Interactions between the Translation Machinery and a Translational preQ1 Riboswitch.

Gene expression is highly regulated with a diversity of regulation at the RNA level. In bacteria, regulation of mRNA translation into protein often occurs through RNA sequence features such as the Shine-Dalgarno (SD) sequence and local structural features. Translational riboswitches in bacteria exemplify such cis-acting regulation. This work look at how structural features of a preQ1 riboswitch effect regulation through interactions with the translation machinery. Broader questions about how individual translational machinery components, such as ribosomal protein S1 and the 30S ribosomal subunit, interact with structured RNAs are also addressed. We sought a more detailed mechanistic view of the interplay between the translational preQ1 riboswitch found in the 5′ UTR of an mRNA from T. tengcongensis, its ligand preQ1, and the SD sequence accessibility. To this end, we developed SiM-KARTS, a generalized strategy to interrogate site-specific structural dynamics of RNA molecules based on probe hybridization kinetics. Intriguingly, we found that the riboswitch expression platform alternates between conformations with differing SD accessibility, which are distinguished by “bursts” of probe binding, the pattern of which is modulated by ligand. This challenges the assumption that riboswitches behave in simple ON/OFF fashion and thus has broader implications for how we think about translational riboswitch regulation. The folding and unfolding of RNA structure influences other cellular processes besides translation. Ribosomal protein S1 performs other roles outside of the context of translation, which are related to its RNA binding or unfolding capacity. We used the well-characterized preQ1 riboswitch as a model pseudoknot to study how S1 interacts with defined, stable tertiary structure. S1 is able to bind and at least partially unfold this pseudoknot in a manner that is limited by RNA structural stability. Lastly, we investigated the influence of S1 on translation of preQ1 riboswitch-containing mRNAs and found that the effects of ligand on translation are not potentiated by the loss of S1. There is, however, a dramatic effect on translational coupling, invoking a role for S1 in polycistronic mRNA translation. These results highlight the need for additional techniques, such as assays at the single molecule level, to monitor early 30S-mRNA interactions during translation.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116677/1/palund_1.pd

The acceptability of promotional policies to teachers and administrators in five selected communities.

Thesis (Ed.M.)--Boston Universit

Elevated cerebral spinal fluid biomarkers in children with mucopolysaccharidosis I-H.

Mucopolysaccharidosis (MPS) type-IH is a lysosomal storage disease that results from mutations in the IDUA gene causing the accumulation of glycosaminoglycans (GAGs). Historically, children with the severe phenotype, MPS-IH (Hurler syndrome) develop progressive neurodegeneration with death in the first decade due to cardio-pulmonary complications. New data suggest that inflammation may play a role in MPS pathophysiology. To date there is almost no information on the pathophysiologic changes within the cerebral spinal fluid (CSF) of these patients. We evaluated the CSF of 25 consecutive patients with MPS-IH. While CSF glucose and total protein were within the normal range, we found a significantly mean elevated CSF opening pressure at 24 cm H2O (range 14-37 cm H2O). We observed a 3-fold elevation in CSF heparan sulfate and a 3-8 fold increase in MPS-IH specific non-reducing ends, I0S0 and I0S6. Cytokine analyses in CSF of children with MPS-IH showed significantly elevated inflammatory markers including: MCP-1 SDF-1a, IL-Ra, MIP-1b, IL-8, and VEGF in comparison to unaffected children. This is the largest report of CSF characteristics in children with MPS-IH. Identification of key biomarkers may provide further insight into the inflammatory-mediated mechanisms related to MPS diseases and perhaps lead to improved targeted therapies

The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts

In response to intracellular signals in Gram-negative bacteria, translational riboswitches—commonly embedded in messenger RNAs (mRNAs)—regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ_1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by ‘bursts’ of probe binding associated with increased SD sequence accessibility. Addition of preQ_1 decreases the lifetime of the SD’s high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation

Intrathecal enzyme replacement for Hurler syndrome: biomarker association with neurocognitive outcomes.

