Transcriptional Bursting Diversifies the Behaviour of a Toggle Switch: Hybrid Simulation of Stochastic Gene Expression

Hybrid models for gene expression combine stochastic and deterministic representations of the underlying biophysical mechanisms. According to one of the simplest hybrid formalisms, protein molecules are produced in randomly occurring bursts of a randomly distributed size while they are degraded deterministically. Here, we use this particular formalism to study two key regulatory motifs—the autoregulation loop and the toggle switch. The distribution of burst times is determined and used as a basis for the development of exact simulation algorithms for gene expression dynamics. For the autoregulation loop, the simulations are compared to an analytic solution of a master equation. Simulations of the toggle switch reveal a number of qualitatively distinct scenarios with implications for the modelling of cell-fate selection.     

Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B

Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic.     

Response of NADPH-Diaphorase-Exhibiting Neurons in the Medullar Reticular Formation to High Spinal Cord Injury

1. The effect of hemisection of the cervical spinal cord on NADPH-diaphorase staining in the reticular nuclei of the rabbit medulla was investigated using histochemical technique.2. A quantitative assessment of somal and neuropil NADPH-diaphorase staining was made by an image analyzer in a selected area of each reticular nucleus of the rabbit medulla.3. On the 7th postsurgery day, the highest up-regulation of somatic NADPH-diapho- rase staining was observed in regions regulating cardiorespiratory processes; however, the highest increase of neuropil NADPH-diaphorase staining was found in the reticular nuclei modulating the tonus of postural muscles.4. The degeneration of non-NADPH-diaphorase-stained neurons was detected throughout the reticular formation of the medulla, but the extent of neuronal death did not correlate with the up-regulation of the NADPH-diaphorase staining in the reticular nuclei of the medulla.5. The findings provide evidence that NADPH-diaphorase-exhibiting neurons are refractory to the hemisection of the cervical spinal cord and that the neuronal up-regulation of NADPH-diaphorase at the medullar level is probably not a causative factor leading to the death of the reticulospinal neurons.     

Inhibitory effect of IGF-I on induced apoptosis in mouse preimplantation embryos cultured in vitro

Insulin-like growth factor I (IGF-I) has been shown to promote mammalian early embryo development. Increased cell division or decreased cell death have been proposed as two main possible mechanisms in its effect. Here we examine the nature of this promoting effect in a model situation. Camptothecin (0.01 microg/ml) and actimomycin D (0.005 microg/ml) were used to induce apoptosis. Four-cell mouse embryos were cultured in vitro to blastocyst stage in the temporary (15 h) presence or absence of apoptotic inductors and in the permanent presence or absence of IGF-I (100 ng/ml). Embryos were assessed by morphological triple staining (Hoechst 33342, propidium iodide, Calcein AM) and comet assay on Day 5, 120 h after administration of hCG. The number of nuclei, the blastocyst formation, the proportion of embryos containing fragmented DNA and the percentage of apoptotic and secondary necrotic nuclei were assessed. Both inductors of apoptosis significantly increased the percentage of apoptotic and secondary necrotic cells and reduced total cell counts (camptothecin, P>0.001; actinomycin D, P>0.001). When IGF-I was added to the culture medium in the presence of an apoptosis inductor, apoptosis incidence was significantly decreased (P<0.001). The addition of IGF-I into control samples also decreased the percentage of apoptotic and secondary necrotic cells. In contrast, IGF-I addition had no significant influence on embryo development (P>0.05). Our data suggest a primary role for IGF-I as an apoptotic survival factor in mouse preimplantation embryos in specific conditions.     

Synthesis of 1-D- and 1-L-myo-inosityl 2-N-acetamido-2-deoxy-alpha-D-glucopyranoside establishes substrate specificity of the Mycobacterium tuberculosis enzyme AcGI deacetylase

Mycothiol (MSH, 1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside) is the principal low molecular weight thiol in actinomycetes. The enzyme 1-D-myo-inosityl 2-N-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase (AcGI deacetylase) is involved in the biosynthesis of MSH and forms the free amine 1-D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside, which is used in the third of four steps of MSH biosynthesis. Here, we report the synthesis of two isomers of AcGI, which contain either 1-L-myo-inositol or 1-D-myo-inositol. These synthetic products were used to investigate substrate specificity of the Mycobacterium tuberculosis enzyme AcGI deacetylase.     

Nuclear import of factors involved in signaling is inhibited in C3H/10T1/2 cells treated with tetradecylthioacetic acid

The non-beta-oxidisable tetradecylthioacetic acid (TTA) is incorporated into cellular membranes when C3H/10T1/2 cells are cultured in TTA-containing medium. We here demonstrate that this alteration in cellular membranes affect the nuclear translocation of proteins involved in signal transduction. Analysis of cellular fatty acid composition shows that TTA and TTA:1n-8 constitute approximately 40 mol% of total fatty acids in cellular/nuclear membranes. Activation of c-fos expression is significantly inhibited in TTA-treated cells but the enzymatic activation of mitogen activated protein kinase (ERK) is not affected. Immunofluorescence and confocal microscopy studies demonstrate that in mitogene-stimulated TTA-treated cells, the translocation of phosphorylated ERK1/2, protein kinase C alpha (PKC alpha), and PKC beta(1) from the cytoplasm into the nucleus is considerably decreased and delayed. Concomitant with a decreased nuclear import, ERK1/2 dephosphorylation is decreased in TTA-treated cells. There is no TTA-induced inhibition of nuclear import of proteins with a classical nuclear localization signal (NLS), as seen by in vitro nuclear import experiments of BSA fused to the NLS from SV40 large T, or in vivo studies of hnRNP A1 nuclear import. The expression levels of Importin alpha, Importin beta, Importin 7, and NTF2 are not altered in the TTA-treated cells. Taken together, our data indicate that TTA treatment causes changes in cellular fatty acid composition that negatively affect NLS-independent mechanisms of protein translocation through the nuclear pore complex.     

Influence of structure of human,rat, and bovine serum albumins on binding properties of photoactive drug hypericin

Fluorescence binding measurements and molecular modeling were employed to study the interaction of hypericin (Hyp) with human (HSA), rat (RSA), and bovine (BSA) serum albumins. Fluorescence emission data show the solubility of Hyp increasing in the order BSA, HSA, and RSA. Molecular modeling was used to construct the detailed structural models of the complexes and to explain the differences in the binding properties of Hyp. It was shown that the structures of Hyp/HSA and Hyp/RSA complexes are more similar and in some aspects different from those found for the Hyp/BSA complex. The role of the amino acid sequence in the IIA subdomains of HSA, RSA, and BSA is discussed to explain the observed differences.