Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces

[EN] Bacteria in the genus Streptomyces are major producers of antibiotics and other pharmacologically active compounds. Genetic and physiological manipulations of these bacteria are important for new drug discovery and production development. An essential part of any ‘genetic toolkit’ is the availability of regulatable promoters. We have adapted the tetracycline (Tc) repressor/operator (TetR/ tetO ) regulatable system from transposon Tn 10 for use in Streptomyces . The synthetic Tc controllable promoter (tcp), tcp830 , was active in a wide range of Streptomyces species, and varying levels of induction were observed after the addition of 1–100 ng/ml of anhydrotetracycline (aTc). Streptomyces coelicolor contained an innate Tc-controllable promoter regulated by a TetR homologue (SCO0253). Both natural and synthetic promoters were active and inducible throughout growth. Using the luxAB genes expressing luciferase as a reporter system, we showed that induction factors of up to 270 could be obtained for tcp830 . The effect of inducers on the growth of S.coelicolor was determined; addition of aTc at concentrations where induction is optimal, i.e. 0.1–1 μg/ml, ranged from no effect on growth rate to a small increase in the lag period compared with cultures with no inducerSIThe authors acknowledge gifts of plasmids and strains from Prof. Leadlay, Prof. Hillen, Prof. Bujard, Dr Herron and Dr Paget. The authors thank Dr Sumby, Dr Ding and Wael Hussein for the construction of several plasmids and vectors. The authors also thank Prof. Williams for the use of Lucy. This work was funded by the BBSRC. Funding to pay the Open Access publication charges for this article was provided by JIS

Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces

Bacteria in the genus Streptomyces are major producers of antibiotics and other pharmacologically active compounds. Genetic and physiological manipulations of these bacteria are important for new drug discovery and production development. An essential part of any ‘genetic toolkit’ is the availability of regulatable promoters. We have adapted the tetracycline (Tc) repressor/operator (TetR/tetO) regulatable system from transposon Tn10 for use in Streptomyces. The synthetic Tc controllable promoter (tcp), tcp830, was active in a wide range of Streptomyces species, and varying levels of induction were observed after the addition of 1–100 ng/ml of anhydrotetracycline (aTc). Streptomyces coelicolor contained an innate Tc-controllable promoter regulated by a TetR homologue (SCO0253). Both natural and synthetic promoters were active and inducible throughout growth. Using the luxAB genes expressing luciferase as a reporter system, we showed that induction factors of up to 270 could be obtained for tcp830. The effect of inducers on the growth of S.coelicolor was determined; addition of aTc at concentrations where induction is optimal, i.e. 0.1–1 μg/ml, ranged from no effect on growth rate to a small increase in the lag period compared with cultures with no inducer

Selective synthesis of phenanthrenes and dihydrophenanthrenes via gold-catalyzed cycloisomerization of biphenyl embedded trienynes

Readily available o′-alkenyl-o-alkynylbiaryls, a particular type of 1,7-enynes, undergo a selective cycloisomerization reaction in the presence of a gold(I) catalyst to give interesting phenanthrene and dihydrophenanthrene derivatives in high yields. The solvent used provokes a switch in the evolution of the gold intermediate and plays a key role in the reaction outcome.Ministerio de Economıá y Competitividad (MINECO), AEI and FEDER (projects CTQ2017- 85263-R and CTQ2016-75023-C2-1-P), Instituto de Salud Carlos III (FEDER funds, ISCIII RETIC REDINREN RD16/ 0009/0015), Junta de Castilla y León and FEDER (BU291P18), and University of Alcalá (projects CCGP2017- EXP/016 and CCG2018/EXP-00

Renal tumouroids: challenges of manufacturing 3D cultures from patient derived primary cells.

Recent advancements in 3D in vitro culture have allowed for the development of cancer tissue models which accurately recapitulate the tumour microenvironment. Consequently, there has been increased innovation in therapeutic drug screening. While organoid cultures show great potential, they are limited by the time scale of their growth in vitro and the dependence upon commercial matrices, such as Matrigel, which do not allow for manipulations of their composition or mechanical properties. Here, we show a straightforward approach for the isolation and culture of primary human renal carcinoma cells and matched non-affected kidney. This approach does not require any specific selection for cancer cells, and allows for their direct culture in amenable 3D collagen-based matrices, with the preservation of cancer cells as confirmed by NGS sequencing. This method allows for culture of patient-derived cancer cells in 3D microenvironment, which can be used for downstream experimentation such as investigation of cell-matrix interaction or drug screening. [Abstract copyright: © 2022. Crown.