PurposeAbnormalities in cerebrospinal fluid (CSF) have been reported in Hurler syndrome, a fatal neurodegenerative lysosomal disorder. While no biomarker has predicted neurocognitive response to treatment, one of these abnormalities, glycosaminoglycan nonreducing ends (NREs), holds promise to monitor therapeutic efficacy. A trial of intrathecal enzyme replacement therapy (ERT) added to standard treatment enabled tracking of CSF abnormalities, including NREs. We evaluated safety, biomarker response, and neurocognitive correlates of change.MethodsIn addition to intravenous ERT and hematopoietic cell transplantation, patients (N = 24) received intrathecal ERT at four peritransplant time points; CSF was evaluated at each point. Neurocognitive functioning was quantified at baseline, 1 year, and 2 years posttransplant. Changes in CSF biomarkers and neurocognitive function were evaluated for an association.ResultsOver treatment, there were significant decreases in CSF opening pressure, biomarkers of disease activity, and markers of inflammation. Percent decrease in NRE from pretreatment to final intrathecal dose posttransplant was positively associated with percent change in neurocognitive score from pretreatment to 2 years posttransplant.ConclusionIntrathecal ERT was safe and, in combination with standard treatment, was associated with reductions in CSF abnormalities. Critically, we report evidence of a link between a biomarker treatment response and neurocognitive outcome in Hurler syndrome

Identification and characterisation of endogenous Avian Leukosis Virus subgroup E (ALVE) insertions in chicken whole genome sequencing data

Background: Endogenous retroviruses (ERVs) are the remnants of retroviral infections which can elicit prolonged genomic and immunological stress on their host organism. In chickens, endogenous Avian Leukosis Virus subgroup E (ALVE) expression has been associated with reductions in muscle growth rate and egg production, as well as providing the potential for novel recombinant viruses. However, ALVEs can remain in commercial stock due to their incomplete identification and association with desirable traits, such as ALVE21 and slow feathering. The availability of whole genome sequencing (WGS) data facilitates high-throughput identification and characterisation of these retroviral remnants. Results: We have developed obsERVer, a new bioinformatic ERV identification pipeline which can identify ALVEs in WGS data without further sequencing. With this pipeline, 20 ALVEs were identified across eight elite layer lines from Hy-Line International, including four novel integrations and characterisation of a fast feathered phenotypic revertant that still contained ALVE21. These bioinformatically detected sites were subsequently validated using new high-throughput KASP assays, which showed that obsERVer was highly precise and exhibited a 0% false discovery rate. A further fifty-seven diverse chicken WGS datasets were analysed for their ALVE content, identifying a total of 322 integration sites, over 80% of which were novel. Like exogenous ALV, ALVEs show site preference for proximity to protein-coding genes, but also exhibit signs of selection against deleterious integrations within genes. Conclusions: obsERVer is a highly precise and broadly applicable pipeline for identifying retroviral integrations in WGS data. ALVE identification in commercial layers has aided development of high-throughput diagnostic assays which will aid ALVE management, with the aim to eventually eradicate ALVEs from high performance lines. Analysis of non-commercial chicken datasets with obsERVer has revealed broad ALVE diversity and facilitates the study of the biological effects of these ERVs in wild and domesticated populations

Halogenation as a tool to tune antimicrobial activity of peptoids

Abstract Antimicrobial peptides have attracted considerable interest as potential new class of antibiotics against multi-drug resistant bacteria. However, their therapeutic potential is limited, in part due to susceptibility towards enzymatic degradation and low bioavailability. Peptoids (oligomers of N -substituted glycines) demonstrate proteolytic stability and better bioavailability than corresponding peptides while in many cases retaining antibacterial activity. In this study, we synthesized a library of 36 peptoids containing fluorine, chlorine, bromine and iodine atoms, which vary by length and level of halogen substitution in position 4 of the phenyl rings. As we observed a clear correlation between halogenation of an inactive model peptoid and its increased antimicrobial activity, we designed chlorinated and brominated analogues of a known peptoid and its shorter counterpart. Short brominated analogues displayed up to 32-fold increase of the activity against S. aureus and 16- to 64-fold against E. coli and P. aeruginosa alongside reduced cytotoxicity. The biological effect of halogens seems to be linked to the relative hydrophobicity and self-assembly properties of the compounds. By small angle X-ray scattering (SAXS) we have demontrated how the self-assembled structures are dependent on the size of the halogen, degree of substitution and length of the peptoid, and correlated these features to their activity