The Idiopathic Pulmonary Fibrosis-Associated Single Nucleotide Polymorphism RS35705950 Is Transcribed in a MUC5B Promoter Associated Long Non-Coding RNA (AC061979.1).

LncRNAs are involved in regulatory processes in the human genome, including gene expression. The rs35705950 SNP, previously associated with IPF, overlaps with the recently annotated lncRNA AC061979.1, a 1712 nucleotide transcript located within the MUC5B promoter at chromosome 11p15.5. To document the expression pattern of the transcript, we processed 3.9 TBases of publicly available RNA-SEQ data across 27 independent studies involving lung airway epithelial cells. Epithelial lung cells showed expression of this putative pancRNA. The findings were independently validated in cell lines and primary cells. The rs35705950 is found within a conserved region (from fish to primates) within the expressed sequence indicating functional importance. These results implicate the rs35705950-containing AC061979.1 pancRNA as a novel component of the MUC5B expression control minicircuitry.

Bacterial Adaptation to Venom in Snakes and Arachnida.

Animal venoms are considered sterile sources of antimicrobial compounds with strong membrane-disrupting activity against multidrug-resistant bacteria. However, venomous bite wound infections are common in developing nations. Investigating the envenomation organ and venom microbiota of five snake and two spider species, we observed venom community structures that depend on the host venomous animal species and evidenced recovery of viable microorganisms from black-necked spitting cobra (Naja nigricollis) and Indian ornamental tarantula (Poecilotheria regalis) venoms. Among the bacterial isolates recovered from N. nigricollis, we identified two venom-resistant, novel sequence types of Enterococcus faecalis whose genomes feature 16 virulence genes, indicating infectious potential, and 45 additional genes, nearly half of which improve bacterial membrane integrity. Our findings challenge the dogma of venom sterility and indicate an increased primary infection risk in the clinical management of venomous animal bite wounds. IMPORTANCE Notwithstanding their 3 to 5% mortality, the 2.7 million envenomation-related injuries occurring annually-predominantly across Africa, Asia, and Latin America-are also major causes of morbidity. Venom toxin-damaged tissue will develop infections in some 75% of envenomation victims, with E. faecalis being a common culprit of disease; however, such infections are generally considered to be independent of envenomation. Here, we provide evidence on venom microbiota across snakes and arachnida and report on the convergent evolution mechanisms that can facilitate adaptation to black-necked cobra venom in two independent E. faecalis strains, easily misidentified by biochemical diagnostics. Therefore, since inoculation with viable and virulence gene-harboring bacteria can occur during envenomation, acute infection risk management following envenomation is warranted, particularly for immunocompromised and malnourished victims in resource-limited settings. These results shed light on how bacteria evolve for survival in one of the most extreme environments on Earth and how venomous bites must be also treated for infections.

Comprehensive analysis of drugs to treat SARS­CoV­2 infection: Mechanistic insights into current COVID­19 therapies (Review).

The major impact produced by the severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) focused many researchers attention to find treatments that can suppress transmission or ameliorate the disease. Despite the very fast and large flow of scientific data on possible treatment solutions, none have yet demonstrated unequivocal clinical utility against coronavirus disease 2019 (COVID­19). This work represents an exhaustive and critical review of all available data on potential treatments for COVID­19, highlighting their mechanistic characteristics and the strategy development rationale. Drug repurposing, also known as drug repositioning, and target based methods are the most used strategies to advance therapeutic solutions into clinical practice. Current in silico, in vitro and in vivo evidence regarding proposed treatments are summarized providing strong support for future research efforts.

A new threat from an old enemy: Re­emergence of coronavirus (Review).

