Crystal Structure Investigations of Small Heat Shock Proteins and Serine Palmitoyltransferase

The content of this thesis concerns the pursuit of protein structures from cradle to grave. This process is illustrated though the progress made on two separate proteins. The first is the serine palmitoyltransferase from Toxoplasma gondii (TgSPT). This protein is of interest as sphingolipid biosynthesis in apicomplexan parasites is currently the subject of study as a possible drug target, most notably in Plasmodium. We begin by looking at the techniques of bioinformatics used to analyse the amino acid sequence of this protein, and by calculation and comparison with other known protein sequences we are able to begin to identify the different domains of the protein and also to begin to identify their respective functions. Bioinformatic techniques are then further used in order to design constructs to be moved forward for overproduction in vivo. A number of different constructs showing significant soluble expression of various truncations of TgSPT are reported, and various techniques relating to overexpression an purification are considered, including high-throughput screening work undertaken at the Oxford Protein Production Facility. The second protein looked at is the small heat shock protein from Methanococcus jannaschii (MjHSP). This protein is used as a more tractable analogue for the human α-B-crystallin. We look here at the wild type protein as well as a mutant mimicking the disease-causing R120G mutation in α-B-crystallin, R107G, and also two different truncation mutants, mimicking the Q151X truncation. The processes relating to crystal growth and optimisation, to data collection, and to data analysis and structure solution are then considered. Finally, a redetermined structure of wild type MjHSP at significantly increased resolution, and the structure of the R107G mutant are reported

The Ursinus Weekly, October 27, 1947

Thespians to stage New York success • Sophs plan dances, class doggie roast • Gen. Arnold makes demand for greater accent on sciences in college curricula • AVC appoints leaders; starts campus clean-up • Ye good olde days in prospect again as school spirit re-appears on campus • Legal society fetes new members; Vice-president Helfferich speaks • May queen to be selected early this semester, Nov. 6 • Gym construction near end; plans for interior finished • Beardwood chemical group elects Pfeiffer president • Reporters added to staff; Lois Cain new sports assistant • Y commission slate opens with discussions, address • New Spanish Club elects officers • Dorms vote to fill WSGA berths • Pre-meds to hear psychiatrist • Aid for Europe? • Grizzlies to play host to victory-hungry PMC • Soccer team bows to Rutgers \u2711\u27, 6-2 • Three teams tied for first in campus football league • Swarthmore interception sets up tally; first frame score gives Garnet 7-0 win • JV hockey team tops Moravian • JV booters thump Hill School • Snell\u27s belles win third straight, 6-1 • Dr. Child addresses English Club • German Club organizes for 1947 • French Club plans activities • New FTA members to be inductedhttps://digitalcommons.ursinus.edu/weekly/1624/thumbnail.jp

The Ursinus Weekly, October 13, 1947

Bears chalk up second straight win, defeat Haverford 6-0: Blydenburg takes pass for touchdown; Gridders maintain unscored-upon pace • General Arnold visits Ursinus for Founders\u27 Day ceremony • Leroy Grayson heads new inter-frat council • Nite game bus tickets available • Swarthmore man guest at AVC meeting tonite • Indian will address forum, Y, on relief • Annex student directs new campus orchestra • Dance and pep rally boost morale on eve before game • Curtain Club holds reception; New members admitted to ranks • Beaver students in food-saving plan similar to Ursinus\u27 • Philosophy student discovers panacea for all problems after two-week study • Hohlfeld to discuss problems of education in British Guiana • Campus briefs: Recorded concerts; Zeta Chi meeting; Inter-frat Council • Annex-dotes • Alumni-society notes • Radium expert enlivens address with experiments, illustrations • Bears to face heavy Moravian 11 Friday • Soccer team bows to Lafayette, 4-1, in opening contest • Campus football resumes, Luther Wilt heads league • Bearettes win practice match • Brotherhood to lead services • Player of the week • Speakers obtained by pre-legal group • Debates start next month • Former Army nurse returns to campus after extensive service in Europe, Asia • Betsy Greene heads English Clubhttps://digitalcommons.ursinus.edu/weekly/3123/thumbnail.jp

Risk algorithm using serial biomarker measurements doubles the number of screen-detected cancers compared with a single-threshold rule in the United Kingdom collaborative trial of ovarian cancer screening

