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Synthetic biology seeks to probe fundamental aspects of biological form and function by construction [i.e., (re)synthesis] rather than deconstruction (analysis). In this sense, biological sciences now follow the lead given by the chemical sciences. Synthesis can complement analytic studies but also allows novel approaches to answering fundamental biological questions and opens up vast opportunities for the exploitation of biological processes to provide solutions for global problems. In this review, we explore aspects of this synthesis paradigm as applied to the chemistry and function of nucleic acids in biological systems and beyond, specifically, in genome resynthesis, synthetic genetics (i.e., the expansion of the genetic alphabet, of the genetic code, and of the chemical make-up of genetic systems), and the elaboration of orthogonal biosystems and components.

To discuss recent advances supporting the role of red blood cells (RBCs) in the host immune response.Over the last century, research has demonstrated that red blood cells exhibit functions beyond oxygen transport, including immune function. Recent work indicates that the nucleic acid sensing receptor, toll-like receptor 9 (TLR9), is expressed on the RBC surface and implicated in innate immune activation and red cell clearance during inflammatory states. In addition to this DNA-sensing role of RBCs, there is growing evidence that RBCs may influence immune function by inducing vascular dysfunction. RBC proteomics and metabolomics have provided additional insight into RBC immune function, with several studies indicating changes to RBC membrane structure and metabolism in response to severe acute respiratory syndrome coronavirus 2 infection. These structural RBC changes may even provide insight into the pathophysiology of the 'long-coronavirus disease 2019' phenomenon. Finally, evidence suggests that RBCs may influence host immune responses via complement regulation. Taken together, these recent findings indicate RBCs possess immune function. Further studies will be required to elucidate better how RBC immune function contributes to the heterogeneous host response during inflammatory states.The appreciation for nongas exchanging, red blood cell immune functions is rapidly growing. A better understanding of these RBC functions may provide insight into the heterogeneity observed in the host immune response to infection and inflammation.

10 pages, 5 figures, 1 table, supporting Information https://pubs.acs.org/doi/10.1021/jacs.2c13043, We study here a DNA oligonucleotide having the ability to form two different i-motif structures whose relative stability depends on pH and temperature. The major species at neutral pH is stabilized by two C:C+ base pairs capped by two minor groove G:C:G:C tetrads. The high pH and thermal stability of this structure are mainly due to the favorable effect of the minor groove tetrads on their adjacent positively charged C:C+ base pairs. At pH 5, we observe a more elongated i-motif structure consisting of four C:C+ base pairs capped by two G:T:G:T tetrads. Molecular dynamics calculations show that the conformational transition between the two structures is driven by the protonation state of key cytosines. In spite of large conformational differences, the transition between the acidic and neutral structures can occur without unfolding of the i-motif. These results represent the first case of a conformational switch between two different i-motif structures and illustrate the dramatic pH-dependent plasticity of this fascinating DNA motif, This investigation was supported by research grants from the Spanish “Ministerio de Ciencia e Innovación” (PID2020-116620GB-I00, RTI2018-096704-B-100, and PID2021-122478NB-I00), the Center of Excellence for HPC H2020 European Commission; “BioExcel-2. Centre of Excellence for Computational Biomolecular Research” (823830); and the Instituto de Salud Carlos III–Instituto Nacional de Bioinformatica (ISCIII PT 17/0009/0007 cofunded by the Fondo Europeo de Desarrollo Regional). This project was cofunded by the European Regional Development Fund under the framework of the ERFD Operative Programme for Catalunya, the Catalan Government AGAUR (SGR2017-134), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNAsequencing analyses of samples frommouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.

Nucleic acid nanotechnology is a powerful tool in the fields of biosensing and nanomedicine owing to their high editability and easy synthesis and modification. Artificial nucleic acid nanostructures have become an emerging research hotspot as gene carriers with low cytotoxicity and immunogenicity for therapeutic approaches. In this review, recent progress in the design and functional mechanisms of nucleic acid-based artificial nano-vectors especially for exogenous siRNA and antisense oligonucleotide delivery is summarized. Different types of DNA nanocarriers, including DNA junctions, tetrahedrons, origami, hydrogels and scaffolds, are introduced. The enhanced targeting strategies to improve the delivery efficacy are demonstrated. Furthermore, RNA based gene nanocarrier systems by self-assembly of short strands, rolling circle transcription, chemical crosslinking and using RNA motifs and DNA-RNA hybrids are demonstrated. Finally, the outlook and potential challenges are highlighted. The nucleic acid-based artificial nanocarriers offer a promising and precise tool for gene delivery and therapy.

In nucleic acid drug discovery, it is extremely important to develop a technology to understand the distribution in target organs and to trace the degradation process in the body in order to optimize the structure and improve the efficiency of the clinical trial process. Since nucleic acid drugs are essentially metabolically degraded into numerous fragments, labeling at the internal position is preferable to that at the terminus. Due to the high molar specific activity of tritium, various approaches for tritium-labeling have been studied for nucleic acid drugs. Nevertheless, a generally-applicable method for tritium labeling of the internal position of a nucleic acid has not been established. In this study, we have demonstrated a new and efficient method for site-specific tritium labeling of the cytosine base at a predefined internal position in nucleic acid drugs. This method was developed by the chemical modification of the cytosine 4-amino group with the pyridinyl vinyl keto group by the functionality-transfer reaction using the reactive oligodeoxynucleotide (ODN), followed by reduction with NaBT

Collection, preservation, and shipment of histological specimens in low-resource settings is challenging. We present a novel method that achieved excellent preservation of placental specimens from rural Mali by using formalin fixation, ethanol dehydration, and long-term storage in a solar-powered freezer. Sample preservation success was 92%, permitting evaluation of current and past malaria infection, anemia, placental maturity, and inflammation. Using RNAscope® hybridization we were able to visualize cell-specific gene expression patterns in the formalin-fixed paraffin-embedded (FFPE) specimens. Additionally, our method entailed mirrored sampling from the two cut faces of a cotyledon, one for the FFPE workflows and the other for storage in RNAlater™ and RNA-seq.

