A dry powder formulation for peripheral lung delivery and absorption of an anti-SARS-CoV-2 ACE2 decoy polypeptide.

One of the strategies proposed for the neutralization of SARS-CoV-2 has been to synthetize small proteins able to act as a decoy towards the virus spike protein, preventing it from entering the host cells. In this work, the incorporation of one of these proteins, LCB1, within a spray-dried formulation for inhalation was investigated. A design of experiments approach was applied to investigate the optimal condition for the manufacturing of an inhalable powder. The lead formulation, containing 6% w/w of LCB1 as well as trehalose and L-leucine as excipients, preserved the physical stability of the protein and its ability to neutralize the virus. In addition, the powder had a fine particle fraction of 58.6% and a very high extra-fine particle fraction (31.3%) which could allow a peripheral deposition in the lung. The in vivo administration of the LCB1 inhalation powder showed no significant difference in the pharmacokinetic from the liquid formulation, indicating the rapid dissolution of the microparticles and the protein capability to translocate into the plasma. Moreover, LCB1 in plasma samples still maintained the ability to neutralize the virus. In conclusion, the optimized spray drying conditions allowed to obtain an inhalation powder able to preserve the protein biological activity, rendering it suitable for a systemic prevention of the viral infection via pulmonary administration.

Time-course transcriptome analysis of a double challenge bleomycin-induced lung fibrosis rat model uncovers ECM homoeostasis-related translationally relevant genes.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an irreversible disorder with a poor prognosis. The incomplete understanding of IPF pathogenesis and the lack of accurate animal models is limiting the development of effective treatments. Thus, the selection of clinically relevant animal models endowed with similarities with the human disease in terms of lung anatomy, cell biology, pathways involved and genetics is essential. The bleomycin (BLM) intratracheal murine model is the most commonly used preclinical assay to evaluate new potential therapies for IPF. Here, we present the findings derived from an integrated histomorphometric and transcriptomic analysis to investigate the development of lung fibrosis in a time-course study in a BLM rat model and to evaluate its translational value in relation to IPF. METHODS: Rats were intratracheally injected with a double dose of BLM (days 0-4) and sacrificed at days 7, 14, 21, 28 and 56. Histomorphometric analysis of lung fibrosis was performed on left lung sections. Transcriptome profiling by RNAseq was performed on the right lung lobes and results were compared with nine independent human gene-expression IPF studies. RESULTS: The histomorphometric and transcriptomic analyses provided a detailed overview in terms of temporal gene-expression regulation during the establishment and repair of the fibrotic lesions. Moreover, the transcriptomic analysis identified three clusters of differentially coregulated genes whose expression was modulated in a time-dependent manner in response to BLM. One of these clusters, centred on extracellular matrix (ECM)-related process, was significantly correlated with histological parameters and gene sets derived from human IPF studies. CONCLUSIONS: The model of lung fibrosis presented in this study lends itself as a valuable tool for preclinical efficacy evaluation of new potential drug candidates. The main finding was the identification of a group of persistently dysregulated genes, mostly related to ECM homoeostasis, which are shared with human IPF.

Time-resolved transcriptomic profiling of the developing rabbit's lungs: impact of premature birth and implications for modelling bronchopulmonary dysplasia.