PURPOSE: Cancer screening strategies have commonly adopted single-biomarker thresholds to identify abnormality. We investigated the impact of serial biomarker change interpreted through a risk algorithm on cancer detection rates. PATIENTS AND METHODS: In the United Kingdom Collaborative Trial of Ovarian Cancer Screening, 46,237 women, age 50 years or older underwent incidence screening by using the multimodal strategy (MMS) in which annual serum cancer antigen 125 (CA-125) was interpreted with the risk of ovarian cancer algorithm (ROCA). Women were triaged by the ROCA: normal risk, returned to annual screening; intermediate risk, repeat CA-125; and elevated risk, repeat CA-125 and transvaginal ultrasound. Women with persistently increased risk were clinically evaluated. All participants were followed through national cancer and/or death registries. Performance characteristics of a single-threshold rule and the ROCA were compared by using receiver operating characteristic curves. RESULTS: After 296,911 women-years of annual incidence screening, 640 women underwent surgery. Of those, 133 had primary invasive epithelial ovarian or tubal cancers (iEOCs). In all, 22 interval iEOCs occurred within 1 year of screening, of which one was detected by ROCA but was managed conservatively after clinical assessment. The sensitivity and specificity of MMS for detection of iEOCs were 85.8% (95% CI, 79.3% to 90.9%) and 99.8% (95% CI, 99.8% to 99.8%), respectively, with 4.8 surgeries per iEOC. ROCA alone detected 87.1% (135 of 155) of the iEOCs. Using fixed CA-125 cutoffs at the last annual screen of more than 35, more than 30, and more than 22 U/mL would have identified 41.3% (64 of 155), 48.4% (75 of 155), and 66.5% (103 of 155), respectively. The area under the curve for ROCA (0.915) was significantly (P = .0027) higher than that for a single-threshold rule (0.869). CONCLUSION: Screening by using ROCA doubled the number of screen-detected iEOCs compared with a fixed cutoff. In the context of cancer screening, reliance on predefined single-threshold rules may result in biomarkers of value being discarded

Synergism of anisotropic and computational NMR methods reveals the likely configuration of phormidolide A.

Characterization of the complex molecular scaffold of the marine polyketide natural product phormidolide A represents a challenge that has persisted for nearly two decades. In light of discordant results arising from recent synthetic and biosynthetic reports, a rigorous study of the configuration of phormidolide A was necessary. This report outlines a synergistic effort employing computational and anisotropic NMR investigation, that provided orthogonal confirmation of the reassigned side chain, as well as supporting a further correction of the C7 stereocenter

A core outcome set for localised prostate cancer effectiveness trials

Objective: To develop a core outcome set (COS) applicable for effectiveness trials of all interventions for localised prostate cancer. Background: Many treatments exist for localised prostate cancer, although it is unclear which offers the optimal therapeutic ratio. This is confounded by inconsistencies in the selection, definition, measurement and reporting of outcomes in clinical trials. Subjects and methods: A list of 79 outcomes was derived from a systematic review of published localised prostate cancer effectiveness studies and semi-structured interviews with 15 prostate cancer patients. A two-stage consensus process involving 118 patients and 56 international healthcare professionals (HCPs) (cancer specialist nurses, urological surgeons and oncologists) was undertaken, consisting of a three-round Delphi survey followed by a face-to-face consensus panel meeting of 13 HCPs and 8 patients. Results: The final COS included 19 outcomes. Twelve apply to all interventions: death from prostate cancer, death from any cause, local disease recurrence, distant disease recurrence/metastases, disease progression, need for salvage therapy, overall quality of life, stress urinary incontinence, urinary function, bowel function, faecal incontinence, sexual function. Seven were intervention-specific: perioperative deaths (surgery), positive surgical margin (surgery), thromboembolic disease (surgery), bothersome or symptomatic urethral or anastomotic stricture (surgery), need for curative treatment (active surveillance), treatment failure (ablative therapy), and side effects of hormonal therapy (hormone therapy). The UK-centric participants may limit the generalisability to other countries, but trialists should reason why the COS would not be applicable. The default position should not be that a COS developed in one country will automatically not be applicable elsewhere. Conclusion: We have established a COS for trials of effectiveness in localised prostate cancer, applicable across all interventions which should be measured in all localised prostate cancer effectiveness trials

FLow and Benthic ECology 4D – FLOWBEC – an overview

This work is funded by NERC/DEFRA (grants NE/J004332/1, NE/J004308/1, NE/J004200/1, NE/J004359/1, NE/J004316/1, NE/J004219/1, NE/J00426X/1, NE/J004294/1). We also like to acknowledge OpenHydro Ltd and Atlantis Resources Ltd for allowing the placement of the FLOWBEC frame in close proximity to their installations at EMEC, and Marine Scotland Science for their support developing and deploying the FLOWBEC frame.Publisher PD

Tumour stage, treatment, and survival of women with high-grade serous tubo-ovarian cancer in UKCTOCS: an exploratory analysis of a randomised controlled trial

Background: In UKCTOCS, there was a decrease in the diagnosis of advanced stage tubo-ovarian cancer but no reduction in deaths in the multimodal screening group compared with the no screening group. Therefore, we did exploratory analyses of patients with high-grade serous ovarian cancer to understand the reason for the discrepancy.// Methods: UKCTOCS was a 13-centre randomised controlled trial of screening postmenopausal women from the general population, aged 50–74 years, with intact ovaries. The trial management system randomly allocated (2:1:1) eligible participants (recruited from April 17, 2001, to Sept 29, 2005) in blocks of 32 using computer generated random numbers to no screening or annual screening (multimodal screening or ultrasound screening) until Dec 31, 2011. Follow-up was through national registries until June 30, 2020. An outcome review committee, masked to randomisation group, adjudicated on ovarian cancer diagnosis, histotype, stage, and cause of death. In this study, analyses were intention-to-screen comparisons of women with high-grade serous cancer at censorship (Dec 31, 2014) in multimodal screening versus no screening, using descriptive statistics for stage and treatment endpoints, and the Versatile test for survival from randomisation. This trial is registered with the ISRCTN Registry, 22488978, and ClinicalTrials.gov, NCT00058032.// Findings: 202 562 eligible women were recruited (50 625 multimodal screening; 50 623 ultrasound screening; 101 314 no screening). 259 (0·5%) of 50 625 participants in the multimodal screening group and 520 (0·5%) of 101 314 in the no screening group were diagnosed with high-grade serous cancer. In the multimodal screening group compared with the no screening group, fewer were diagnosed with advanced stage disease (195 [75%] of 259 vs 446 [86%] of 520; p=0·0003), more had primary surgery (158 [61%] vs 219 [42%]; p<0·0001), more had zero residual disease following debulking surgery (119 [46%] vs 157 [30%]; p<0·0001), and more received treatment including both surgery and chemotherapy (192 [74%] vs 331 [64%]; p=0·0032). There was no difference in the first-line combination chemotherapy rate (142 [55%] vs 293 [56%]; p=0·69). Median follow-up from randomisation of 779 women with high-grade serous cancer in the multimodal and no screening groups was 9·51 years (IQR 6·04–13·00). At censorship (June 30, 2020), survival from randomisation was longer in women with high-grade serous cancer in the multimodal screening group than in the no screening group with absolute difference in survival of 6·9% (95% CI 0·4–13·0; p=0·042) at 18 years (21% [95% CI 15·6–26·2] vs 14% [95% CI 10·5–17·4]).// Interpretation: To our knowledge, this is the first evidence that screening can detect high-grade serous cancer earlier and lead to improved short-term treatment outcomes compared with no screening. The potential survival benefit for women with high-grade serous cancer was small, most likely due to only modest gains in early detection and treatment improvement, and tumour biology. The cumulative results of the trial suggest that surrogate endpoints for disease-specific mortality should not currently be used in screening trials for ovarian cancer

Tumour stage, treatment, and survival of women with high-grade serous tubo-ovarian cancer in UKCTOCS: an exploratory analysis of a randomised controlled trial

