Peroxiredoxins : a model for a self-assembling nanoscale system.

The formation of large, complex structures from small building blocks through self-assembly is widely seen in proteins and provides a tool for the creation of functional nanoscale devices. However, the factors controlling protein self-assembly are complex and often poorly understood. Peroxiredoxins are a large family of proteins, many of which are able to form a variety of large structures from a small, basic unit. This assembly has been shown to be strongly influenced by the redox state of the enzyme, which functions as a switch, controlling self-assembly. This thesis uses a protein from this family, human peroxiredoxin 3 (hPrx3) as a model system to investigate whether the self-assembly properties of hPrx3 can be influenced by rational protein engineering. Three forms of hPrx3 were purified and examined. These were the wild type and two variants: a mutant (S78A) and a His-tagged form. Size exclusion chromatography showed that each form showed a different ratio of dimers and larger species. Both variants showed preference for larger species, especially in the His-tagged form. This was shown to be partially dependent on metal binding in the His-tagged form. Larger species formed from multiple rings were also identified. SAXS measurement indicated that in the wild type enzyme, higher order species were dodecameric rings. For the His-tagged variant, SAXS measurement showed that the species observed had a different structure than that of the wild type. Electron microscopy showed that higher order structures seen in both wild type hPrx3 and His-tagged hPrx3 were ring shaped, with dimensions consistent with dodecamers. A competitive assay showed that the wild type, with kcat/km values near 2 x 10⁷, consistent with published results. Both variant forms showed evidence of slightly higher activity than the wild type, indicating a link between activity and assembly. A peroxiredoxin from the thermophilic bacteria Thermus aquaticus, TaqPrx was also examined, in an attempt to investigate a peroxiredoxin capable of self-assembly at high temperatures, which would be very useful for a nanoscale device. TaqPrx was cloned, purified and examined, however, no evidence of self-assembly was observed. Protein modelling and dynamic light scattering measurement indicated that the protein purified was monomeric and had a structure. Sparse matrix crystal screening identified conditions that allowed crystal formation, although strongly diffracting crystals were not produced. A novel assay for peroxiredoxin activity was developed, and suggested that TaqPrx shows peroxiredoxin activity. This thesis shows that peroxiredoxins are a useful model system for the investigation of how protein self-assembly is controlled, and how it can be influenced by protein engineering

Deciphering the origin and evolution of Hepatitis B viruses by means of a family of non-enveloped fish viruses

Hepatitis B viruses (HBVs), which are enveloped viruses with reverse-transcribed DNA genomes, constitute the family Hepadnaviridae. An outstanding feature of HBVs is their streamlined genome organization with extensive gene overlap. Remarkably, the ∼1,100 bp open reading frame (ORF) encoding the envelope proteins is fully nested within the ORF of the viral replicase P. Here, we report the discovery of a diversified family of fish viruses, designated nackednaviruses, which lack the envelope protein gene, but otherwise exhibit key characteristics of HBVs including genome replication via protein-primed reverse-transcription and utilization of structurally related capsids. Phylogenetic reconstruction indicates that these two virus families separated more than 400 million years ago before the rise of tetrapods. We show that HBVs are of ancient origin, descending from non-enveloped progenitors in fishes. Their envelope protein gene emerged de novo, leading to a major transition in viral lifestyle, followed by co-evolution with their hosts over geologic eras

The relation of physical comorbidity and multimorbidity to fibromyalgia, widespread pain and fibromyalgia-related variables

Objective To investigate the relation of physical (non-psychological) comorbidity and multimorbidity to quantitative measures of fibromyalgia and musculoskeletal pain. Methods We studied 12, 215 patients in a research databank with quantitative measures of fibromyalgia related variables (FMV) that included binary determinations of fibromyalgia and widespread pain, and constituent variables of fibromyalgia diagnosis that included the widespread pain index (WPI), symptom severity score (SSS) and the polysymptomatic distress scale (PSD). We assessed 10 self-reported comorbid conditions, and covariates that included age, sex, body mass index, hypertension, smoking history and total household income. We used nearest neighbor matching and regression adjustment treatment effects models to measure the effect of comorbidities on FMV. Results We found a positive association between FMV and the probability of having each comorbid condition. Patients with ≥1 comorbidities had PSD, WPI and SSS increases of 3.0 (95% CI 2.7-3.3), 1.8 (95% CI 1.6-2.0) and 1.2 (95% CI 1.1-1.3) units, and an increase in FM prevalence from 20.4% to 32.6%. As the number of comorbid conditions present increased from 1 to 4 or more, PSD, WPI, SSS and fibromyalgia percent increased stepwise. For patients with ≥4 conditions, the predicted prevalence of fibromyalgia was 55.2%. Conclusion Fibromyalgia and FMV are associated with increase in the number of comorbidities, and the association can be measured quantitatively. However, the association with widespread pain and fibromyalgia may be an effect of man-made definitions of widespread pain and fibromyalgia, as comorbidity increases are also present with sub-syndromal levels of widespread pain and fibromyalgia