BACKGROUND: Premature birth, perinatal inflammation, and life-saving therapies such as postnatal oxygen and mechanical ventilation are strongly associated with the development of bronchopulmonary dysplasia (BPD); these risk factors, alone or combined, cause lung inflammation and alter programmed molecular patterns of normal lung development. The current knowledge on the molecular regulation of lung development mainly derives from mechanistic studies conducted in newborn rodents exposed to postnatal hyperoxia, which have been proven useful but have some limitations. METHODS: Here, we used the rabbit model of BPD as a cost-effective alternative model that mirrors human lung development and, in addition, enables investigating the impact of premature birth per se on the pathophysiology of BPD without further perinatal insults (e.g., hyperoxia, LPS-induced inflammation). First, we characterized the rabbit's normal lung development along the distinct stages (i.e., pseudoglandular, canalicular, saccular, and alveolar phases) using histological, transcriptomic and proteomic analyses. Then, the impact of premature birth was investigated, comparing the sequential transcriptomic profiles of preterm rabbits obtained at different time intervals during their first week of postnatal life with those from age-matched term pups. RESULTS: Histological findings showed stage-specific morphological features of the developing rabbit's lung and validated the selected time intervals for the transcriptomic profiling. Cell cycle and embryo development, oxidative phosphorylation, and WNT signaling, among others, showed high gene expression in the pseudoglandular phase. Autophagy, epithelial morphogenesis, response to transforming growth factor ß, angiogenesis, epithelium/endothelial cells development, and epithelium/endothelial cells migration pathways appeared upregulated from the 28th day of gestation (early saccular phase), which represents the starting point of the premature rabbit model. Premature birth caused a significant dysregulation of the inflammatory response. TNF-responsive, NF-κB regulated genes were significantly upregulated at premature delivery and triggered downstream inflammatory pathways such as leukocyte activation and cytokine signaling, which persisted upregulated during the first week of life. Preterm birth also dysregulated relevant pathways for normal lung development, such as blood vessel morphogenesis and epithelial-mesenchymal transition. CONCLUSION: These findings establish the 28-day gestation premature rabbit as a suitable model for mechanistic and pharmacological studies in the context of BPD.

Deregulated intracellular pathways define novel molecular targets for HBV-specific CD8 T cell reconstitution in chronic hepatitis B.

BACKGROUND & AIMS: In chronic HBV infection, elevated reactive oxygen species levels derived from dysfunctional mitochondria can cause increased protein oxidation and DNA damage in exhausted virus-specific CD8 T cells. The aim of this study was to understand how these defects are mechanistically interconnected to further elucidate T cell exhaustion pathogenesis and, doing so, to devise novel T cell-based therapies. METHODS: DNA damage and repair mechanisms, including parylation, CD38 expression, and telomere length were studied in HBV-specific CD8 T cells from chronic HBV patients. Correction of intracellular signalling alterations and improvement of antiviral T cell functions by the NAD precursor nicotinamide mononucleotide and by CD38 inhibition was assessed. RESULTS: Elevated DNA damage was associated with defective DNA repair processes, including NAD-dependent parylation, in HBV-specific CD8 cells of chronic HBV patients. NAD depletion was indicated by the overexpression of CD38, the major NAD consumer, and by the significant improvement of DNA repair mechanisms, and mitochondrial and proteostasis functions by NAD supplementation, which could also improve the HBV-specific antiviral CD8 T cell function. CONCLUSIONS: Our study delineates a model of CD8 T cell exhaustion whereby multiple interconnected intracellular defects, including telomere shortening, are causally related to NAD depletion suggesting similarities between T cell exhaustion and cell senescence. Correction of these deregulated intracellular functions by NAD supplementation can also restore antiviral CD8 T cell activity and thus represents a promising potential therapeutic strategy for chronic HBV infection. IMPACT AND IMPLICATIONS: Correction of HBV-specific CD8 T cell dysfunction is believed to represent a rational strategy to cure chronic HBV infection, which however requires a deep understanding of HBV immune pathogenesis to identify the most important targets for functional T cell reconstitution strategies. This study identifies a central role played by NAD depletion in the intracellular vicious circle that maintains CD8 T cell exhaustion, showing that its replenishment can correct impaired intracellular mechanisms and reconstitute efficient antiviral CD8 T cell function, with implications for the design of novel immune anti-HBV therapies. As these intracellular defects are likely shared with other chronic virus infections where CD8 exhaustion can affect virus clearance, these results can likely also be of pathogenetic relevance for other infection models.

TFIIIC as a Potential Epigenetic Modulator of Histone Acetylation in Human Stem Cells.

