Three open reading frames (ORFs) have been located downstream ofcefE in the cephamycin C gene cluster of Streptomyces clavuligerus. ORF13 (pcd) encodes a 496-amino-acid protein (molecular weight [MW], 52,488) with an N-terminal amino acid sequence identical to that of pure piperideine-6-carboxylate dehydrogenase. ORF14 (cmcT) encodes a 523-amino-acid protein (MW, 54,232) analogous to Streptomyces proteins for efflux and resistance to antibiotics. ORF15 (pbp74) encodes a high molecular weight penicillin-binding protein (MW, 74,094).

The nine biosynthesis genes of the Nocardia lactamdurans cephamycin cluster are expressed as three different mRNAs initiating at promoters latp, cefDp, andpcbABp, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (latp andcefDp) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (Streptomyces antibiotic regulatory proteins) family. The lat gene is transcribed in a single monocistronic transcript initiating at latp. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (pcbAB, pcbC,cmcI, cmcJ, cefF, andcmcH) started at pcbABp. A third polycistronic mRNA corresponding to the cefD and cefE genes started at cefDp.

Rationale: Viral infections are complex processes based on an intricate network of molecular interactions. The infectious agent hijacks components of the cellular machinery for its profit, circumventing the natural defense mechanisms triggered by the infected cell. The successful completion of the replicative viral cycle within a cell depends on the function of viral components versus the cellular defenses. Non-coding RNAs (ncRNAs) are important cellular modulators, either promoting or preventing the progression of viral infections. Among these ncRNAs, the long non-coding RNA (lncRNA) family is especially relevant due to their intrinsic functional properties and ubiquitous biological roles. Specific lncRNAs have been recently characterized as modulators of the cellular response during infection of human host cells by single stranded RNA viruses. However, the role of host lncRNAs in the infection by human RNA coronaviruses such as SARS-CoV-2 remains uncharacterized.

Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field.COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu).

SummaryMicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provides an exciting avenue towards antiviral therapeutics. From patient transcriptomic data, we have discovered a circulating miRNA, miR-2392, that is directly involved with SARS-CoV-2 machinery during host infection. Specifically, we found that miR-2392 was key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia as well as promoting many symptoms associated with COVID-19 infection. We demonstrate miR-2392 is present in the blood and urine of COVID-19 patients tested, but not detected in COVID-19 negative patients. These findings indicate the potential for developing a novel, minimally invasive, COVID-19 detection method. Lastly, using both in vitro human and in vivo hamster models, we have developed a novel miRNA-based antiviral therapeutic targeting miR-2392 that significantly reduces SARS-CoV-2 viability and may potentially inhibit a COVID-19 disease state in the host.

The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in Nocardia lactamdurans, in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the α-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the pcbAB gene and to a lesser extent also the lat transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the cefD promoter was not affected. Coupling of the lat and pcbABpromoters to the reporter xylE gene showed that expression from the lat and pcbAB promoters was increased by addition of diaminopropane in Streptomyces lividans. Intracellular accumulation of diamines in Nocardia may be a signal to trigger antibiotic production.

An expression and purification cassette containing the aminoglycoside phosphotransferase gene (aph) as selective marker has been constructed in the Escherichia coli vector pULHis2. DNA fragments inserted in the cassette can be easily subcloned in pIJ699 to give vectors for overexpression of genes in Streptomyces and purification of proteins by a one-step procedure. The expression system uses the thiostrepton-inducible promoter tipA for expression and a six histidine coding nucleotide sequence that is fused in frame to the foreign gene inserted in the polylinker. The pULHis2-derived expression vector has been used satisfactorily to express and to purify the P7 and P8 proteins of Nocardia lactamdurans which carry out the methoxylation of cephalosporin C to 7-methoxycephalosporin C.

No abstract