Nucleic acid damage from oxidative stress (NA-OXS) may be a molecular mechanism driving the severely increased morbidity and mortality from somatic causes in adults with psychiatric disorders.To systematically retrieve and analyze data on NA-OXS across the psychiatric disorder diagnostic spectrum.The PubMed, Embase, and PsycINFO databases were searched from inception to November 16, 2021. A hand search of reference lists of relevant articles was also performed.Key study inclusion criteria in this meta-analysis were as follows: adult human study population, measurement of any marker of DNA or RNA damage from oxidative stress, and either a (1) cross-sectional design comparing patients with psychiatric disorders (any diagnosis) with a control group or (2) prospective intervention. Two authors screened the studies, and 2 senior authors read the relevant articles in full and assessed them for eligibility.The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines were followed. Two authors performed data extraction independently, and a senior coauthor was consulted in cases of disagreement. Data were synthesized with random-effects and multilevel meta-analyses.The predefined hypothesis was that individuals with psychiatric disorders have increased NA-OXS levels. The main outcome was the standardized mean differences (SMDs) among patients and controls in nucleic acid oxidation markers compared across diagnostic groups. Analyses were divided into combinations of biological matrices and nucleic acids.Eighty-two studies fulfilled the inclusion criteria, comprising 205 patient vs control group comparisons and a total of 10 151 patient and 10 532 control observations. Overall, the data showed that patients with psychiatric disorders had higher NA-OXS levels vs controls across matrices and molecules. Pooled effect sizes ranged from moderate for urinary DNA markers (SMD = 0.44 [95% CI, 0.20-0.68]; P .001) to very large for blood cell DNA markers (SMD = 1.12 [95% CI, 0.69-1.55; P .001). Higher NA-OXS levels were observed among patients with dementias followed by psychotic and bipolar disorders. Sensitivity analyses excluding low-quality studies did not materially alter the results. Intervention studies were few and too heterogenous for meaningful meta-analysis.The results of this meta-analysis suggest that there is an association with increased NA-OXS levels in individuals across the psychiatric disorder diagnostic spectrum. NA-OXS may play a role in the somatic morbidity and mortality observed among individuals with psychiatric disorders.

Fifty years ago, the first atomic-resolution structure of a nucleic acid double helix, the mini-duplex (ApU)

Background RNA extraction is a step that precedes several molecular techniques. The fibrous tissue, more specifically the dura mater, has several limitations in routine protocols, and lacks optimization protocols to overcome these problems. Objective To test stock reagents and purification kits, optimizing commercial kit protocols for RNA extraction from the dura mater. Methods Dura mater samples were obtained from eight Wistar rats and maintained in two different stabilizers. The samples were purified using four different protocols, and the RNA was evaluated for the yield and purity in NanoDrop 2000 (Thermo Scientific, Wilmington, DE, United States). Beta-actin gene was used for analyzing gene expression, since is one of the most used reference genes. Results The RNA preservation was similar in both stabilizers. The addition of an incubation step prior the purification protocols allowed better tissue digestion and RNA recovery. The RNA purified using the protocols membrane-based showed higher quality than liquid-liquid purification. This impact was observed in the 3-week evaluation using RT-qPCR. Conclusion Stabilizers are efficient for RNA preservation and membrane-based purification protocols are more suitable for RNA recovery from dura mater tissue, allowing the evaluation of gene expression in this type of tissue. Adaptations in the dura mater RNA extraction protocol differ from the pre-established protocols because it takes into account the peculiarity of fibrous tissue and low cellularity. In addition to providing a low-cost mechanism, based on techniques that are part of the laboratory routine, it is possible to improve the quality of the extracted material, ensuring greater efficiency in the use of subsequent techniques. Resumo Antecedentes A extração de RNA é uma etapa que antecede várias técnicas moleculares. O tecido fibroso, mais especificamente a dura-máter, apresenta várias limitações nos protocolos de rotina e carece de protocolos de otimização para superar estes problemas. Objetivo Testar reagentes de estoque e kits de purificação, otimizando protocolos de kits comerciais para extração de RNA da dura-máter. Métodos Amostras de dura-máter foram obtidas de oito ratos Wistar e mantidas em dois estabilizadores diferentes. As amostras foram purificadas em quatro protocolos diferentes e o RNA foi avaliado quanto ao rendimento e pureza no NanoDrop 2000 (Thermo Scientific, Wilmington, DE, United States). O gene da beta-actina foi utilizado para analisar a expressão gênica, uma vez que é um dos genes de referência mais utilizados. Resultados A preservação do RNA foi semelhante em ambos os estabilizadores. A adição de uma etapa de incubação antes dos protocolos de purificação permitiu uma melhor digestão do tecido e recuperação de RNA. O RNA purificado pelos protocolos baseados em membrana apresentou qualidade superior ao da purificação líquido-líquido. Este impacto foi observado na avaliação de três semanas usando RT-qPCR. Conclusão Os estabilizadores são eficientes para preservação do RNA e os protocolos de purificação baseados em membrana são mais adequados para recuperação de RNA do tecido da dura-máter, permitindo a avaliação da expressão gênica neste tipo de tecido. As adaptações no protocolo de extração de RNA da dura-máter diferem dos protocolos preestabelecidos porque leva em consideração a peculiaridade do tecido fibroso e com baixa celularidade. Além de fornecer um mecanismo de baixo custo, baseado em técnicas que fazem parte da rotina laboratorial, é possível melhorar a qualidade do material extraído, garantindo maior eficácia no uso de técnicas subsequentes.

Fatty acid vesicles may have played a role in the origin of life as a major structural component of protocells, with the potential for encapsulation of genetic materials. Vesicles that grew and divided more rapidly than other vesicles could have had a selective advantage. Fatty acid vesicles grow by incorporating additional fatty acids from micelles, and certain prebiotic molecules (e.g., sugars, nucleobases, and amino acids) can bind to fatty acid vesicles and stabilize them. Here, we investigated whether the presence of a variety of biomolecules affects the overall growth of vesicles composed of decanoic acid, a prebiotically plausible fatty acid, upon micelle addition. We tested 31 molecules, including 15 dipeptides, 7 amino acids, 6 nucleobases or nucleosides, and 3 sugars. We find that the initial radius and final radius of vesicles are largely unaffected by the presence of the additional compounds. However, three dipeptides enhanced the initial rates of growth compared to control vesicles with no small molecules added; another three dipeptides decreased the initial rates of growth. We conclude that vesicles can indeed grow in the presence of a wide range of molecules likely to have been involved in the origin of life. These results imply that vesicles would have been able to grow in complex and heterogeneous chemical environments. We find that the molecules that enhance the initial growth rate tend to have hydrophobic groups (e.g., leucine), which may interact with the lipid membrane to affect growth rate; furthermore, the molecules that cause the largest decrease in initial growth rate are dipeptides containing a serine residue, which contains a hydroxyl group that could potentially hydrogen-bond with the fatty acid carboxylate groups.