Regulation of histone acetylation dictates patterns of gene expression and hence cell identity. Due to their clinical relevance in cancer biology, understanding how human embryonic stem cells (hESCs) regulate their genomic patterns of histone acetylation is critical, but it remains largely to be investigated. Here, we provide evidence that acetylation of histone H3 lysine-18 (H3K18ac) and lysine-27 (H3K27ac) is only partially established by p300 in stem cells, while it represents the main histone acetyltransferase (HAT) for these marks in somatic cells. Our analysis reveals that whereas p300 marginally associated with H3K18ac and H3K27ac in hESCs, it largely overlapped with these histone marks upon differentiation. Interestingly, we show that H3K18ac is found at "stemness" genes enriched in RNA polymerase III transcription factor C (TFIIIC) in hESCs, whilst lacking p300. Moreover, TFIIIC was also found in the vicinity of genes involved in neuronal biology, although devoid of H3K18ac. Our data suggest a more complex pattern of HATs responsible for histone acetylations in hESCs than previously considered, suggesting a putative role for H3K18ac and TFIIIC in regulating "stemness" genes as well as genes associated with neuronal differentiation of hESCs. The results break ground for possible new paradigms for genome acetylation in hESCs that could lead to new avenues for therapeutic intervention in cancer and developmental diseases.

An Optimized Workflow for the Discovery of New Antimicrobial Compounds Targeting Bacterial RNA Polymerase Complex Formation.

Bacterial resistance represents a major health problem worldwide and there is an urgent need to develop first-in-class compounds directed against new therapeutic targets. We previously developed a drug-discovery platform to identify new antimicrobials able to disrupt the protein-protein interaction between the ß' subunit and the σ70 initiation factor of bacterial RNA polymerase, which is essential for transcription. As a follow-up to such work, we have improved the discovery strategy to make it less time-consuming and more cost-effective. This involves three sequential assays, easily scalable to a high-throughput format, and a subsequent in-depth characterization only limited to hits that passed the three tests. This optimized workflow, applied to the screening of 5360 small molecules from three synthetic and natural compound libraries, led to the identification of six compounds interfering with the ß'-σ70 interaction, and thus was capable of inhibiting promoter-specific RNA transcription and bacterial growth. Upon supplementation with a permeability adjuvant, the two most potent transcription-inhibiting compounds displayed a strong antibacterial activity against Escherichia coli with minimum inhibitory concentration (MIC) values among the lowest (0.87-1.56 µM) thus far reported for ß'-σ PPI inhibitors. The newly identified hit compounds share structural feature similarities with those of a pharmacophore model previously developed from known inhibitors.

An RNA Polymerase III General Transcription Factor Engages in Cell Type-Specific Chromatin Looping.

Transcription factors (TFs) bind DNA in a sequence-specific manner and are generally cell type-specific factors and/or developmental master regulators. In contrast, general TFs (GTFs) are part of very large protein complexes and serve for RNA polymerases' recruitment to promoter sequences, generally in a cell type-independent manner. Whereas, several TFs have been proven to serve as anchors for the 3D genome organization, the role of GTFs in genome architecture have not been carefully explored. Here, we used ChIP-seq and Hi-C data to depict the role of TFIIIC, one of the RNA polymerase III GTFs, in 3D genome organization. We find that TFIIIC genome occupancy mainly occurs at specific regions, which largely correspond to Alu elements; other characteristic classes of repetitive elements (REs) such as MIR, FLAM-C and ALR/alpha are also found depending on the cell's developmental origin. The analysis also shows that TFIIIC-enriched regions are involved in cell type-specific DNA looping, which does not depend on colocalization with the master architectural protein CTCF. This work extends previous knowledge on the role of TFIIIC as a bona fide genome organizer whose action participates in cell type-dependent 3D genome looping via binding to REs.

Targeting p53 and histone methyltransferases restores exhausted CD8+ T cells in HCV infection.