The new outbreak of coronavirus from December 2019 has brought attention to an old viral enemy and has raised concerns as to the ability of current protection measures and the healthcare system to handle such a threat. It has been known since the 1960s that coronaviruses can cause respiratory infections in humans; however, their epidemic potential was understood only during the past two decades. In the present review, we address current knowledge on coronaviruses from a short history to epidemiology, pathogenesis, clinical manifestation of the disease, as well as treatment and prevention strategies. Although a great amount of research and efforts have been made worldwide to prevent further outbreaks of coronavirus­associated disease, the spread and lethality of the 2019 outbreak (COVID­19) is proving to be higher than previous epidemics on account of international travel density and immune naivety of the population. Only strong, joint and coordinated efforts of worldwide healthcare systems, researchers, and pharmaceutical companies and receptive national leaders will succeed in suppressing an outbreak of this scale.

Measuring the Action of Oligonucleotide Therapeutics in the Lung at the Cell Type-Specific Level by Tissue Disruption and Cell Sorting (TDCS).

The clinical potential of DNA and RNA-targeting therapeutics for airways disease has been hampered by the poor translation of promising drug candidates from cell culture to in vivo models and the clinic. For example, classical preclinical approaches routinely report 20-60% target knockdown effects in the lung, where 1 or 2 log effects are observed in isolated cell cultures in vitro. Preparation of monocellular suspensions of tissues by mechanoenzymatic disruption followed by cell sorting (TDCS) after in vivo drug dosing, however, can offer pharmacokinetic and pharmacodynamic insights on the effects of drugs to precise cell subpopulations. Moreover, this can be reliably achieved with up to 66% fewer animals than standard in vivo pharmacology approaches due to lower data variance afforded through analytics on defined, viable cell numbers. Here we describe the TDCS methodology for the isolation of total lung epithelia, lung macrophages, and epithelium/macrophage-depleted cell fractions from mouse lungs using a two-stage sorting process of immunomagnetic bead separation followed by flow cytometric sorting using fluorescent antibodies against well-established surface markers such as F4/80, CD11b, and CD326. Validated antibodies for additional cell types and markers are also provided.

RACE-SEQ and Population-Wide Polymorphism Susceptibility Testing for Endonucleolytically Active, RNA-Targeting Therapeutics.

High-throughput sequencing of the products of 5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) reactions (RACE-SEQ) enables the mapping and digital enumeration of expected and novel 5' ends in RNA molecules. The resulting data are essential in documenting the mechanism of action and precision of endonucleolytically active, RNA-targeting drugs such as RNase H-active antisense or small interfering RNA. When applied to error-prone replication systems such as RNA viruses or in vitro RNA replicon systems, the method can additionally report the relative susceptibility of known and unknown polymorphisms to a prospective sequence-specific drug, making it a powerful tool in patient selection and stratification as well as resistance prediction.We describe the preparation of sequencing libraries for ultra-high depth 5' RLM-RACE analysis on two popular second-generation high-throughput sequencing platforms (Illumina, Ion Torrent) and supply a detailed bioinformatics analysis pipeline for target site activity definition and enumeration. We further illustrate how the pipeline can be simply modified to generate polymorphism-specific drug susceptibility data from in vitro replicon experiments (RACE-SEQ-MM), in a patient-free manner, to cover both known and unknown target site variants in the population.

The emerging field of venom-microbiomics for exploring venom as a microenvironment, and the corresponding Initiative for Venom Associated Microbes and Parasites (iVAMP).

Venom is a known source of novel antimicrobial natural products. The substantial, increasing number of these discoveries have unintentionally culminated in the misconception that venom and venom-producing glands are largely sterile environments. Culture-dependent and -independent studies on the microbial communities in venom microenvironments reveal the presence of archaea, algae, bacteria, fungi, protozoa, and viruses. Venom-centric microbiome studies are relatively sparse to date with the adaptive advantages that venom-associated microbes might offer to their hosts, or that hosts might provide to venom-associated microbes, remaining largely unknown. We highlight the potential for the discovery of venom microbiomes within the adaptive landscape of venom systems. The considerable number of convergently evolved venomous animals, juxtaposed with the comparatively few known studies to identify microbial communities in venom, provides new possibilities for both biodiversity and therapeutic discoveries. We present an evidence-based argument for integrating microbiology as part of venomics (i.e., venom-microbiomics) and introduce iVAMP, the Initiative for Venom Associated Microbes and Parasites (https://ivamp-consortium.github.io/), as a growing collaborative consortium. We express commitment to the diversity, inclusion and scientific collaboration among researchers interested in this emerging subdiscipline through expansion of the iVAMP consortium.