Strategies utilizing the CRISPR/Cas nucleases Cas13 and Cas12 have shown great promise in the development of highly sensitive and rapid diagnostic assays for the detection of pathogenic nucleic acids. The most common approaches utilizing fluorophore-quencher molecular beacons require strand amplification strategies or highly sensitive optical setups to overcome the limitations of the readout. Here, we demonstrate a flexible strategy for assembling highly luminescent and colorimetric quantum dot-nucleic acid hairpin (QD-HP) molecular beacons for use in CRISPR/Cas diagnostics. This strategy utilizes a chimeric peptide-peptide nucleic acid (peptide-PNA) to conjugate fluorescently labeled DNA or RNA hairpins to ZnS-coated QDs. QDs are particularly promising alternatives for molecular beacons due to their greater brightness, strong UV absorbance with large emission offset, exceptional photostability, and potential for multiplexing due to their sharp emission peaks. Using Förster resonance energy transfer (FRET), we have developed ratiometric reporters capable of pM target detection (without nucleotide amplification) for both target DNA and RNA, and we further demonstrated their capabilities for multiplexing and camera-phone detection. The flexibility of this system is imparted by the dual functionality of the QD as both a FRET donor and a central nanoscaffold for arranging nucleic acids and fluorescent acceptors on its surface. This method also provides a generalized approach that could be applied for use in other CRISPR/Cas nuclease systems.

Nucleic acid therapeutics represent a major advance toward treating diseases at their root cause. However, nucleic acids are prone to degradation by serum endonucleases, clearance through the immune system, and rapid degradation in complex medium. To overcome these barriers, nucleic acids frequently include chemical modifications to improve stability or decrease immune responses. Lipid nanoparticles (LNPs) have enabled a dramatic reduction in the dose required to achieve a therapeutic effect by protecting these nucleic acids and improving their intracellular delivery. It has been assumed thus far that nonspecific ionic interactions drive LNP formation and chemical modifications to the nucleic acid backbone to confer improved stability do not impact LNP delivery in any way. Here, we demonstrate that these chemical modifications do impact LNP morphology substantially, and phosphorothioate modifications produce stronger interactions with ionizable amino lipids, resulting in enhanced entrapment. This work represents a major first step toward greater understanding of the interaction between the lipid components and nucleic acids within an LNP.

Long-lived emissive nucleic acid probes are widely used in biochemical analysis due to their programmable structures, high signal-to-background ratio, and high sensitivity. Homogeneous detection based on long-lived emissive nucleic acid probes is often achieved through Förster resonance energy transfer (FRET), which suffers from the limitation of a narrow effective distance range. Herein, a new strategy of accessing nucleic acid hybridization-responsive luminescent probes is presented. The photoluminescence (PL) of a Lumi4-Tb complex internally modified with DNA is switched on by nucleic acid hybridization, after which the PL is increased up to 20 times. PL lifetime analysis revealed a possible mechanism of luminescence enhancement. Due to the flexibility of single-stranded nucleic acid chains, the bases and phosphate groups can coordinate with the Tb(III), which reduces the stability of the Tb complex and results in weak PL. After hybridization, the rigid double helix structure suppresses the coordination between Tb(III) and the bases or phosphate groups, causing luminescence enhancement. As the DNA sequence can be freely designed, an array of probes for different DNA or RNA targets can be created with the same Tb complex. Moreover, the novel probe design can afford pM detection limits of DNA or RNA without any nucleic acid amplification and exhibits great potential for nucleic acid detection in clinical diagnosis.

Abstract: An early diagnosis has the potential to greatly decrease cancer mortality. For that purpose, specific cancer biomarkers have been molecularly targeted by aptamer sequences to enable an accurate and rapid detection. Aptamer-based biosensors for cancer diagnostics are a promising alternative to those using antibodies, due to their high affinity and specificity to the target molecules and advantageous production. Synthetic nucleic acid aptamers are generated by in vitro Systematic Evolution of Ligands by Exponential enrichment (SELEX) methodologies that have been improved over the years to enhance the efficacy and shorten the selection process. Aptamers have been successfully applied in electrochemical, optical, photoelectrochemical and piezoelectrical-based detection strategies. These aptasensors comprise a sensitive, accurate and inexpensive option for cancer detection being used as point-of-care devices. This review highlights the recent advances in cancer biomarkers, achievements and optimizations made in aptamer selection, as well as the different aptasensors developed for the detection of several cancer biomarkers.

An ideal molecular diagnostic method should be sensitive, specific, low cost, rapid, portable, and easy to operate. Traditional nucleic acid detection methods based mainly on PCR technology have not only high sensitivity and specificity, but also some limitations, such as the need for expensive equipment and skilled technicians, being both time and labor intensive, and difficult to implement in some regions. However, with the continuous development of CRISPR-Cas technology and its application in molecular diagnosis, new approaches have been used for the construction of molecular diagnostic systems. In this review, we discuss recent advances in CRISPR-based molecular diagnostic technologies and highlight the revolution they bring to the field of molecular diagnostics.

The nasopharyngeal swab is the gold standard collection method for COVID-19, but is invasive and painful, subsequently resulting in poor patient acceptance. This investigation explores the process of developing and validating an alternative respiratory pathogen collection device that relies on a nasopharyngeal irrigation mechanic. The primary objective was to determine if sufficient pathological sampling can be achieved by mechanism of nasopharyngeal irrigation that is proportionate to the nasopharyngeal swab method.The study device was designed using Shapr3D modeling software and fabricated on a fused deposition modeling printer. Fifteen participants were enrolled with each receiving a saline nasopharyngeal washing using the study device. Specimen adequacy was evaluated by two real-time reverse transcriptase polymerase chain reaction (PCR) testing methods to identify the presence of the human RNase P gene. Results were evaluated quantitatively through interpretation of the PCR cycle threshold (Ct).All 15 specimens tested positive for the presence of RNaseP, demonstrating specimen cellularity, adequate extraction of nucleic acids, and the absence of inhibitors to amplification. The mean CThe importance of early diagnostic testing and its role in countermeasures for communicable diseases such as COVID-19 is well established in the literature. Innovation to bolster our testing infrastructure is more important now than ever. This study was successful in developing and validating an alternative nasopharyngeal respiratory pathogen collection device that utilizes fluid debridement as its core mechanic. Data from this pilot study demonstrated the study device was successful in producing high-quality specimens for PCR testing. Feedback from the study participants was also in favor of the study device when compared to the nasopharyngeal swab.

