An Interplay among FIS, H-NS, and Guanosine Tetraphosphate Modulates Transcription of the Escherichia coli cspA Gene under Physiological Growth Conditions

CspA, the most characterized member of the csp gene family of Escherichia coli, is highly expressed not only in response to cold stress, but also during the early phase of growth at 37°C. Here, we investigate at molecular level the antagonistic role played by the nucleoid proteins FIS and H-NS in the regulation of cspA expression under non-stress conditions. By means of both probing experiments and immunological detection, we demonstrate in vitro the existence of binding sites for these proteins on the cspA regulatory region, in which FIS and H-NS bind simultaneously to form composite DNA-protein complexes. While the in vitro promoter activity of cspA is stimulated by FIS and repressed by H-NS, a compensatory effect is observed when both proteins are added in the transcription assay. Consistently with these findings, inactivation of fis and hns genes reversely affect the in vivo amount of cspA mRNA. In addition, by means of strains expressing a high level of the alarmone guanosine tetraphosphate ((p)ppGpp) and in vitro transcription assays, we show that the cspA promoter is sensitive to (p)ppGpp inhibition. The (p)ppGpp-mediated expression of fis and hns genes is also analyzed, thus clarifying some aspects of the regulatory loop governing cspA transcription

Interaction of the main cold shock protein CS7.4 (CspA) of Escherichia coli with the promoter region of hns

Escherichia coli protein CS7.4 (CspA), homologous to the class of eukaryotic Y-box DNA-binding proteins, is a cold shock transcriptional activator of at least two genes, hns and gyrA. It was demonstrated that all or nearly all the elements necessary for the stimulation of hns transcription by CS7.4 protein are located in the proximal 110 bp DNA fragment of this gene with no additional elements being present in a longer fragment (660 bp) extending further upstream from the hns promoter. Protein CS7.4 bound strongly to the 110 bp segment of the hns promoter in crude extracts of cold shocked cells, but the purified protein displayed a weak interaction with the same DNA fragment. Purified CS7.4 protein also caused increased or decreased accessibility to DNase I at different sites of the 110 bp fragment of hns but the majority of these effects was seen only in the presence of RNA polymerase. Since gel shift experiments showed that protein CS7.4 stimulated the binding of RNA polymerase to the promoter of hns and since it is known that there are similarities between CS7.4 and ssDNA-binding proteins, we suggest that formation of the open complex by the RNA polymerase or protein-protein contacts between CS7.4 and the RNA polymerase are prerequisites for and/or the effects of the interaction of CS7.4 with its DNA target. The presence of a conserved CCAAT element in the hns promoter region, on the other hand, was found not to be stringently required for cold shock activation since expression of E coli of an hns-cat fusion containing the Proteus vulgaris hns promoter lacking a CCAAT box increased over four-fold after cold shock

Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions

The most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is > or = 50 microM during early exponential growth at 37 degrees C; therefore, its designation as a major cold-shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-to-late exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5' untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA)

Re-examining the contested good: proceedings from a postgraduate workshop on good food

Following the 2017 postgraduate research workshop hosted by the SOAS Food Studies Centre, in collaboration with University of Warwick Food GRP, this article brings together nine research briefs written by various participants. Inspired by the workshop's provocative theme, “What Is Good Food?”, each author explores how food categories are shaped and negotiated in different contexts and across scales. In this multi-authored article, the question of “good” food is first presented as contingent upon nutritional, economic, political, ritual, or moral conditions. Each author then reveals how globally defined notions of food's goodness are often resisted on the ground by producers and consumers, beyond the notions of ethics or “alternative” food movements that have often been the emphasis of previous literature dealing with the topic of good food. Taken together, this article scrutinizes the effects of various hierarchies of power and invites readers to reassess why and how good food continues to be a contested category