Renal tumouroids: challenges of manufacturing 3D cultures from patient derived primary cells

Recent advancements in 3D in vitro culture have allowed for the development of cancer tissue models which accurately recapitulate the tumour microenvironment. Consequently, there has been increased innovation in therapeutic drug screening. While organoid cultures show great potential, they are limited by the time scale of their growth in vitro and the dependence upon commercial matrices, such as Matrigel, which do not allow for manipulations of their composition or mechanical properties. Here, we show a straightforward approach for the isolation and culture of primary human renal carcinoma cells and matched non-affected kidney. This approach does not require any specific selection for cancer cells, and allows for their direct culture in amenable 3D collagen-based matrices, with the preservation of cancer cells as confirmed by NGS sequencing. This method allows for culture of patient-derived cancer cells in 3D microenvironment, which can be used for downstream experimentation such as investigation of cell-matrix interaction or drug screening

Hypothesized role of pregnancy hormones on HER2+breast tumor development

Breast cancer incidence rates have declined among older but not younger women; the latter are more likely to be diagnosed with breast cancers carrying a poor prognosis. Epidemiological evidence supports an increase in breast cancer incidence following pregnancy with risk elevated as much as 10 years post-partum. We investigated the association between years since last full-term pregnancy at the time of diagnosis (10 years) and breast tumor subtype in a case series of premenopausal Hispanic women (n = 627). Participants were recruited in the United States, Mexico, and Spain. Cases with known estrogen receptor (ER), progesterone receptor (PR), and HER2 status, with one or more full-term pregnancies >/=1 year prior to diagnosis were eligible for this analysis. Cases were classified into three tumor subtypes according to hormone receptor (HR+ = ER+ and/or PR+; HR- = ER- and PR-) expression and HER2 status: HR+/HER2-, HER2+ (regardless of HR), and triple negative breast cancer. Case-only odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated for HER2+ tumors in reference to HR+/HER2- tumors. Participants were pooled in a mixed-effects logistic regression model with years since pregnancy as a fixed effect and study site as a random effect. When compared to HR+/HER2- cases, women with HER2+ tumors were more likely be diagnosed in the post-partum period of 45 years) did not materially alter our results (OR = 1.78; 95 % CI, 1.08-2.93). These findings support the novel hypothesis that factors associated with the post-partum breast, possibly hormonal, are involved in the development of HER2+ tumors

Intracellular Mechanical Drugs Induce Cell-Cycle Altering and Cell Death

Current advances in materials science have demonstrated that extracellular mechanical cues can define cell function and cell fate. However, a fundamental understanding of the manner in which intracellular mechanical cues affect cell mechanics remains elusive. How intracellular mechanical hindrance, reinforcement, and supports interfere with the cell cycle and promote cell death is described here. Reproducible devices with highly controlled size, shape, and with a broad range of stiffness are internalized in HeLa cells. Once inside, they induce characteristic cell-cycle deviations and promote cell death. Device shape and stiffness are the dominant determinants of mechanical impairment. Device structural support to the cell membrane and centering during mitosis maximize their effects, preventing spindle centering, and correct chromosome alignment. Nanodevices reveal that the spindle generates forces larger than 114 nN which overcomes intracellular confinement by relocating the device to a less damaging position. By using intracellular mechanical drugs, this work provides a foundation to defining the role of intracellular constraints on cell function and fate, with relevance to fundamental cell mechanics and nanomedicine.Peer reviewe

Proteome from patients with metabolic syndrome is regulated by quantity and quality of dietary lipids

Background: Metabolic syndrome is a multi-component disorder associated to a high risk of cardiovascular disease. Its etiology is the result of a complex interaction between genetic and environmental factors, including dietary habits. We aimed to identify the target proteins modulated by the long-term consumption of four diets differing in the quality and quantity of lipids in the whole proteome of peripheral blood mononuclear cells (PBMC). Results: A randomized, controlled trial conducted within the LIPGENE study assigned 24 MetS patients for 12 weeks each to 1 of 4 diets: a) high-saturated fatty acid (HSFA), b) high-monounsaturated fatty acid (HMUFA), c) low-fat, high-complex carbohydrate diets supplemented with placebo (LFHCC) and d) low-fat, high-complex carbohydrate diets supplemented with long chain (LC) n-3 polyunsaturated fatty acids (PUFA) (LFHCC n-3). We analyzed the changes induced in the proteome of both nuclear and cytoplasmic fractions of PBMC using 2-D proteomic analysis. Sixty-seven proteins were differentially expressed after the long-term consumption of the four diets. The HSFA diet induced the expression of proteins responding to oxidative stress, degradation of ubiquitinated proteins and DNA repair. However, HMUFA, LFHCC and LFHCC n-3 diets down-regulated pro-inflammatory and oxidative stress-related proteins and DNA repairing proteins. Conclusion: The long-term consumption of HSFA, compared to HMUFA, LFHCC and LFHCC n-3, seems to increase the cardiovascular disease (CVD) risk factors associated with metabolic syndrome, such as inflammation and oxidative stress, and seem lead to DNA damage as a consequence of high oxidative stress

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality