Signatures of criticality arise in simple neural population models with correlations

Large-scale recordings of neuronal activity make it possible to gain insights into the collective activity of neural ensembles. It has been hypothesized that neural populations might be optimized to operate at a 'thermodynamic critical point', and that this property has implications for information processing. Support for this notion has come from a series of studies which identified statistical signatures of criticality in the ensemble activity of retinal ganglion cells. What are the underlying mechanisms that give rise to these observations? Here we show that signatures of criticality arise even in simple feed-forward models of retinal population activity. In particular, they occur whenever neural population data exhibits correlations, and is randomly sub-sampled during data analysis. These results show that signatures of criticality are not necessarily indicative of an optimized coding strategy, and challenge the utility of analysis approaches based on equilibrium thermodynamics for understanding partially observed biological systems.Comment: 36 pages, LaTeX; added journal reference on page 1, added link to code repositor

Comparative Genomic Analysis of Antimicrobial-Resistant Escherichia coli from South American Camelids in Central Germany

South American camelids (SAC) are increasingly kept in Europe in close contact with humans and other livestock species and can potentially contribute to transmission chains of epizootic, zoonotic and antimicrobial-resistant (AMR) agents from and to livestock and humans. Consequently, SAC were included as livestock species in the new European Animal Health Law. However, the knowledge on bacteria exhibiting AMR in SAC is too scarce to draft appropriate monitoring and preventive programs. During a survey of SAC holdings in central Germany, 39 Escherichia coli strains were isolated from composite fecal samples by selecting for cephalosporin or fluoroquinolone resistance and were here subjected to whole-genome sequencing. The data were bioinformatically analyzed for strain phylogeny, detection of pathovars, AMR genes and plasmids. Most (33/39) strains belonged to phylogroups A and B1. Still, the isolates were highly diverse, as evidenced by 28 multi-locus sequence types. More than half of the isolates (23/39) were genotypically classified as multidrug resistant. Genes mediating resistance to trimethoprim/sulfonamides (22/39), aminoglycosides (20/39) and tetracyclines (18/39) were frequent. The most common extended-spectrum-β-lactamase gene was bla CTX-M-1 (16/39). One strain was classified as enteropathogenic E. coli . The positive results indicate the need to include AMR bacteria in yet-to-be-established animal disease surveillance protocols for SAC

A novel MCF-10A line allowing conditional oncogene expression in 3D culture

Introduction Non-transformed mammary epithelial cell lines such as MCF-10A recapitulate epithelial morphogenesis in three-dimensional (3D) tissue culture by forming acinar structures. They represent an important tool to characterize the biological properties of oncogenes and to model early carcinogenic events. So far, however, these approaches were restricted to cells with constitutive oncogene expression prior to the set-up of 3D cultures. Although very informative, this experimental setting has precluded the analysis of effects caused by sudden oncoprotein expression or withdrawal in established epithelial cultures. Here, we report the establishment and use of a stable MCF-10A cell line (MCF-10Atet) fitted with a novel and improved doxycycline (dox)-regulated expression system allowing the conditional expression of any transgene. Methods MCF-10Atet cells were generated by stable transfection with pWHE644, a vector expressing a second generation tetracycline-regulated transactivator and a novel transcriptional silencer. In order to test the properties of this new repressor/activator switch, MCF-10Atet cells were transfected with a second plasmid, pTET-HABRAF-IRES-GFP, which responds to dox treatment with the production of a bi-cistronic transcript encoding hemagglutinin-tagged B-Raf and green fluorescent protein (GFP). This improved conditional expression system was then characterized in detail in terms of its response to various dox concentrations and exposure times. The plasticity of the phenotype provoked by oncogenic B-RafV600E in MCF-10Atet cells was analyzed in 3D cultures by dox exposure and subsequent wash-out. Results MCF-10Atet cells represent a tightly controlled, conditional gene expression system. Using B-RafV600E as a model oncoprotein, we show that its sudden expression in established 3D cultures results in the loss of acinar organization, the induction of an invasive phenotype and hallmarks of epithelial-to-mesenchymal transition (EMT). Importantly, we show for the first time that this severe transformed phenotype can be reversed by dox wash-out and concomitant termination of oncogene expression. Conclusions Taken together, we have generated a stable MCF-10A subline allowing tight dox-controlled and reversible expression of any transgene without the need to modify its product by introducing artificial dimerization or ligand-binding domains. This system will be very valuable to address phenomena such as EMT, oncogene addiction, oncogene-induced senescence and drug resistance