Nucleic acid therapeutics are a versatile class of sequence-programmable drugs that offer a robust and clinically viable strategy to modulate expression or correct genetic defects contributing to disease. The majority of drugs currently on the market target proteins; however, proteins only represent a subset of possible disease targets. Nucleic acid therapeutics allow intuitive engagement with genome sequences providing a more direct way to target many diseases at their genetic root cause. Their clinical success depends on platform technologies which can support durable and well tolerated pharmacological activity in a given tissue. Nucleic acid drugs possess a potent combination of target specificity and adaptability required to advance drug development for many diseases. As these therapeutic technologies mature, their clinical applications can also expand access to personalized therapies for patients with rare or solo genetic diseases. Spaceflight crew members exposed to the unique hazards of spaceflight, especially those related to galactic cosmic radiation (GCR) exposure, represent another patient subset who may also benefit from nucleic acid drugs as countermeasures. In this review, we will discuss the various classes of RNA- and DNA-targeted nucleic acid drugs, provide an overview of their present-day clinical applications, and describe major strategies to improve their delivery, safety, and overall efficacy.

Non-healing wounds have long been the subject of scientific and clinical investigations. Despite breakthroughs in understanding the biology of delayed wound healing, only limited advances have been made in properly treating wounds. Recently, research into nucleic acids (NAs) such as small-interfering RNA (siRNA), microRNA (miRNA), plasmid DNA (pDNA), aptamers, and antisense oligonucleotides (ASOs) has resulted in the development of a latest therapeutic strategy for wound healing. In this regard, dendrimers, scaffolds, lipid nanoparticles, polymeric nanoparticles, hydrogels, and metal nanoparticles have all been explored as NA delivery techniques. However, the translational possibility of NA remains a substantial barrier. As a result, different NAs must be identified, and their distribution method must be optimized. This review explores the role of NA-based therapeutics in various stages of wound healing and provides an update on the most recent findings in the development of NA-based nanomedicine and biomaterials, which may offer the potential for the invention of novel therapies for this long-term condition. Further, the challenges and potential for miRNA-based techniques to be translated into clinical applications are also highlighted.

The oral route is the most convenient and simplest mode of administration. Nevertheless, orally administration of some commonly used therapeutic drugs, such as polypeptides, therapeutic proteins, small-molecule drugs, and nucleic acids, remains a major challenge due to the harsh gastrointestinal environment and the limited oral bioavailability. Extracellular vesicles (EVs) are diverse, nanoscale phospholipid vesicles that are actively released by cells and play crucial roles in intercellular communications. Some EVs have been shown to survive with the gastrointestinal tract (GIT) and can cross biological barriers. The potential of EVs to cross the GIT barrier makes them promising natural delivery carriers for orally administered drugs. Here, we introduce the uniqueness of EVs and their feasibility as oral drug delivery vehicles (ODDVs). Then we provide a general description of the different cellular EVs based oral drug delivery systems (ODDSs) currently under study and emphasize the contribution of endogenous features and multifunctional properties of EVs to the delivery performance. The current obstacles of moving EVs based ODDSs from bench to bedside are also discussed.

Presently, three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved against oncogene addicted EGFR-mutant non-small cell lung cancer (NSCLC). Patients with actionable EGFR mutations invariably develop resistance. This resistance can be intrinsic (primary) or acquired (secondary).This was a retrospective study carried out between January 2016 and April 2021 analysing 486 samples of NSCLC for primary and secondary resistance to first- (erlotinib, gefitinb), second- (afatinib) and/or third-generation (osimertinib) TKIs in EGFR-mutant NSCLCs by next generation sequencing (NGS). Tissue NGS was carried out using the Thermofischer Ion Torrent™ Oncomine™ Focus 52 gene assay; liquid biopsy NGS was carried out using the Oncomine Lung Cell-Free Total Nucleic Acid assay. All cases were previously tested for a single EGFR gene with the Therascreen® EGFR RGQ PCR kit.The results were divided into four groups: (i) group 1: primary resistance to first- and/or second-generation TKIs. This group, with 21 cases, showed EGFR exon 20 insertions, dual, complex mutations and variant of unknown significance, de novo MET gene amplification besides other mutations. (ii) Group 2: primary resistance to third-generation TKIs. This group showed two cases, with one showing dual EGFR mutation (L858R and E709A) and EGFR gene amplification. (iii) Group 3: secondary resistance to first- and second-generation TKIs. This group had 27 cases, which were previously reported negative for EGFR T790M by single gene testing. Significant findings were MET gene amplification in four cases, with one also showing MET exon 14 skipping mutation. Three cases showed small cell change and one showed loss of primary mutation. (iv) Group 4: secondary resistance to third-generation TKIs. The latter group was further subgrouped into group 4A: secondary resistance to osimertinib (third-generation TKI) when offered as second-line therapy after first- and second-generation TKIs on detection of T790M mutation. This group had 15 cases. EGFR T790M mutation was lost in 10 (10/15; 67%) cases and was retained in five cases. Patients with T790M loss experienced early resistance (6.9 months versus 12.6 months mean, P = 0.0024) compared with cases that retained T790M. Two cases gained MET amplification as the resistance mechanisms. Other mutations that were found when EGFR T790M was lost were in FGFR3, KRAS, PIK3CA, CTNNB1, BRAF genes. One case had EML4-ALK translocation. Two cases showed driver EGFR deletion 19, retained T790M and C797S mutation in Cis form. Group 4B: secondary resistance to osimertinib (when given as first-line therapy) in EGFR-mutant NSCLC. This group had three cases. The duration of osimertinib treatment ranged from 11 to 17 months. Two patients showed additional C797S mutation along with primary EGFR mutation.This study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs.

