BioVersys Works to Bring Antibiotic Resistance to an End

BioVersys, founded in 2008, is working on bringing a technology for screening and for the development of 'transcriptional regulator inhibiting compounds' (TRICs) to patients in order to overcome antibiotic resistance. The co-founders share their view on what makes successful scientists pursue a career as start-up entrepreneurs rather than a classic academic career. They describe the history and milestones of their company, and how their everyday work differs from that of peers in an academic or industrial research setting

Rational design of a small molecule-responsive intramer controlling transgene expression in mammalian cells

Aptamers binding proteins or small molecules have been shown to be versatile and powerful building blocks for the construction of artificial genetic switches. In this study, we present a novel aptamer-based construct regulating the Tet Off system in a tetracycline-independent manner thus achieving control of transgene expression. For this purpose, a TetR protein-inhibiting aptamer was engineered for use in mammalian cells, enabling the RNA-responsive control of the tetracycline-dependent transactivator (tTA). By rationally attaching the theophylline aptamer as a sensor, the inhibitory TetR aptamer and thus tTA activity became dependent on the ligand of the sensor aptamer. Addition of the small molecule theophylline resulted in enhanced binding to the corresponding protein in vitro and in inhibition of reporter gene expression in mammalian cell lines. By using aptamers as adaptors in order to control protein activity by a predetermined small molecule, we present a simple and straightforward approach for future applications in the field of Chemical Biology. Moreover, aptamer-based control of the widely used Tet system introduces a new layer of regulation thereby facilitating the construction of more complex gene network

A modular degron library for synthetic circuits in mammalian cells

Tight control over protein degradation is a fundamental requirement for cells to respond rapidly to various stimuli and adapt to a fluctuating environment. Here we develop a versatile, easy-to-handle library of destabilizing tags (degrons) for the precise regulation of protein expression profiles in mammalian cells by modulating target protein half-lives in a predictable manner. Using the well-established tetracycline gene-regulation system as a model, we show that the dynamics of protein expression can be tuned by fusing appropriate degron tags to gene regulators. Next, we apply this degron library to tune a synthetic pulse-generating circuit in mammalian cells. With this toolbox we establish a set of pulse generators with tailored pulse lengths and magnitudes of protein expression. This methodology will prove useful in the functional roles of essential proteins, fine-tuning of gene-expression systems, and enabling a higher complexity in the design of synthetic biological systems in mammalian cells

A synthetic low-frequency mammalian oscillator

Circadian clocks have long been known to be essential for the maintenance of physiological and behavioral processes in a variety of organisms ranging from plants to humans. Dysfunctions that subvert gene expression of oscillatory circadian-clock components may result in severe pathologies, including tumors and metabolic disorders. While the underlying molecular mechanisms and dynamics of complex gene behavior are not fully understood, synthetic approaches have provided substantial insight into the operation of complex control circuits, including that of oscillatory networks. Using iterative cycles of mathematical model-guided design and experimental analyses, we have developed a novel low-frequency mammalian oscillator. It incorporates intronically encoded siRNA-based silencing of the tetracycline-dependent transactivator to enable the autonomous and robust expression of a fluorescent transgene with periods of 26 h, a circadian clock-like oscillatory behavior. Using fluorescence-based time-lapse microscopy of engineered CHO-K1 cells, we profiled expression dynamics of a destabilized yellow fluorescent protein variant in single cells and real time. The novel oscillator design may enable further insights into the system dynamics of natural periodic processes as well as into siRNA-mediated transcription silencing. It may foster advances in design, analysis and application of complex synthetic systems in future gene therapy initiatives

Dissecting Colistin Resistance Mechanisms in Extensively Drug-Resistant Acinetobacter baumannii Clinical Isolates

