in Clinical Specimens for Pathogen Identification and Genome-Wide Analysis.

A large number of viruses can individually and concurrently cause various respiratory illnesses. Metagenomic sequencing using next-generation sequencing (NGS) technology is capable of identifying a variety of pathogens. Here, we describe a method using a large panel of oligo probes to enrich sequence targets of 34 respiratory DNA and RNA viruses that reduces non-viral reads in NGS data and achieves high performance of sequencing-based pathogen identification. The approach can be applied to total nucleic acids purified from respiratory swabs stored in viral transport medium. Illumina TruSeq RNA Access Library procedure is used in targeted sequencing of respiratory viruses. The samples are subjected to RNA fragmentation, random reverse transcription, random PCR amplification, and ligation with barcoded library adaptors. The libraries are pooled and subjected to two rounds of enrichments by using a large panel of oligos designed to capture whole genomes of 34 respiratory viruses. The enriched libraries are amplified and sequenced using Illumina MiSeq sequencing system and reagents. This method can achieve viral detection sensitivity comparable with molecular assay and obtain partial to complete genome sequences for each virus to allow accurate genotyping and variant analysis.

Comprehensive viral enrichment enables sensitive respiratory virus genomic identification and analysis by next generation sequencing.

Next generation sequencing (NGS) technologies have revolutionized the genomics field and are becoming more commonplace for identification of human infectious diseases. However, due to the low abundance of viral nucleic acids (NAs) in relation to host, viral identification using direct NGS technologies often lacks sufficient sensitivity. Here, we describe an approach based on two complementary enrichment strategies that significantly improves the sensitivity of NGS-based virus identification. To start, we developed two sets of DNA probes to enrich virus NAs associated with respiratory diseases. The first set of probes spans the genomes, allowing for identification of known viruses and full genome sequencing, while the second set targets regions conserved among viral families or genera, providing the ability to detect both known and potentially novel members of those virus groups. Efficiency of enrichment was assessed by NGS testing reference virus and clinical samples with known infection. We show significant improvement in viral identification using enriched NGS compared to unenriched NGS. Without enrichment, we observed an average of 0.3% targeted viral reads per sample. However, after enrichment, 50%-99% of the reads per sample were the targeted viral reads for both the reference isolates and clinical specimens using both probe sets. Importantly, dramatic improvements on genome coverage were also observed following virus-specific probe enrichment. The methods described here provide improved sensitivity for virus identification by NGS, allowing for a more comprehensive analysis of disease etiology.

Ion release and in vitro enamel fluoride uptake associated with pit and fissure sealants containing microencapsulated remineralizing agents.

PURPOSE: To determine if pit-and-fissure sealants with microencapsulated remineralizing agents with sustained release of fluoride, calcium and phosphate ions could promote enamel fluoride uptake by demineralized tooth structure. METHODS: Sealants that contained 5 w/w% microcapsules with aqueous solutions of 5M Ca(NO3)2 or 0.8M NaF or 6.0M K2HPO4 or a mixture of all three were prepared. Ion release profiles were measured as a function of time. Enamel fluoride uptake by demineralized tooth structure was determined. RESULTS: Sustained release of fluoride, calcium and phosphate ions from a sealant was demonstrated. Fluoride uptake by demineralized enamel was significantly increased compared to a control sealant manufactured without microcapsules (P< 0.01). Bovine enamel that contained 2.2±2.1 µg F/g of enamel prior to exposure to a sealant without microcapsules had 2.3±0.5 after 90 days. Enamel exposed to sealant with 5w/% NaF microcapsules went from 3.5±3.5 µg F/g of enamel prior to exposure to 148±76 after 90 days. Enamel exposed to sealant with 2 w/w% NaF, 2 w/w% Ca(NO3)2 and 1 w/w% K2HPO4 microcapsules went from 1.7±0.7 µg F/g of enamel prior to exposure to 190±137 after 90 days. CLINICAL SIGNIFICANCE: Sealants with encapsulated remineralizing agents were capable of releasing biologically available fluoride, calcium, and phosphate ions. Incorporation of these microcapsules in pit and fissure sealants is a promising method for remineralization determined by enamel fluoride uptake measurements.

SSB recruitment of Exonuclease I aborts template-switching in Escherichia coli.

Misalignment of a nascent strand and the use of an alternative template during DNA replication, a process termed "template-switching", can give rise to frequent mutations and genetic rearrangements. Mutational hotspots are frequently found associated with imperfect inverted repeats ("quasipalindromes" or "QPs") in many organisms, including bacteriophage, bacteria, yeast and mammals. Evidence suggests that QPs mutate by a replication template-switch whereby one copy of the inverted repeat templates synthesis of the other. To study quasipalindrome-associated mutagenesis ("QPM") more systematically, we have engineered mutational reporters in the lacZ gene of Escherichia coli, that revert to Lac+ specifically by QPM. We and others have shown that QPM is more efficient during replication of the leading strand than it is on the lagging strand. We have previously shown that QPM is elevated and that the leading-strand bias is lost in mutants lacking the major 3' ssDNA exonucleases, ExoI and ExoVII. This suggests that one or both of these exonucleases more efficiently abort template-switches on the lagging strand. Here, we show that ExoI is primarily responsible for this bias and that its ability to be recruited by single-strand DNA binding protein plays a critical role in QPM avoidance and strand bias. In addition to these stand-alone exonucleases, loss of the 3' proofreading exonuclease activity of the replicative DNA polymerase III also greatly elevates QPM. This may be because template-switching is initiated by base misincorporation, leading to polymerase dissociation and subsequent nascent strand misalignment; alternatively or additionally, the proofreading exonuclease may scavenge displaced 3' DNA that would otherwise be free to misalign.

Complete genome sequence of a KI polyomavirus isolated from an otherwise healthy child with severe lower respiratory tract infection.

Unbiased, deep sequencing of a nasal specimen from an otherwise healthy 13-month-old boy hospitalized in intensive care revealed high gene expression and the complete genome of a novel isolate of KI polyomavirus (KIPyV). Further investigation detected minimal gene expression of additional viruses, suggesting that KIPyV was potentially the causal agent. Analysis of the complete genome of isolate NMKI001 revealed it is different from all previously reported genomes and contains two amino acid differences as compared to the closest virus isolate, Stockholm 380 (EF127908). J. Med. Virol. 89:926-930, 2017. © 2016 Wiley Periodicals, Inc.

Complete Genome Sequence of a Novel Human WU Polyomavirus Isolate Associated with Acute Respiratory Infection.

We report here the complete genome sequence of a WU polyomavirus (WUPyV) isolate, NM040708, collected from a patient with an acute respiratory infection in New Mexico. The double-stranded DNA (dsDNA) genome of NM040708 is 5,229 bp in length and differs from the WUPyV reference with accession no. NC_009539 by 6 nucleotides and 2 amino acids.

Plant compartment and biogeography affect microbiome composition in cultivated and native Agave species.

Desert plants are hypothesized to survive the environmental stress inherent to these regions in part thanks to symbioses with microorganisms, and yet these microbial species, the communities they form, and the forces that influence them are poorly understood. Here we report the first comprehensive investigation of the microbial communities associated with species of Agave, which are native to semiarid and arid regions of Central and North America and are emerging as biofuel feedstocks. We examined prokaryotic and fungal communities in the rhizosphere, phyllosphere, leaf and root endosphere, as well as proximal and distal soil samples from cultivated and native agaves, through Illumina amplicon sequencing. Phylogenetic profiling revealed that the composition of prokaryotic communities was primarily determined by the plant compartment, whereas the composition of fungal communities was mainly influenced by the biogeography of the host species. Cultivated A. tequilana exhibited lower levels of prokaryotic diversity compared with native agaves, although no differences in microbial diversity were found in the endosphere. Agaves shared core prokaryotic and fungal taxa known to promote plant growth and confer tolerance to abiotic stress, which suggests common principles underpinning Agave-microbe interactions.

Ion release, fluoride charge of and adhesion of an orthodontic cement paste containing microcapsules.

OBJECTIVES: Dental materials capable of releasing calcium, phosphate and fluoride are of great interest for remineralization. Microencapsulated aqueous solutions of these ions in orthodontic cement demonstrate slow, sustained release by passive diffusion through a permeable membrane without the need for dissolution or etching of fillers. The potential to charge a dental material formulated with microencapsulated water with fluoride by toothbrushing with over the counter toothpaste and the effect of microcapsules on cement adhesion to enamel was determined. METHODS: Orthodontic cements that contained microcapsules with water and controls without microcapsules were brushed with over-the-counter toothpaste and fluoride release was measured. Adhesion measurements were performed loading orthodontic brackets to failure. Cements that contained microencapsulated solutions of 5.0M Ca(NO3)2, 0.8M NaF, 6.0MK2HPO4 or a mixture of all three were prepared. Ion release profiles were measured as a function of time. RESULTS: A greater fluoride charge and re-release from toothbrushing was demonstrated compared to a control with no microcapsules. Adhesion of an orthodontic cement that contained microencapsulated remineralizing agents was 8.5±2.5MPa compared to the control without microcapsules which was of 8.3±1.7MPa. Sustained release of fluoride, calcium and phosphate ions from cement formulated with microencapsulated remineralizing agents was demonstrated. CONCLUSIONS: Orthodontic cements with microcapsules show a release of bioavailable fluoride, calcium, and phosphate ions near the tooth surface while having the ability to charge with fluoride and not effect the adhesion of the material to enamel. Incorporation of microcapsules in dental materials is promising for promoting remineralization.

Molecular Evidence of Sexual Transmission of Ebola Virus.

A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiologic data, the genomic analysis provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD. (Funded by the Defense Threat Reduction Agency and others.).