Hepatitis C virus infection (HCV) represents a unique model to characterize, from early to late stages of infection, the T cell differentiation process leading to exhaustion of human CD8+ T cells. Here we show that in early HCV infection, exhaustion-committed virus-specific CD8+ T cells display a marked upregulation of transcription associated with impaired glycolytic and mitochondrial functions, that are linked to enhanced ataxia-telangiectasia mutated (ATM) and p53 signaling. After evolution to chronic infection, exhaustion of HCV-specific T cell responses is instead characterized by a broad gene downregulation associated with a wide metabolic and anti-viral function impairment, which can be rescued by histone methyltransferase inhibitors. These results have implications not only for treatment of HCV-positive patients not responding to last-generation antivirals, but also for other chronic pathologies associated with T cell dysfunction, including cancer.

Expression of DinJ-YafQ System of Lactobacillus casei Group Strains in Response to Food Processing Stresses.

Toxin-antitoxin (TA) systems are widely distributed in bacterial genomes and are involved in the adaptive response of microorganisms to stress conditions. Few studies have addressed TA systems in Lactobacillus and their role in the adaptation to food environments and processes. In this work, for six strains belonging to L. casei group isolated from dairy products, the expression of DinJ-YafQ TA system was investigated after exposure to various food-related stresses (nutrient starvation, low pH, high salt concentration, oxidative stress, and high temperature), as well as to the presence of antibiotics. In particular, culturability and DinJ-YafQ expression were evaluated for all strains and conditions by plate counts and RT qPCR. Among all the food-related stress conditions, only thermal stress was capable to significantly affect culturability. Furthermore, exposure to ampicillin significantly decreased the culturability of two L. rhamnosus strains. The regulation of DinJ-YafQ TA system resulted strain-specific; however, high temperature was the most significant stress condition able to modulate DinJ-YafQ expression. The increasing knowledge about TA systems activity and regulation might offer new perspectives to understand the mechanisms that L. casei group strains exploit to adapt to different niches or production processes.

Alu RNA Modulates the Expression of Cell Cycle Genes in Human Fibroblasts.

Alu retroelements, whose retrotransposition requires prior transcription by RNA polymerase III to generate Alu RNAs, represent the most numerous non-coding RNA (ncRNA) gene family in the human genome. Alu transcription is generally kept to extremely low levels by tight epigenetic silencing, but it has been reported to increase under different types of cell perturbation, such as viral infection and cancer. Alu RNAs, being able to act as gene expression modulators, may be directly involved in the mechanisms determining cellular behavior in such perturbed states. To directly address the regulatory potential of Alu RNAs, we generated IMR90 fibroblasts and HeLa cell lines stably overexpressing two slightly different Alu RNAs, and analyzed genome-wide the expression changes of protein-coding genes through RNA-sequencing. Among the genes that were upregulated or downregulated in response to Alu overexpression in IMR90, but not in HeLa cells, we found a highly significant enrichment of pathways involved in cell cycle progression and mitotic entry. Accordingly, Alu overexpression was found to promote transition from G1 to S phase, as revealed by flow cytometry. Therefore, increased Alu RNA may contribute to sustained cell proliferation, which is an important factor of cancer development and progression.

New Antimicrobials Targeting Bacterial RNA Polymerase Holoenzyme Assembly Identified with an in Vivo BRET-Based Discovery Platform.

Bacterial resistance represents a major health threat worldwide, and the development of new therapeutics, including innovative antibiotics, is urgently needed. We describe a discovery platform, centered on in silico screening and in vivo bioluminescence resonance energy transfer in yeast cells, for the identification of new antimicrobials that, by targeting the protein-protein interaction between the ß'-subunit and the initiation factor σ70 of bacterial RNA polymerase, inhibit holoenzyme assembly and promoter-specific transcription. Out of 34 000 candidate compounds, we identified seven hits capable of interfering with this interaction. Two derivatives of one of these hits proved to be effective in inhibiting transcription in vitro and growth of the Gram-positive pathogens Staphylococcus aureus and Listeria monocytogenes. Upon supplementation of a permeability adjuvant, one derivative also effectively inhibited Escherichia coli growth. On the basis of the chemical structures of these inhibitors, we generated a ligand-based pharmacophore model that will guide the rational discovery of increasingly effective antibacterial agents.

Identification and first characterization of DinJ-YafQ toxin-antitoxin systems in Lactobacillus species of biotechnological interest.