Peroxiredoxin is a versatile self-assembling tecton for protein nanotechnology

\u3cp\u3eThe potential for protein tectons to be used in nanotechnology is increasingly recognized, but the repertoire of stable proteins that assemble into defined shapes in response to an environmental trigger is limited. Peroxiredoxins (Prxs) are a protein family that shows an amazing array of supramolecular assemblies, making them attractive tectons. Human Prx3 (hPrx3) forms toroidal oligomers characteristic of the Prx family, but no structure has been solved to date. Here we report the first 3-D structure of this protein, derived from single-particle analysis of TEM images, establishing a dodecameric structure. This result was supported by SAXS measurements. We also present the first detailed structure of a double toroidal Prx from a higher organism determined by SPA. Guided by these structures, variants of the protein were designed to facilitate controlled assembly of protein nanostructures through the association of the toroids. We observed an enhanced population of stacked toroids, as seen by TEM; nanocages and interlocked toroids were also visible. Low pH was successfully predicted to generate long ordered nanotubes. Control over the length of the tubes was gained by adding ammonium sulfate to the assembly buffer. These versatile assembly properties demonstrate the considerable potential of hPrx3 as a tecton for protein nanotechnology.\u3c/p\u3

All-cause and cause-specific mortality in persons with fibromyalgia and widespread pain: An observational study in 35,248 persons with rheumatoid arthritis, non-inflammatory rheumatic disorders and clinical fibromyalgia

Purpose: Studies of the relation of fibromyalgia (FM) and widespread pain (WSP) to mortality have differed as to the presence or absence of an association and the extent of cause-specific mortality. However, no studies have investigated which definitions of FM and WSP associate with mortality, nor of FM mortality in other diseases. We investigated these issues and the meaning of mortality in patients with FM. Methods: We used Cox regression to study 35,248 rheumatic disease patients with up to 16 years of mortality follow-up in all patients and separately in those with diagnoses of rheumatoid arthritis (RA) (N = 26,458), non-inflammatory rheumatic disorders (NIRMD) (N = 5,167) and clinically diagnosed FM (N = 3,659). We applied 2016 FM criteria and other FM and WSP criteria to models adjusted for age and sex as well as to models that included a full range of covariates, including comorbid disease and functional status. We estimated the degree of explained of variance (R2) as a measure of predictive ability. Results: We found positive associations between al‘l definitions of FM and WSP and all-cause mortality, with relative risks (RR)s ranging from 1.19 (95%CI 1.15–1.24) for American College of Rheumatology (ACR) 1990 WSP to 1.38 (1.31–1.46) in age and sex adjusted revised 2016 criteria (FM 2016). However, in full covariate models the FM 2016 RR reduced further to 1.15 (1.09–1.22). The association with mortality was noted with RA (1.52 (1.43–1.61)), NIRMD (1.43 (1.24–1.66)) and clinical FM (1.41 (1.14–1.75) - where 37% of FM diagnosed patients did not satisfy FM 2016 criteria. In the all-patient analyses, the age and sex explained variation (R2) was 0.255, increasing to 0.264 (4.4%) when FM 2016 criteria were added, and to 0.378 in a full covariate model. Death causes related to FM 2016 status included accidents, 1.45 (1.11–1.91); diabetes 1.78 (1.16–2,71); suicide, 3.01 (1.55–5.84) and hypertensive related disorders, 3.01 (1.55–5.84). Cancer deaths were less common 0.77 (0.68–0.88). Conclusions: FM is weakly associated with mortality within all criteria definitions of FM and WSP examined (3.4% of explained variance), and across all diseases (RA, NIRMD, clinical FM) equally. Clinical and criteria-defined FM had different mortality outcomes. We found no evidence for a positive association of cancer and FM or WSP

Peroxiredoxin is a Versatile Self-Assembling Tecton for Protein Nanotechnology

The potential for protein tectons to be used in nanotechnology is increasingly recognized, but the repertoire of stable proteins that assemble into defined shapes in response to an environmental trigger is limited. Peroxiredoxins (Prxs) are a protein family that shows an amazing array of supramolecular assemblies, making them attractive tectons. Human Prx3 (hPrx3) forms toroidal oligomers characteristic of the Prx family, but no structure has been solved to date. Here we report the first 3-D structure of this protein, derived from single-particle analysis of TEM images, establishing a dodecameric structure. This result was supported by SAXS measurements. We also present the first detailed structure of a double toroidal Prx from a higher organism determined by SPA. Guided by these structures, variants of the protein were designed to facilitate controlled assembly of protein nanostructures through the association of the toroids. We observed an enhanced population of stacked toroids, as seen by TEM; nanocages and interlocked toroids were also visible. Low pH was successfully predicted to generate long ordered nanotubes. Control over the length of the tubes was gained by adding ammonium sulfate to the assembly buffer. These versatile assembly properties demonstrate the considerable potential of hPrx3 as a tecton for protein nanotechnology

Physics Opportunities for the Fermilab Booster Replacement

This white paper presents opportunities afforded by the Fermilab Booster Replacement and its various options. Its goal is to inform the design process of the Booster Replacement about the accelerator needs of the various options, allowing the design to be versatile and enable, or leave the door open to, as many options as possible. The physics themes covered by the paper include searches for dark sectors and new opportunities with muons