Threose nucleic acid (TNA) is considered a potential RNA progenitor due to its chemical simplicity, base pairing property, and capability of folding into a functional tertiary structure. However, it is unknown whether the functional property can be maintained during transition from TNA to RNA. Here, we use a toggle

Electrostatic potentials around macromolecules in the presence of mobile charges are difficult to assess especially for highly charged systems. Here, we report measurements of local electrostatic potentials around DNA by paramagnetic NMR. Through quantitative analysis of NMR paramagnetic relaxation enhancement arising from positively charged or neutral paramagnetic cosolutes, we were able to determine local electrostatic potentials around

A DNA triplex has the advantages of improved nanostructure stability and pH environment responsiveness compared with single-stranded and double-stranded nucleic acids. However, sequence stability and low design efficiency hinder the application of DNA triplexes. Therefore, a DNA triplex design approach (TripDesign) based on interaction forces is proposed. First, we present the stacking force constraint, torsional stress constraint, and G-quadruplex motif constraint and then use an improved memetic algorithm to design triplex sequences under combinatorial constraints. Finally, to quantify the process of triplex formation, we also explore the minimum length of the triplex-forming oligos (TFOs) required to form the triplex and the factors that produce depletion in cyclic pH-jump experiments. The experimental results show that the sequences produced by TripDesign have high stability and reversibility, and the proposed approach achieves efficient and automatic sequence design. In addition, this study characterizes multiple basic parameters of DNA triplex formation and promotes the wider application of DNA triplexes in nanotechnology.

Important roles of humoral tumor immunity are often pointed out; however, precise profiles of dominant antigens and developmental mechanisms remain elusive. We systematically investigated the humoral antigens of dominant intratumor immunoglobulin clones found in human cancers. We found that approximately half of the corresponding antigens were restricted to strongly and densely negatively charged polymers, resulting in simultaneous reactivities of the antibodies to both densely sulfated glycosaminoglycans (dsGAGs) and nucleic acids (NAs). These anti-dsGAG/NA antibodies matured and expanded via intratumoral immunological driving force of innate immunity via NAs. These human cancer-derived antibodies exhibited acidic pH-selective affinity across both antigens and showed specific reactivity to diverse spectrums of human tumor cells. The antibody-drug conjugate exerted therapeutic effects against multiple cancers in vivo by targeting cell surface dsGAG antigens. This study reveals that intratumoral immunological reactions propagate tumor-oriented immunoglobulin clones and demonstrates a new therapeutic modality for the universal treatment of human malignancies.

Existing nucleic acid and antigen profiling methods for COVID-19 diagnosis fail to simultaneously meet the demands in sensitivity and detection speed, hampering them from being a comprehensive way for epidemic prevention and control. Thus, effective screening of COVID-19 requires a simple, fast, and sensitive method. Here, we report a rapid assay for ultrasensitive and highly specific profiling of COVID-19 associated antigen. The assay is based on a binding-induced DNA assembly on a nanoparticle scaffold that acts by fluorescence translation. By binding two aptamers to a target protein, the protein brings the DNA regions into close proximity, forming closed-loop conformation and resulting in the formation of the fluorescence translator. Using this assay, saliva nucleocapsid protein (N protein) has been profiled quantitatively by converting the N protein molecule information into a fluorescence signal. The fluorescence intensity is enhanced with increasing N protein concentration caused by the metal enhanced fluorescence using a simple, specific, and fast profiling assay within 3 min. On this basis, the assay enables a high recognition ratio and a limit of detection down to 150 fg mL

The rapid identification of bacterial pathogens in clinical samples like blood, urine, pus, and sputum is the need of the hour. Conventional bacterial identification methods like culturing and nucleic acid-based amplification have limitations like poor sensitivity, high cost, slow turnaround time, etc. Raman spectroscopy, a label-free and noninvasive technique, has overcome these drawbacks by providing rapid biochemical signatures from a single bacterium. Raman spectroscopy combined with chemometric methods has been used effectively to identify pathogens. However, a robust approach is needed to utilize Raman features for accurate classification while dealing with complex data sets such as spectra obtained from clinical isolates, showing high sample-to-sample heterogeneity. In this study, we have used Raman spectroscopy-based identification of pathogens from clinical isolates using a deep transfer learning approach at the single-cell level resolution. We have used the data-augmentation method to increase the volume of spectra needed for deep-learning analysis. Our ResNet model could specifically extract the spectral features of eight different pathogenic bacterial species with a 99.99% classification accuracy. The robustness of our model was validated on a set of blinded data sets, a mix of cultured and noncultured bacterial isolates of various origins and types. Our proposed ResNet model efficiently identified the pathogens from the blinded data set with high accuracy, providing a robust and rapid bacterial identification platform for clinical microbiology.

Estimation of residual risk (RR) is necessary to understand status of blood safety with current testing strategies and need to improve it further. These estimates from India are lacking till date. Present study was aimed to estimate RR and incident rate (IR) of transfusion transmitted infectious disease (TTIDs) in Indian setting.Blood donor demographic data, serological testing results and nucleic acid testing (NAT) screening results were collected retrospectively from January 2015 to June 2017. Mathematical modelling using NAT yield/window period model was done to estimate IR and RR of TTIDs. Data was compared between different groups based on age-group, gender, donation repeatability and type of donor.A total 106 119 blood donors donated 109 441 units with overall TTID reactivity of 2.22%. HBV was most prevalent TTID (1.26%) with RR of 16.1 per million followed by HCV and HIV with RR of 4.4 and 3.1 per million donations, respectively. NAT testing variably reduced RR ranging from 69.4% to 96.1% depending on TTID. Younger, repeat and voluntary donors had significantly lower prevalence of TTIDs compared to older, first time and replacement donors.Gaps in the blood safety could be bridged significantly by implementing NAT testing and using quality serological assays. Comparatively high RR despite using quality serological assays and ID-NAT testing highlights need to develop long term strategies to improve blood safety by focusing on improving donor pool by recruiting regular voluntary donors among youth and imparting knowledge of healthy practices.

The use of CRISPR/Cas9 systems in genome editing has been limited by the inability to efficiently deliver the key editing components to and across tissues and cell membranes, respectively. Spherical nucleic acids (SNAs) are nanostructures that provide privileged access to both but have yet to be explored as a means of facilitating gene editing. Herein, a new class of CRISPR SNAs are designed and evaluated in the context of genome editing. Specifically, Cas9 ProSNAs comprised of Cas9 cores densely modified with DNA on their exteriors and preloaded with single-guide RNA were synthesized and evaluated for their genome editing capabilities in the context of multiple cell lines. The radial orientation of the DNA on the Cas9 protein surface enhances cellular uptake, without the need for electroporation or transfection agents. In addition, the Cas9 proteins defining the cores of the ProSNAs were fused with GALA peptides on their N-termini and nuclear localization signals on their C-termini to facilitate endosomal escape and maximize nuclear localization and editing efficiency, respectively. These constructs were stable against protease digestion under conditions that fully degrade the Cas9 protein, when not transformed into an SNA, and used to achieve genome editing efficiency between 32 and 47%. Taken together, these novel constructs and advances point toward a way of significantly broadening the scope of use and impact of CRISPR-Cas9 genome editing systems.