DinJ-YafQ is a type II TA system comprising the ribosome-dependent RNase YafQ toxin and the DinJ antitoxin protein. Although the module has been extensively characterized in Escherichia coli, little information is available for homologous systems in lactic acid bacteria. In this study, we employed bioinformatics tools to identify DinJ-YafQ systems in Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus species, commonly used in biotechnological processes. Among a total of nineteen systems found, two TA modules from Lactobacillus paracasei and two modules from Lactobacillus rhamnosus wild strains were isolated and their activity was verified by growth assays in Escherichia coli either in liquid and solid media. The RNase activity of the YafQ toxins was verified in vivo by probing mRNA dynamics and metabolism with single-cell Thioflavin T fluorescence. Our findings demonstrate that, albeit DinJ-YafQ TA systems are widely distributed in lactic acid bacteria, only few are fully functional, while others have lost toxicity even though they maintain high sequence identity with wild type YafQ and a likely functional antitoxin protein.

Correction: Identification of an early transcriptomic signature of insulin resistance and related diseases in lymphomonocytes of healthy subjects.

[This corrects the article DOI: 10.1371/journal.pone.0182559.].

Interpreting and integrating big data in non-coding RNA research.

In the last two decades, we have witnessed an impressive crescendo of non-coding RNA studies, due to both the development of high-throughput RNA-sequencing strategies and an ever-increasing awareness of the involvement of newly discovered ncRNA classes in complex regulatory networks. Together with excitement for the possibility to explore previously unknown layers of gene regulation, these advancements led to the realization of the need for shared criteria of data collection and analysis and for novel integrative perspectives and tools aimed at making biological sense of very large bodies of molecular information. In the last few years, efforts to respond to this need have been devoted mainly to the regulatory interactions involving ncRNAs as direct or indirect regulators of protein-coding mRNAs. Such efforts resulted in the development of new computational tools, allowing the exploitation of the information spread in numerous different ncRNA data sets to interpret transcriptome changes under physiological and pathological cell responses. While experimental validation remains essential to identify key RNA regulatory interactions, the integration of ncRNA big data, in combination with systematic literature mining, is proving to be invaluable in identifying potential new players, biomarkers and therapeutic targets in cancer and other diseases.

Pezizomycetes genomes reveal the molecular basis of ectomycorrhizal truffle lifestyle.

Tuberaceae is one of the most diverse lineages of symbiotic truffle-forming fungi. To understand the molecular underpinning of the ectomycorrhizal truffle lifestyle, we compared the genomes of Piedmont white truffle (Tuber magnatum), Périgord black truffle (Tuber melanosporum), Burgundy truffle (Tuber aestivum), pig truffle (Choiromyces venosus) and desert truffle (Terfezia boudieri) to saprotrophic Pezizomycetes. Reconstructed gene duplication/loss histories along a time-calibrated phylogeny of Ascomycetes revealed that Tuberaceae-specific traits may be related to a higher gene diversification rate. Genomic features in Tuber species appear to be very similar, with high transposon content, few genes coding lignocellulose-degrading enzymes, a substantial set of lineage-specific fruiting-body-upregulated genes and high expression of genes involved in volatile organic compound metabolism. Developmental and metabolic pathways expressed in ectomycorrhizae and fruiting bodies of T. magnatum and T. melanosporum are unexpectedly very similar, owing to the fact that they diverged ~100 Ma. Volatile organic compounds from pungent truffle odours are not the products of Tuber-specific gene innovations, but rely on the differential expression of an existing gene repertoire. These genomic resources will help to address fundamental questions in the evolution of the truffle lifestyle and the ecology of fungi that have been praised as food delicacies for centuries.

Author Correction: A family of archaea-like carboxylesterases preferentially expressed in the symbiotic phase of the mycorrhizal fungus Tuber melanosporum.

A correction to this article has been published and is linked from the HTML and the PDF versions of this paper. The error has been fixed in the paper.