A novel antifungal property for the Bacillus licheniformis ComX pheromone and its possible role in inter-kingdom cross-talk.

Quorum sensing molecules (QSMs) regulate, through a chemical communication process, multiple complex systems in bacterial and some fungal populations on the basis of cell density. The bacterial QSMs involved in inter-kingdom cross-talk may exhibit antagonistic activity against fungi. This provides an important opportunity for biocontrol of fungal invasion in plants. It has been shown that cultures of Bacillus spp. inhibit fungal growth. Here, we explore the inhibitory potential of the industrial workhorse Bacillus licheniformis NCIMB-8874 and its QSM (ComX pheromone) on the growth of Aspergillus flavus, a cereal, legume, and nut crop pathogen. Our studies show that ComX filtered extracts from cultures of B. licheniformis can cause a significant reduction in the growth of A. flavus NRRL 3357 and ESP 15 at a concentration as low as 13 µg ml-1. This work evidences, for the first time, the inter-kingdom utility of the bacterial quorum sensing ComX pheromone indicating potential antifungal food security against A. flavus.

Profiling the Mismatch Tolerance of Argonaute 2 through Deep Sequencing of Sliced Polymorphic Viral RNAs.

Low allelic and clonal variability among endogenous RNAi targets has focused mismatch tolerance studies to RNAi-active guide strands. However, the inherent genomic instability of RNA viruses such as hepatitis C virus (HCV) gives rise to quasi-species mutants within discrete clones: this facilitates mismatch tolerance studies from a target perspective. We recently quantified the slicing imprecision of Argonaute 2 using small interfering RNA (siRNA) analogs of the DNA-directed RNAi drug TT-034 and next-generation sequencing of 5' RNA ligase-mediated rapid amplification of cDNA ends (RACE-SEQ). Here, we present an open-source, customizable, and computationally light RACE-SEQ bioinformatic pipeline, describing adaptations that semiquantitatively report the impact of RNAi hybridization site mismatches from the target perspective. The analysis shows that Argonaute 2 has a substitution-specific, 3- to 5-log activity window between fully complementary targets and targets with mismatches across positions 10-11. It further focuses the endonucleotic Slicer imprecision around positions 13-17, demonstrating its dependence on guide strand central region complementarity, and potentiation by even a single mismatch. We further propose pharmacogenomics value in testing endogenous targets using recombinant replicon systems and RACE-SEQ to report the pharmacodynamics of sequence-specific oligonucleotide therapeutics against all possible polymorphisms in a population, in a minimally biased, patient-free manner.

Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood.

The West African Ebola virus outbreak underlined the importance of delivering mass diagnostic capability outside the clinical or primary care setting in effectively containing public health emergencies caused by infectious disease. Yet, to date, there is no solution for reliably deploying at the point of need the gold standard diagnostic method, real time quantitative reverse transcription polymerase chain reaction (RT-qPCR), in a laboratory infrastructure-free manner. In this proof of principle work, we demonstrate direct performance of RT-qPCR on fresh blood using far-red fluorophores to resolve fluorogenic signal inhibition and controlled, rapid freeze/thawing to achieve viral genome extraction in a single reaction chamber assay. The resulting process is entirely free of manual or automated sample pre-processing, requires no microfluidics or magnetic/mechanical sample handling and thus utilizes low cost consumables. This enables a fast, laboratory infrastructure-free, minimal risk and simple standard operating procedure suited to frontline, field use. Developing this novel approach on recombinant bacteriophage and recombinant human immunodeficiency virus (HIV; Lentivirus), we demonstrate clinical utility in symptomatic EBOV patient screening using live, infectious Filoviruses and surrogate patient samples. Moreover, we evidence assay co-linearity independent of viral particle structure that may enable viral load quantification through pre-calibration, with no loss of specificity across an 8 log-linear maximum dynamic range. The resulting quantitative rapid identification (QuRapID) molecular diagnostic platform, openly accessible for assay development, meets the requirements of resource-limited countries and provides a fast response solution for mass public health screening against emerging biosecurity threats.

Genomic and molecular characterization of a novel quorum sensing molecule in Bacillus licheniformis.

Quorum sensing molecules (QSMs) are involved in the regulation of complicated processes helping bacterial populations respond to changes in their cell-density. Although the QS gene cluster (comQXPA) has been identified in the genome sequence of some bacilli, the QS system B. licheniformis has not been investigated in detail, and its QSM (ComX pheromone) has not been identified. Given the importance of this antagonistic bacterium as an industrial workhorse, this study was aimed to elucidate B. licheniformis NCIMB-8874 QS. The results obtained from bioinformatics studies on the whole genome sequence of this strain confirmed the presence of essential quorum sensing-related genes. Although polymorphism was verified in three proteins of this cluster, ComQ, precursor-ComX and ComP, the transcription factor ComA was confirmed as the most conserved protein. The cell-cell communication of B. licheniformis NCIMB-8874 was investigated through further elucidation of the ComX pheromone as 13-amino acid peptide. The peptide sequence of the pheromone has been described through biochemical characterisation.

Clinical potential of oligonucleotide-based therapeutics in the respiratory system.

The discovery of an ever-expanding plethora of coding and non-coding RNAs with nodal and causal roles in the regulation of lung physiology and disease is reinvigorating interest in the clinical utility of the oligonucleotide therapeutic class. This is strongly supported through recent advances in nucleic acids chemistry, synthetic oligonucleotide delivery and viral gene therapy that have succeeded in bringing to market at least three nucleic acid-based drugs. As a consequence, multiple new candidates such as RNA interference modulators, antisense, and splice switching compounds are now progressing through clinical evaluation. Here, manipulation of RNA for the treatment of lung disease is explored, with emphasis on robust pharmacological evidence aligned to the five pillars of drug development: exposure to the appropriate tissue, binding to the desired molecular target, evidence of the expected mode of action, activity in the relevant patient population and commercially viable value proposition.

Ebola Check: Delivering molecular diagnostics at the point of need.

The 2013-5 global outbreak of Ebolavirus disease brought to sharp focus the need for diagnostic capacity to be equitably available on a global scale: from the most under-developed areas of resource-limited countries in West Africa to high volume international travel hubs in Europe and the USA. Quick detection of the causal agent of disease is pivotal to containment, contact tracing and clinical action to protect healthcare workers, communities and patients. Nucleic acid testing (NAT) by real time reverse transcription quantitative polymerase chain reaction (RT-PCR) has emerged as the preferred method for reliable patient status confirmation. Presently, this is served through advanced clinical molecular laboratory testing in a <8hr manual process that requires 3.5ml venous blood samples. To meet the demand in West Africa, this has necessitated large-scale mobile laboratory and volunteer biomedical scientist deployment: a solution that has proven eventually adequate, albeit temporary against future re-emergence of this and other haemorrhagic fever disease agents prevalent in the region. The EbolaCheck consortium was formed in August 2014 to address the need for delivering NAT at the point of care. We have developed a novel platform technology that can QUantitatively, RAPidly IDentify (QuRapID) known RNA or DNA targets in viruses, bacteria, or eukaryotic cells directly in crude biofluids, including whole blood, in under 40min using a 5 microliter sample. The portable, battery-operated system lacks microfluidics, pumps or other sensitive/high cost parts making it suitable for the environmental and economic challenges of resource-limited countries. The simple, safe, 5-step sample-to-answer process requires minimal training and informs frontline healthcare workers of diagnostic status, whilst reporting remotely epidemiologically relevant results. Data on biosafety level 2 surrogate Ebolavirus templates presented in encapsulated or enveloped viruses indicate performance comparable to clinical laboratory testing and utility beyond filoviruses. Emerging performance data on live Ebolavirus, non-human primate disease model and patient samples, as well as future development plans will be discussed.

Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

Safe, long-term hepatic expression of anti-HCV shRNA in a nonhuman primate model.

The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a "single-shot " therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.

Transcriptome analysis shows activation of circulating CD8+ T cells in patients with severe asthma.

BACKGROUND: Although previous studies have implicated tissue CD4(+) T cells in the development and maintenance of the inflammatory response in asthmatic patients, little is known about the role of CD8(+) T cells. There is now accumulating evidence that microRNAs and other noncoding RNAs are important regulators of T-cell function. OBJECTIVES: We sought to use transcriptomics to determine the activation state of circulating CD4(+) and CD8(+) T cells in patients with nonsevere and severe asthma. METHODS: mRNA and noncoding RNA expression in circulating T cells was measured by means of microarray, quantitative real-time PCR, or both. RESULTS: Comparison of mRNA expression showed widespread changes in the circulating CD8(+) but not CD4(+) T cells from patients with severe asthma. No changes were observed in the CD4(+) and CD8(+) T cells in patients with nonsevere asthma versus those in healthy control subjects. Bioinformatics analysis showed that the changes in CD8(+) T-cell mRNA expression were associated with multiple pathways involved in T-cell activation. As with mRNAs, we also observed widespread changes in expression of noncoding RNA species, including natural antisense, pseudogenes, intronic long noncoding RNAs (lncRNAs), and intergenic lncRNAs in CD8(+) T cells from patients with severe asthma. Measurement of the microRNA expression profile showed selective downregulation of miR-28-5p in CD8(+) T cells and reduction of miR-146a and miR-146b in both CD4(+) and CD8(+) T cells. CONCLUSIONS: Severe asthma is associated with the activation of circulating CD8(+) T cells but not CD4(+) T cells. This response is correlated with the downregulation of miR-146a/b and miR-28-5p, as well as changes in the expression of multiple species of lncRNA that might regulate CD8(+) T-cell function.

Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

Measuring the action of CPP-siRNA conjugates in the lung.

Two of the most promising and complex areas in biologics development, either as research tools or potential therapeutics, are cell-penetrating peptides (CPPs) and RNA interference (RNAi) modulators. Consequently, the combined application of these technologies in pursuit of improved delivery profiles for RNAi cargoes presents its own unique challenges. Direct access to the targeted tissue is luxury not always available to the researcher; however, the example of lung presents an excellent opportunity for presenting methodologies relevant to understanding the local impact of CPP-conjugated RNAi modulators. This chapter therefore expands upon updated protocols established on the study of the function of endogenous RNAi and the utility of CPPs in the delivery of short interfering RNA (siRNA) to therapeutically relevant cells in the lung. Methods for sample collection, preservation, and processing are provided with a view to facilitate qualitative and quantitative analysis of delivery. In addition, a protocol for mapping siRNA delivery by in situ hybridisation is provided.

Targeting the lung using siRNA and antisense based oligonucleotides.

The accessibility to topical administration through inhalation, combined with its large surface area, has led to speculation that the lung might offer an ideal target for the application of oligonucleotide based therapeutics. In this review, we shall critically examine the challenges facing antisense and siRNA based approaches for target validation in vivo and as potential therapeutics. In particular, we shall discuss the antisense and siRNA based approaches in relation to factors such as delivery, distribution, stability, off-target effects, unwanted immune responses and the selection of the optimum mRNA targets.