Chemical and biological interfaces are of various types, which could be between two materials of the same and/or different states, two phases of the same material, biological substrates and the outer environment, surfactant or polymeric membranes and the bulk, and so forth. Small-molecule guests frequently interact with such interfaces that decide their functionalities. The structural and behavioral properties undergo considerable characteristic changes, which control their final course of action in the targeted application. This Perspective will discuss mainly the chemical interfaces constituted by the surfactants, polymers, lipids, and nucleic acids and their impacts on the dynamics of small-molecule guests. Some specific and interesting phenomena and future prospects will be elucidated in this Perspective.

The recent outbreak of one of the RNA viruses (2019-nCoV) has affected most of the population and the fatalities reported may label it as a modern-day scourge. Active research on RNA virus infections and vaccine development had more commercial impact which leads to an increase in patent filings. Patents are a goldmine of information whose mining yields crucial technological inputs for further research. In this research article, we have investigated both the patent applications and granted patents, to identify the technological trends and their impact on 2019-nCoV infection using biotechnology-related keywords such as genes, proteins, nucleic acid etc. related to the RNA virus infection. In our research, patent analysis was majorly focused on prospecting for patent data related to the RNA virus infections. Our patent analysis specifically identified spike protein (S protein) and nucleocapsid proteins (N proteins) as the most actively researched macromolecules for vaccine and/or drug development for diagnosis and treatment of RNA virus based infectious diseases. The outcomes of this patent intelligence study will be useful for the researchers who are actively working in the area of vaccine or drug development for RNA virus-based infections including 2019-nCoV and other emerging and re-emerging viral infections in the near future.

Chitosan (CS)-based polyplexes are produced by spontaneous electrostatic association with nucleic acids using CS in excess. Interactions of positively charged polyplexes, and the unbound CS, with negatively charged blood components limit the applicable dosage of such polymeric nanoparticles (NPs) and development of formulations with improved hemocompatibility and transfection efficiency is needed. Here, we introduce a strategy based on Tangential Flow Filtration (TFF) to remove unbound CS, concentrate polyplexes and subsequently coat with hyaluronic acid (HA) to improve hemocompatibility and bioactivity. Optimal TFF conditions were established. A library of HA with different molecular weights and degrees of sulfation was used at different carboxyl + sulfate to phosphate ratios for polyplex coating, bioactivity and hemocompatibility assessment. A systematic optimization of TFF conditions allowed for purification of polylpexes from excess unbound CS and subsequent coating with HA. Except for high molecular weight HA, for which macroscopic aggregation was observed, both sulfated and non-sulfated HAs resulted in small sized and homogenous coated complexes. However, sulfated HAs displayed higher stability during the second filtration process indicating their stronger binding affinity to polyplexes. Finally, we found that low molecular weight HA-coated polyplexes have equivalent silencing efficiency in vitro and improved hemocompatibility compared to uncoated polyplexes.

The unprecedented emergence of Japanese encephalitis (JE) in mainland Australia represents an outbreak of high clinical and public health significance. JE is a zoonosis spread by mosquitoes and is one of the most important causes of endemic viral encephalitis in South-East Asia and the Indian subcontinent. While occasional cases of human Japanese encephalitis virus (JEV) infection have occurred in far north Australia, its detection in pigs and the substantial number of locally acquired human cases across multiple jurisdictions in early 2022 prompted the declaration of this outbreak as a Communicable Disease Incident of National Significance. Laboratory testing for JEV is complex, and most cases are diagnosed by serology, for which interpretation is difficult. This review provides a comprehensive outline of currently available methods for JEV diagnosis including serology, nucleic acid amplification testing, virus isolation, sequencing and metagenomics. The relative advantages and disadvantages of the diagnostic tests are presented, as well as their value in clinical and public health contexts. This review also explores the role of mosquito, veterinary and human surveillance as part of the laboratory response to JEV. As JEV may become endemic in Australia, a collaborative and coordinated One Health approach involving animal, human and environmental health is required for optimal disease response and control.

Nucleic acid-based therapy emerges as a powerful weapon for the treatment of tumors thanks to its direct, effective, and lasting therapeutic effect. Encouragingly, continuous nucleic acid-based drugs have been approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Despite the tremendous progress, there are few nucleic acid-based drugs for brain tumors in clinic. The most challenging problems lie on the instability of nucleic acids, difficulty in traversing the biological barriers, and the off-target effect. Herein, nucleic acid-based therapy for brain tumor is summarized considering three aspects: (i) the therapeutic nucleic acids and their applications in clinical trials; (ii) the various administration routes for nucleic acid delivery and the respective advantages and drawbacks. (iii) the strategies and carriers for improving stability and targeting ability of nucleic acid drugs. This review provides thorough knowledge for the rational design of nucleic acid-based drugs against brain tumor.

Nucleic acid drugs hold great promise for potential treatment of a variety of diseases. But efficient delivery is still the major challenge impeding translation. Nanoformulations based on polymers and lipids require preparation processes such as microfluidic mixing, spray drying or final filling, where pumping is a crucial step. Here, we studied the effect of pumping on the component and overall loss of a binary polyplex formulation made of DNA and polyethyleneimine (PEI). We varied tubing length and material with a focus on subsequent spray drying. Interestingly, product loss increased with the length of silicon tubing. Losses of DNA were prevented by using Pumpsil. The following spray drying process did not affect DNA content but caused PEI loss. Characterization of the different tubing materials revealed similar hydrophobicity of all tubing materials and showed neutral Pumpsil® surface charge, negative Santoprene™ surface charge, and a positive Silicon surface charge. Hence, adsorption of DNA onto tubing material was concluded to be the root cause for DNA loss after pumping and is based upon an interplay of ionic and hydrophobic interactions between polyplexes and tubing material. Overall, selecting the appropriate tubing material for processing nucleic acid nanoparticles is key to achieving satisfactory product quality.

The introduction of the exact nuclear Overhauser enhancement (eNOE) methodology to solution-state nuclear magnetic resonance (NMR) spectroscopy results in tighter distance restraints from NOEs than in convention analysis. These improved restraints allow for higher resolution in structure calculation and even the disentanglement of different conformations of macromolecules. While initial work primarily focused on technical development of the eNOE, structural studies aimed at the elucidation of spatial sampling in proteins and nucleic acids were published in parallel prior to 2018. The period of 2018-2022 saw a continued series of technical innovation, but also major applications addressing biological questions. Here, we review both aspects, covering topics from the implementation of non-uniform sampling of NOESY buildups, novel pulse sequences, adaption of the eNOE to solid-state NMR, advances in eNOE data analysis, and innovations in structural ensemble calculation, to applications to protein, RNA, and DNA structure elucidation.

Extracellular vesicles (EVs) are membrane-enclosed particles released from almost all cell types. EVs mediate intercellular communication by delivering their surface and luminal cargoes, including nucleic acids, proteins, and lipids, which reflect the pathophysiological conditions of their cellular origins. Hepatocytes and hepatic non-parenchymal cells utilize EVs to regulate a wide spectrum of biological events inside the liver and transfer them to distant organs through systemic circulation. The liver also receives EVs from multiple organs and integrates these extrahepatic signals that participate in pathophysiological processes. EVs have recently attracted growing attention for their crucial roles in maintaining and regulating hepatic homeostasis. This review summarizes the roles of EVs in intrahepatic and interorgan communications under different pathophysiological conditions of the liver, with a focus on chronic liver diseases including nonalcoholic steatohepatitis, alcoholic hepatitis, viral hepatitis, liver fibrosis, and hepatocellular carcinoma. This review also discusses recent progress for potential therapeutic applications of EVs by targeting or enhancing EV-mediated cellular communication for the treatment of liver diseases.

Helicases are essential for nearly all nucleic acid processes across the tree of life, yet detailed understanding of how they couple ATP hydrolysis to translocation and unwinding remains incomplete because their small (∼300 picometer), fast (∼1 ms) steps are difficult to resolve. Here, we use Nanopore Tweezers to observe single Escherichia coli RecQ helicases as they translocate on and unwind DNA at ultrahigh spatiotemporal resolution. Nanopore Tweezers simultaneously resolve individual steps of RecQ along the DNA and conformational changes of the helicase associated with stepping. Our data reveal the mechanochemical coupling between physical domain motions and chemical reactions that together produce directed motion of the helicase along DNA. Nanopore Tweezers measurements are performed under either assisting or opposing force applied directly on RecQ, shedding light on how RecQ responds to such forces in vivo. Determining the rates of translocation and physical conformational changes under a wide range of assisting and opposing forces reveals the underlying dynamic energy landscape that drives RecQ motion. We show that RecQ has a highly asymmetric energy landscape that enables RecQ to maintain velocity when encountering molecular roadblocks such as bound proteins and DNA secondary structures. This energy landscape also provides a mechanistic basis making RecQ an ‘active helicase,’ capable of unwinding dsDNA as fast as it translocates on ssDNA. Such an energy landscape may be a general strategy for molecular motors to maintain consistent velocity despite opposing loads or roadblocks.

Switching of G-quadruplex (G4) structures between variant types of folding has been proved to be a versatile tool for regulation of genomic expression and development of nucleic acid-based constructs. Various specific ligands have been developed to target G4s in K+ solution with therapeutic prospects. Although G4 structures have been reported to be converted by sequence modification or a unimolecular ligand binding event in K+-deficient conditions, switching G4s towards non-G4 folding continues to be a great challenge due to the stability of G4 in physiological K+ conditions. Herein, we first observed the G4 switching towards parallel-stranded duplex (psDNA) by multimolecular ligand binding (namely ligand clustering) to overcome the switching barrier in K+. Purine-rich sequences (e.g. those from the KRAS promoter region) can be converted from G4 structures to dimeric psDNAs using molecular rotors (e.g. thioflavin T and thiazole orange) as initiators. The formed psDNAs provided multiple binding sites for molecular rotor clustering to favor subsequent structures with stability higher than the corresponding G4 folding. Our finding provides a clue to designing ligands with the competency of molecular rotor clustering to implement an efficient G4 switching.

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with backbone structures composed of non-ribose or non-deoxyribose sugars. Phosphonomethylthreosyl nucleic acid (pTNA), a type of XNA that does not base pair with DNA or RNA, has been suggested as a possible genetic material for storing synthetic biology information in cells. A critical step in this process is the synthesis of XNA episomes using laboratory-evolved polymerases to copy DNA information into XNA. Here, we investigate the polymerase recognition of pTNA nucleotides using X-ray crystallography to capture the post-catalytic complex of engineered polymerases following the sequential addition of two pTNA nucleotides onto the 3'-end of a DNA primer. High-resolution crystal structures reveal that the polymerase mediates Watson-Crick base pairing between the extended pTNA adducts and the DNA template. Comparative analysis studies demonstrate that the sugar conformation and backbone position of pTNA are structurally more similar to threose nucleic acid than DNA even though pTNA and DNA share the same six-atom backbone repeat length. Collectively, these findings provide new insight into the structural determinants that guide the enzymatic synthesis of an orthogonal genetic polymer, and may lead to the discovery of new variants that function with enhanced activity. ispartof: NUCLEIC ACIDS RESEARCH vol:50 issue:17 pages:9663-9674 ispartof: location:England status: published

The improper conformation of oligonucleotides on gold nanoparticle surfaces is caused by unintended base adsorption, which hinders DNA hybridization and lowers colloidal stability. In this work, we treated spherical nucleic acids with Br

The target of rapamycin (TOR) protein kinase is a master regulator of cell growth in all eukaryotes, from unicellular yeast and algae to multicellular animals and plants. Target of rapamycin balances the synthesis and degradation of proteins, lipids, carbohydrates and nucleic acids in response to nutrients, growth factors and cellular energy to promote cell growth. Among nutrients, amino acids (AAs) and glucose are central regulators of TOR activity in evolutionary distant eukaryotes such as mammals, plants and algae. However, these organisms obtain the nutrients through totally different metabolic processes. Although photosynthetic eukaryotes can use atmospheric COsub2/subas the sole carbon (C) source for all reactions in the cell, heterotrophic organisms get nutrients from other sources of organic C including glucose. Here, we discuss the impact of autotrophic and heterotrophic metabolism on the nutrient regulation of TOR, focusing on the role of AAs and C sources upstream of this signaling pathway.

The refractive index (RI) is a fundamental physical property of materials. Although measurement of the RI of biological nanoparticles (BNPs) in aqueous media is of great importance to basic research and biomedical applications, it is hampered by their tiny size, large intrinsic heterogeneity, and weak scattering. Here, we report the development of a label-free technique that can determine the RI of individual viruses and small extracellular vesicles (sEVs) with high precision and an analysis rate up to 10 000 particles per minute. This was achieved via the combination of high-sensitivity light-scattering detection by nanoflow cytometry (nFCM) and the Mie theory calculation. With the measured RIs for T7 virions, T7 capsids, and sEVs, the concentrations of nucleic acid in viral particles and protein in the lumen of sEVs were estimated. Furthermore, building upon a simplified core-shell model, the RIs of sEVs ranging from 40 to 200 nm were obtained. By using these RIs, a statistically robust size distribution of sEVs was acquired in minutes with accuracy and resolution matched closely with those of cryo-TEM measurements. Our approach could become an important tool in the RI determination of single BNPs.

Engineered protein switches have been widely applied in cell-based protein sensors and point-of-care diagnosis for the rapid and simple analysis of a wide variety of proteins, metabolites, nucleic acids, and enzymatic activities. Currently, these protein switches are based on two main types of switching mechanisms to transduce the target binding event to a quantitative signal, through a change in the optical properties of fluorescent molecules and the activation of enzymatic activities. In this paper, we introduce a new affinity-tunable protein switch strategy in which the binding of a small-molecule target with the protein activates the streptavidin-biotin interaction to generate a readout signal. In the absence of a target, the biotinylated protein switch forms a closed conformation where the biotin is positioned in close proximity to the protein, imposing a large steric hindrance to prevent the effective binding with streptavidin. In the presence of the target molecule, this steric hindrance is removed, thereby exposing the biotin for streptavidin binding to produce strong fluorescent signals. With this modular sensing concept, various sulfonamide, methotrexate, and trimethoprim drugs can be selectively detected on the cell surface of native and genetically engineered cells using different fluorescent dyes and detection techniques.

Massive diagnostic testing has been performed for appropriate screening and identification of COVID-19 cases in the ongoing global pandemic. However, the environmental impacts of COVID-19 diagnostics have been least considered. In this paper, the environmental impacts of the COVID-19 nucleic acid diagnostics were assessed by following a full cradle-to-grave life-cycle approach. The corresponding life-cycle anthology was established to provide quantitative analysis. Moreover, three alternative scenarios, i.e., material substitution, improved waste treatment, and electric vehicle (EV)-based transportation, were further proposed to discuss the potential environmental mitigation and conservation strategies. It was estimated that the life cycle of a single COVID-19 nucleic acid diagnostic test in China would lead to the emission of 612.9 g CO

Diagnosing hepatitis E infection usually involves specific IgM testing, but sensitivity/specificity concerns mean many guidelines and practices include confirmatory tests. We studied whether additional information confirmatory tests provide justifies their use.We examined 9131 records of anti-hepatitis E IgM assays, 7615 of IgG assays, and 1726 of RT-PCR assays from our regional laboratory, spanning October 2014-October 2018. We paired 495 IgM assay results with a RT-PCR result. We examined whether IgM results predicted PCR results, reviewed discrepant pairs, and investigated the correlation between IgG and PCR results in patients with strongly reactive IgM assays.Anti-hepatitis E IgM titres are bimodal. A high cut-off value optimises prediction of RNA detectability. 7/404 low-IgM samples had detectable RNA, 6 from immunosuppressed patients. 26/91 high-IgM samples did not have detectable RNA. In high-IgM samples, RNA detectability was not associated with IgG titre (one-tailed Mann-Whitney U test, p = 0.14).In immunocompetent patients, tests beyond IgM seldom add clinically useful information. In patients with immunocompromise, IgM and RNA could contribute information. Additional tests' extra costs/intervention delays cannot be justified. IgM assay cut-offs should reflect titres' bimodal distribution, with values standardised using international units.

We sought to determine if corticosteroid administration is associated with a SARS-CoV-2 nucleic acid test-positive result and to describe therapies administered to SARS-CoV-2 infected children.We collected cross-sectional data from participants recruited in 41 pediatric emergency departments (ED) in 10 countries between March 2020 and June 2021. Participants were18 years old, had signs or symptoms of, or risk factors for acute SARS-CoV-2 infection, and had nucleic acid testing performed. To determine if SARS-CoV-2 test status was independently associated with corticosteroid administration, we used a multivariable conditional logistic regression model matched by study site to compare treatments administered based on SARS-CoV-2 test and disposition status. This analysis was repeated for the subgroup of study participants who were hospitalized.30.3% (3,121/10,315) of participants were SARS-CoV-2-positive. Although remdesivir was more commonly administered to SARS-CoV-2-positive children, use was infrequent (25/3120 [0.8%] vs 1/7188 [0.01%]; P = .001). Corticosteroid use was less common among SARS-CoV-2-positive children (219/3120 [7.0%] vs 759/7190 [10.6%]; P.001). Among hospitalized children, there were no differences in provision of inotropes, respiratory support, chest drainage or extracorporeal membrane oxygenation between groups. Corticosteroid administration was associated with age, history of asthma, wheezing, study month, hospitalization and intensive care unit admission; it was not associated with a positive SARS-CoV-2 test result overall (aOR: 0.91; 95%CI: 0.74, 1.12) or among the subgroup of those hospitalized (aOR: 1.04; 95%CI: 0.75, 1.44).Few disease-specific treatments are provided to SARS-CoV-2-positive children; clinical trials evaluating therapies in children are urgently needed.

Porcine respiratory disease complex, which is caused by a combination of pathogens, including swine influenza A virus (SIV) and porcine reproductive respiratory syndrome virus (PRRSV), results in significant economic losses in pig production systems. Nasopharynx-associated lymphoid tissue (NALT) plays an important role in the uptake of pathogens and defence of the nasal mucosa in rodents and humans. We characterized NALT M cells in pigs and detected SIV antigen and PRRSV nucleic acid in NALT using histopathological, immunohistochemical and in-situ hybridization analyses. All SIV- and PRRSV-positive cases examined had suppurative nasopharyngitis and pneumonia. M cells were detected by immunohistochemistry and the distribution of M cells showed an increase in the middle section of NALT. SIV antigen was detected in M cells and PRRSV nucleic acid was demonstrated in the cytoplasm of macrophages in NALT. We believe that SIV and PRRSV infection in the upper respiratory tract induces local immunosuppression and these results confirm that swine NALT is a location for virus replication and may be strongly associated with the development of pneumonia in pigs.