Molecular detection of a potentially toxic diatom species

A few diatom species produce toxins that affect human and animal health. Among these, members of the Pseudo-nitzschia genus were the first diatoms unambiguously identified as producer of domoic acid, a neurotoxin affecting molluscan shell-fish, birds, marine mammals, and humans. Evidence exists indicating the involvement of another diatom genus, Amphora, as a potential producer of domoic acid. We present a strategy for the detection of the diatom species Amphora coffeaeformis based on the development of species-specific oligonucleotide probes and their application in microarray hybridization experiments. This approach is based on the use of two marker genes highly conserved in all diatoms, but endowed with sufficient genetic divergence to discriminate diatoms at the species level. A region of approximately 450 bp of these previously unexplored marker genes, coding for elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), was used to design oligonucleotide probes that were tested for specificity in combination with the corresponding fluorescently labeled DNA targets. The results presented in this work suggest a possible use of this DNA chip technology for the selective detection of A. coffeaeformis in environmental settings where the presence of this potential toxin producer may represent a threat to human and animal health. In addition, the same basic approach can be adapted to a wider range of diatoms for the simultaneous detection of microorganisms used as biomarkers of different water quality levels

The Inhibitory Effect of ddC on Human Immunodeficiency Virus Replication Diminishes in Cells that are Chronically Exposed to the Drug

One possible explanation for the failure of human immunodeficiency virus type 1 (HIV-1) antiretroviral inhibitors to block the clinical progression of the infection may be a failure to maintain adequate drug levels at the site of viral replication. We have previously found that exposure of human monoblastoid cells (U937) for several months to a therapeutically relevant concentration (0.1 μM) of 2′,3′-dideoxycytidine (zalcitabine, ddC) allowed the isolation of a drug-resistant cell line characterized by a normal drug transport but a reduced ability to accumulate 2′,3′-dideoxycytidine 5′-triphosphate (the active antiretroviral form of the drug). In this paper we show that the drug-resistant cells were indistinguishable from normal cells in terms of surface CD4 receptors. The susceptibility of parental and ddC-resistant U937 cells to infection by HIV-1 was similar, as measured by proviral DNA formation. However, HIV-1 p24 production and the number of infectious virus particles produced were significantly lower in the drug-resistant compared to control cells. Addition of 0.1 μM ddC inhibited viral production by up to 92% in the control cells but had no effect on ddC-resistant cells. Thus, human cells exposed to therapeutically relevant ddC concentrations for several months show a reduced ddC anabolism and allow ddC-sensitive HIV-1 to replicate in the presence of inhibitory ddC concentrations

The complexity of the relationship between neuropsychological deficits and impairment in everyday tasks after stroke

Background and purpose: A large body of research reports that stroke patients are debilitated in terms of daily independence after dismissal from the hospital unit. Patients struggle with the use of daily objects or performing complex actions. Differences between individual deficits of patients are often associated with the site of the brain damage. However, clinical studies suggest that patients exhibit varied constellations of action-associated difficulties and neuropsychological deficits. There is a lack of conclusive evidence indicating how different neuropsychological symptoms link to the impaired ability to perform activities of daily living (ADL). Materials and methods: To further address this matter, in this study we compared the behavior of patients with left brain damage (LBD) and right brain damage (RBD) following stroke in two naturalistic task scenarios (tea making and document filing),and compared the committed action errors to the neuropsychological screening results. Results: We observed mild to severe impairments in both the LBD and RBD groups amounting to 37-55% of failure rate in attainment of action goal. Interestingly, the performance on both tasks was not correlated to each other, suggesting that the tasks involved a different set of higher cognitive functions. Despite similar behavioral manifestations, in the LBD group poor task performance was related to deficits in praxis performance and unilateral tactile and visual extinction. The presence of aphasia did not correlate with task performance, except for a link between low scores in Aachen aphasia test scales and misestimation error in the tea making task. In the RBD group, difficulties with performance were primarily linked to deficit in praxis and unilateral visual extinction. Conclusions: Despite similar behavior, the underlying mechanisms of the deficits after stroke might be different (in patients with LBD and RBD) and reveal complex interlinks of cognitive networks involved in the ability to carry on everyday tasks

A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri

The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation

Peers and Instructors as Sources of Distraction from a Cognitive Load Perspective

Framed by literature regarding classroom interactions that affect students’ cognitive processing, this study provided an integrative approach to understanding distracting instructor and student communication. Participants qualitatively reported on either a distracting peer (n = 90) or instructor (n = 127). The responses were coded using anti-citizenship behaviors and instructor misbehaviors. One additional category emerged that extends the instructor misbehavior literature. Participants completed a new distraction scale and a cognitive load scale. Our results revealed differences in frequencies for each behavior, but all instructor and student behaviors were equally distracting and had similar negative influences on students’ cognitive load. Implications for instructors to manage these distracting behaviors are discussed