Nosocomial infections with; Acinetobacter baumannii; are a global problem in intensive care units with high mortality rates. Increasing resistance to first- and second-line antibiotics has forced the use of colistin as last-resort treatment, and increasing development of colistin resistance in; A. baumannii; has been reported. We evaluated the transcriptional regulator PmrA as potential drug target to restore colistin efficacy in; A. baumannii; Deletion of; pmrA; restored colistin susceptibility in 10 of the 12 extensively drug-resistant; A. baumannii; clinical isolates studied, indicating the importance of PmrA in the drug resistance phenotype. However, two strains remained highly resistant, indicating that PmrA-mediated overexpression of the phosphoethanolamine (PetN) transferase PmrC is not the exclusive colistin resistance mechanism in; A. baumannii; A detailed genetic characterization revealed a new colistin resistance mechanism mediated by genetic integration of the insertion element IS; AbaI; upstream of the PmrC homolog EptA (93% identity), leading to its overexpression. We found that; eptA; was ubiquitously present in clinical strains belonging to the international clone 2, and IS; AbaI; integration upstream of; eptA; was required to mediate the colistin-resistant phenotype. In addition, we found a duplicated IS; AbaI; -; eptA; cassette in one isolate, indicating that this colistin resistance determinant may be embedded in a mobile genetic element. Our data disprove PmrA as a drug target for adjuvant therapy but highlight the importance of PetN transferase-mediated colistin resistance in clinical strains. We suggest that direct targeting of the homologous PetN transferases PmrC/EptA may have the potential to overcome colistin resistance in; A. baumannii; IMPORTANCE; The discovery of antibiotics revolutionized modern medicine and enabled us to cure previously deadly bacterial infections. However, a progressive increase in antibiotic resistance rates is a major and global threat for our health care system. Colistin represents one of our last-resort antibiotics that is still active against most Gram-negative bacterial pathogens, but increasing resistance is reported worldwide, in particular due to the plasmid-encoded protein MCR-1 present in pathogens such as; Escherichia coli; and; Klebsiella pneumoniae; Here, we showed that colistin resistance in; A. baumannii; , a top-priority pathogen causing deadly nosocomial infections, is mediated through different avenues that result in increased activity of homologous phosphoethanolamine (PetN) transferases. Considering that MCR-1 is also a PetN transferase, our findings indicate that PetN transferases might be the Achilles heel of superbugs and that direct targeting of them may have the potential to preserve the activity of polymyxin antibiotics

Rational design of a small molecule-responsive intramer controlling transgene expression in mammalian cells

Aptamers binding proteins or small molecules have been shown to be versatile and powerful building blocks for the construction of artificial genetic switches. In this study, we present a novel aptamer-based construct regulating the Tet Off system in a tetracycline-independent manner thus achieving control of transgene expression. For this purpose, a TetR protein-inhibiting aptamer was engineered for use in mammalian cells, enabling the RNA-responsive control of the tetracycline-dependent transactivator (tTA). By rationally attaching the theophylline aptamer as a sensor, the inhibitory TetR aptamer and thus tTA activity became dependent on the ligand of the sensor aptamer. Addition of the small molecule theophylline resulted in enhanced binding to the corresponding protein in vitro and in inhibition of reporter gene expression in mammalian cell lines. By using aptamers as adaptors in order to control protein activity by a predetermined small molecule, we present a simple and straightforward approach for future applications in the field of Chemical Biology. Moreover, aptamer-based control of the widely used Tet system introduces a new layer of regulation thereby facilitating the construction of more complex gene networks.ISSN:1362-4962ISSN:0301-561

Dyrk1A Potentiates Steroid Hormone-Induced Transcription via the Chromatin Remodeling Factor Arip4

Dyrk1A, a mammalian homolog of the Drosophila minibrain gene, encodes a dual-specificity kinase, involved in neuronal development and in adult brain physiology. In humans, a third copy of DYRK1A is present in Down syndrome (trisomy 21) and has been implicated in the etiology of mental retardation. To further understand this pathology, we searched for Dyrk1A-interacting proteins and identified Arip4 (androgen receptor-interacting protein 4), a SNF2-like steroid hormone receptor cofactor. Mouse hippocampal and cerebellar neurons coexpress Dyrk1A and Arip4. In HEK293 cells and hippocampal neurons, both proteins are colocalized in a speckle-like nuclear subcompartment. The functional interaction of Dyrk1A with Arip4 was analyzed in a series of transactivation assays. Either Dyrk1A or Arip4 alone displays an activating effect on androgen receptor- and glucocorticoid receptor-mediated transactivation, and Dyrk1A and Arip4 together act synergistically. These effects are independent of the kinase activity of Dyrk1A. Inhibition of endogenous Dyrk1A and Arip4 expression by RNA interference showed that both proteins are necessary for the efficient activation of androgen receptor- and glucocorticoid receptor-dependent transcription. As Dyrk1A is an activator of steroid hormone-regulated transcription, the overexpression of DYRK1A in persons with Down syndrome may cause rather broad changes in the homeostasis of steroid hormone-controlled cellular events