Management of a Primordial Problem: Redox-sensitive Transcriptional Regulation in Methanosarcina acetivorans

The primordial Earth which hosted the first forms of life was an environment free of oxygen. Early organisms utilized metabolisms dependent upon anaerobic conditions and incorporated systems to which oxygen is deleterious. As the content of oxygen in Earth\u27s atmosphere increased, anaerobic organisms had to acquire methods to sense and combat oxygen and reactive oxygen species. Several mechanisms were advantageous to such anaerobic organisms which correlated transcriptional processes with the redox state of the cell so that energy may be conserved and oxygen stress recovery genes activated during periods of oxidative stress. Iron sulfur (Fe-S) cluster cofactors incorporated within RNA polymerase (RNAP) may sense oxygen to globally regulate transcription. Methanosarcina acetivorans, a methanogenic archaeon, offers an opportunity to study an RNAP with two Fe-S clusters within an organism of a phylogenetically and metabolically diverse group. An in vitro transcription system for M. acetivorans could be used to investigate the effects Fe-S cluster integrity on RNAP activity, which would require the components involved in promoter-specific transcription: RNAP, TATA-binding protein (TBP), and transcription factor B (TFB). This work describes the purification of M. acetivorans TBP and TFB for the development of such a system. M. acetivorans also possesses the methanogen-specific redox-sensitive transcriptional regulator MsvR. This work provides evidence of a physiologically-relevant reducing partner for MsvR. As of yet, M. acetivorans MsvR has only been observed to bind to its own gene. This work investigates the other potential gene targets for MsvR

Letter to the Editor re "Four essential drugs needed for quality care of the dying: a Delphi-study based international expert consensus opinion"

High-quality patient care can be defined as an approach that minimizes harm whilst aligning with people's expectations. Dying people and their relatives have articulated that they expect health care providers to manage physical and psychological symptoms well, with expectations even higher when such care is delivered by specialist services. Despite excellent intentions, palliative care clinicians and researchers have done little to improve systematically the evidence base for prescribing when people are actually dying. Few data exist to inform clinicians' understanding of how people's actual experiences align with their articulated wishes. Symptoms are managed based on relatives' and staff's assumptions of the experience of the dying person, with a “good death” often being seen as quiet and calm. Achieving this often requires sedation, for which the dying person will very rarely have given consent. This requires consideration, especially when evidence suggests people facing death will forgo symptom control to remain as alert and interactive for as long as possible. We ignore patients' wishes at our peril if we are to be truly patient centered

End-of-life research: do we need to build proxy consent into all clinical trial protocols studying the terminal phase?

Research into symptoms that occur at the end of life is paramount for ensuring we provide the best possible care for patients in the terminal phase, yet obtaining informed consent from the study participant is not possible at the time these symptoms occur. Importantly, these questions cannot be answered in any clinical population and defining the net clinical effect of medications used, for example, for noisy respiratory secretions is crucial if the quality of care is to be further improved

Secondary Structural Analysis of Shigella flexneri Invasion Plasmid Antigen B (IPAB)

This study focuses on the secondary structure of invasion plasmid antigen B (IpaB) of Shigella flexneri and the effects on it by the binding of invasion plasmid gene C (IpgC) along with sites of interaction with IpgC. IpgC is the molecular chaperone of IpaB that is responsible for keeping IpaB from associating with invasion plasmid antigen C (IpaC) or degrading before it is secreted. A library of single tryptophan containing mutants was created by replacing the native tryptophan (Trp) at position 105 with phenylalanine (Phe) and using this as a template to make twelve mutants by inserting Trp at places throughout the length of the protein. Fluorescence spectroscopy utilizing the naturally fluorescent properties of Trp was used to determine areas of interaction with IpgC and the effects of IpgC's binding on IpaB's secondary structure and topology. Invasion and hemolysis assays showed that the native Trp at residue 105 could be replaced with Phe and used as a template to make single insertion mutants. The same assays showed that several of the mutants had no effect on the protein's function while some of the mutants showed no invasiveness or hemolysis but no significant structural changes. Three mutants were determined to be problematic and their use was discontinued. The emission maximum scans showed that many of the mutants were not affected by the binding of IpgC. Two mutants plus the wildtype IpaB displayed significant spectral shifts to the red, indicating that the environment around that Trp became more polar (more hydrophilic) when not bound to IpgC. The thermal unfolding analysis showed that IpaB was more stable when in complex with IpgC than when alone except in one case (for the mutant I553W). The Frster resonance energy transfer (FRET) studies provided information to determine the tertiary structure features of IpaB. These data support the hypothesis that IpaB is a highly structured and complex molecule that is more thermally stable when bound to its chaperone IpgC.Department of Biochemistry and Molecular Biolog