Using unique ORFan genes as strain-specific identifiers for Escherichia coli

Background Bacterial identification at the strain level is a much-needed, but arduous and challenging task. This study aimed to develop a method for identifying and differentiating individual strains among multiple strains of the same bacterial species. The set used for testing the method consisted of 17 Escherichia coli strains picked from a collection of strains isolated in Germany, Spain, the United Kingdom and Vietnam from humans, cattle, swine, wild boars, and chickens. We targeted unique or rare ORFan genes to address the problem of selective and specific strain identification. These ORFan genes, exclusive to each strain, served as templates for developing strain-specific primers. Results Most of the experimental strains (14 out of 17) possessed unique ORFan genes that were used to develop strain-specific primers. The remaining three strains were identified by combining a PCR for a rare gene with a selection step for isolating the experimental strains. Multiplex PCR allowed the successful identification of the strains both in vitro in spiked faecal material in addition to in vivo after experimental infections of pigs and recovery of bacteria from faecal material. In addition, primers for qPCR were also developed and quantitative readout from faecal samples after experimental infection was also possible. Conclusions The method described in this manuscript using strain-specific unique genes to identify single strains in a mixture of strains proved itself efficient and reliable in detecting and following individual strains both in vitro and in vivo, representing a fast and inexpensive alternative to more costly methods.Peer Reviewe

A Novel TetR-Regulating Peptide Turns off rtTA-Mediated Activation of Gene Expression

Conditional regulation of gene expression is a powerful and indispensable method for analyzing gene function. The “Tet-On” system is a tool widely used for that purpose. Here, the transregulator rtTA mediates expression of a gene of interest after addition of the small molecule effector doxycycline. Although very effective in rapidly turning on gene expression, the system is hampered by the long half-life of doxycycline which makes shutting down gene expression rapidly very difficult to achieve. We isolated an rtTA-binding peptide by in vivo selection that acts as a doxycycline antagonist and leads to rapid and efficient shut down of rtTA-mediated reporter gene expression in a human cell line. This peptide represents the basis for novel effector molecules which complement the “Tet-system” by enabling the investigator to rapidly turn gene expression not just on at will, but now also off

Evidence for Contemporary Switching of the O-Antigen Gene Cluster between Shiga Toxin-Producing Escherichia coli Strains Colonizing Cattle

Shiga toxin-producing Escherichia coli (STEC) comprise a group of zoonotic enteric pathogens with ruminants, especially cattle, as the main reservoir. O-antigens are instrumental for host colonization and bacterial niche adaptation. They are highly immunogenic and, therefore, targeted by the adaptive immune system. The O-antigen is one of the most diverse bacterial cell constituents and variation not only exists between different bacterial species, but also between individual isolates/strains within a single species. We recently identified STEC persistently infecting cattle and belonging to the different serotypes O156:H25 (n = 21) and O182:H25 (n = 15) that were of the MLST sequence types ST300 or ST688. These STs differ by a single nucleotide in purA only. Fitness-, virulence-associated genome regions, and CRISPR/CAS (clustered regularly interspaced short palindromic repeats/CRISPR associated sequence) arrays of these STEC O156:H25 and O182:H25 isolates were highly similar, and identical genomic integration sites for the stx converting bacteriophages and the core LEE, identical Shiga toxin converting bacteriophage genes for stx1a, identical complete LEE loci, and identical sets of chemotaxis and flagellar genes were identified. In contrast to this genomic similarity, the nucleotide sequences of the O-antigen gene cluster (O-AGC) regions between galF and gnd and very few flanking genes differed fundamentally and were specific for the respective serotype. Sporadic aEPEC O156:H8 isolates (n = 5) were isolated in temporal and spatial proximity. While the O-AGC and the corresponding 5′ and 3′ flanking regions of these aEPEC isolates were identical to the respective region in the STEC O156:H25 isolates, the core genome, the virulence associated genome regions and the CRISPR/CAS elements differed profoundly. Our cumulative epidemiological and molecular data suggests a recent switch of the O-AGC between isolates with O156:H8 strains having served as DNA donors. Such O-antigen switches can affect the evaluation of a strain's pathogenic and virulence potential, suggesting that NGS methods might lead to a more reliable risk assessment

Clostridioides difficile in South American Camelids in Germany: First Insights into Molecular and Genetic Characteristics and Antimicrobial Resistance

Little is known about zoonotic pathogens and their antimicrobial resistance in South American camelids (SAC) in Germany including Clostridioides (C.) difficile. The aim of this study was to investigate prevalence, molecular characteristics and antimicrobial resistance of C. difficile in SAC. Composite SAC faecal samples were collected in 43 husbandries in Central Germany and cultured for C. difficile. Toxinotyping and ribotyping was done by PCR. Whole genome sequencing was performed with Illumina® Miseq™. The genomes were screened for antimicrobial resistance determinants. Genetic relatedness of the isolates was investigated using core genome multi locus sequence typing (cgMLST) and single nucleotide polymorphism analysis. Antimicrobial susceptibility testing was done using the Etest® method. Eight C. difficile isolates were recovered from seven farms. The isolates belonged to different PCR ribotypes. All isolates were toxinogenic. cgMLST revealed a cluster containing isolates recovered from different farms. Seven isolates showed similar resistance gene patterns. Different phenotypic resistance patterns were found. Agreement between phenotypic and genotypic resistance was identified only in some cases. Consequently, SAC may act as a reservoir for C. difficile. Thus, SAC may pose a risk regarding zoonotic transmission of toxinogenic, potentially human-pathogenic and resistant C. difficile isolates

Improved single-chain transactivators of the Tet-On gene expression system

BACKGROUND: The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application. RESULTS: We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used. CONCLUSION: We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used