A family of archaea-like carboxylesterases preferentially expressed in the symbiotic phase of the mychorrizal fungus Tuber melanosporum.

An increasing number of esterases is being revealed by (meta) genomic sequencing projects, but few of them are functionally/structurally characterized, especially enzymes of fungal origin. Starting from a three-member gene family of secreted putative "lipases/esterases" preferentially expressed in the symbiotic phase of the mycorrhizal fungus Tuber melanosporum ("black truffle"), we show here that these enzymes (TmelEST1-3) are dimeric, heat-resistant carboxylesterases capable of hydrolyzing various short/medium chain p-nitrophenyl esters. TmelEST2 was the most active (kcat = 2302 s-1 for p-nitrophenyl-butyrate) and thermally stable (T50 = 68.3 °C), while TmelEST3 was the only one displaying some activity on tertiary alcohol esters. X-ray diffraction analysis of TmelEST2 revealed a classical α/ß hydrolase-fold structure, with a network of dimer-stabilizing intermolecular interactions typical of archaea esterases. The predicted structures of TmelEST1 and 3 are overall quite similar to that of TmelEST2 but with some important differences. Most notably, the much smaller volume of the substrate-binding pocket and the more acidic electrostatic surface profile of TmelEST1. This was also the only TmelEST capable of hydrolyzing feruloyl-esters, suggestinng a possible role in root cell-wall deconstruction during symbiosis establishment. In addition to their potential biotechnological interest, TmelESTs raise important questions regarding the evolutionary recruitment of archaea-like enzymes into mesophilic subterranean fungi such as truffles.

Identification of an early transcriptomic signature of insulin resistance and related diseases in lymphomonocytes of healthy subjects.

Insulin resistance is considered to be a pathogenetic mechanism in several and diverse diseases (e.g. type 2 diabetes, atherosclerosis) often antedating them in apparently healthy subjects. The aim of this study is to investigate with a microarray based approach whether IR per se is characterized by a specific pattern of gene expression. For this purpose we analyzed the transcriptomic profile of peripheral blood mononuclear cells in two groups (10 subjects each) of healthy individuals, with extreme insulin resistance or sensitivity, matched for BMI, age and gender, selected within the MultiKnowledge Study cohort (n = 148). Data were analyzed with an ad-hoc rank-based classification method. 321 genes composed the gene set distinguishing the insulin resistant and sensitive groups, within which the "Adrenergic signaling in cardiomyocytes" KEGG pathway was significantly represented, suggesting a pattern of increased intracellular cAMP and Ca2+, and apoptosis in the IR group. The same pathway allowed to discriminate between insulin resistance and insulin sensitive subjects with BMI >25, supporting his role as a biomarker of IR. Moreover, ASCM pathway harbored biomarkers able to distinguish healthy and diseased subjects (from publicly available data sets) in IR-related diseases involving excitable cells: type 2 diabetes, chronic heart failure, and Alzheimer's disease. The altered gene expression profile of the ASCM pathway is an early molecular signature of IR and could provide a common molecular pathogenetic platform for IR-related disorders, possibly representing an important aid in the efforts aiming at preventing, early detecting and optimally treating IR-related diseases.

Screening for Protein-Protein Interaction Inhibitors Using a Bioluminescence Resonance Energy Transfer (BRET)-Based Assay in Yeast.

The bioluminescence resonance energy transfer (BRET) technology is a widely used live cell-based method for monitoring protein-protein interactions as well as conformational changes within proteins or molecular complexes. Considering the emergence of protein-protein interactions as a new promising class of therapeutic targets, we have adapted the BRET method in budding yeast. In this technical note, we describe the advantages of using this simple eukaryotic model rather than mammalian cells to perform high-throughput screening of chemical compound collections: genetic tractability, tolerance to solvent, rapidity, and no need of expensive robotic systems. Here, the HDM2/p53 interaction, related to cancer, is used to highlight the interest of this technology in yeast. Sharing the protocol of this BRET-based assay with the scientific community will extend its application to other protein-protein interactions, even though it is toxic for mammalian cells, in order to discover promising therapeutic candidates.