Background: In UKCTOCS, there was a decrease in the diagnosis of advanced stage tubo-ovarian cancer but no reduction in deaths in the multimodal screening group compared with the no screening group. Therefore, we did exploratory analyses of patients with high-grade serous ovarian cancer to understand the reason for the discrepancy. Methods: UKCTOCS was a 13-centre randomised controlled trial of screening postmenopausal women from the general population, aged 50–74 years, with intact ovaries. The trial management system randomly allocated (2:1:1) eligible participants (recruited from April 17, 2001, to Sept 29, 2005) in blocks of 32 using computer generated random numbers to no screening or annual screening (multimodal screening or ultrasound screening) until Dec 31, 2011. Follow-up was through national registries until June 30, 2020. An outcome review committee, masked to randomisation group, adjudicated on ovarian cancer diagnosis, histotype, stage, and cause of death. In this study, analyses were intention-to-screen comparisons of women with high-grade serous cancer at censorship (Dec 31, 2014) in multimodal screening versus no screening, using descriptive statistics for stage and treatment endpoints, and the Versatile test for survival from randomisation. This trial is registered with the ISRCTN Registry, 22488978, and ClinicalTrials.gov, NCT00058032. Findings: 202 562 eligible women were recruited (50 625 multimodal screening; 50 623 ultrasound screening; 101 314 no screening). 259 (0·5%) of 50 625 participants in the multimodal screening group and 520 (0·5%) of 101 314 in the no screening group were diagnosed with high-grade serous cancer. In the multimodal screening group compared with the no screening group, fewer were diagnosed with advanced stage disease (195 [75%] of 259 vs 446 [86%] of 520; p=0·0003), more had primary surgery (158 [61%] vs 219 [42%]; p<0·0001), more had zero residual disease following debulking surgery (119 [46%] vs 157 [30%]; p<0·0001), and more received treatment including both surgery and chemotherapy (192 [74%] vs 331 [64%]; p=0·0032). There was no difference in the first-line combination chemotherapy rate (142 [55%] vs 293 [56%]; p=0·69). Median follow-up from randomisation of 779 women with high-grade serous cancer in the multimodal and no screening groups was 9·51 years (IQR 6·04–13·00). At censorship (June 30, 2020), survival from randomisation was longer in women with high-grade serous cancer in the multimodal screening group than in the no screening group with absolute difference in survival of 6·9% (95% CI 0·4–13·0; p=0·042) at 18 years (21% [95% CI 15·6–26·2] vs 14% [95% CI 10·5–17·4]). Interpretation: To our knowledge, this is the first evidence that screening can detect high-grade serous cancer earlier and lead to improved short-term treatment outcomes compared with no screening. The potential survival benefit for women with high-grade serous cancer was small, most likely due to only modest gains in early detection and treatment improvement, and tumour biology. The cumulative results of the trial suggest that surrogate endpoints for disease-specific mortality should not currently be used in screening trials for ovarian cancer. Funding: National Institute for Health Research, Medical Research Council, Cancer Research UK, The Eve Appeal

Warm ice giant GJ 3470b - II. Revised planetary and stellar parameters from optical to near-infrared transit photometry

It is important to explore the diversity of characteristics of low-mass, low-density planets to understand the nature and evolution of this class of planets. We present a homogeneous analysis of 12 new and 9 previously published broad-band photometric observations of the Uranus-sized extrasolar planet GJ 3470b, which belongs to the growing sample of sub-Jovian bodies orbiting M dwarfs. The consistency of our analysis explains some of the discrepancies between previously published results and provides updated constraints on the planetary parameters. Our data are also consistent with previous transit observations of this system. The physical properties of the transiting system can only be constrained as well as the host star is characterized, so we provide new spectroscopic measurements of GJ 3470 from 0.33 to 2.42 μm to aid our analysis. We find R* = 0.48 ± 0.04 R⊙, M* = 0.51 ± 0.06 M⊙, and T_(eff) = 3652 ± 50K for GJ 3470, along with a rotation period of 20.70 ± 0.15 d and an R-band amplitude of 0.01 mag, which is small enough that current transit measurements should not be strongly affected by stellar variability. However, to report definitively whether stellar activity has a significant effect on the light curves, this requires future multiwavelength, multi-epoch studies of GJ 3470. We also present the most precise orbital ephemeris for this system: To = 2455983.70472 ± 0.00021BJD_(TDB), P = 3.336 6487^(+0.0000043)_(−0.0000033)  d, and we see no evidence for transit timing variations greater than 1 min. Our reported planet to star radius ratio is 0.076 42 ± 0.000 37. The physical parameters of this planet are R_p = 3.88 ± 0.32 R⊕ and M_p = 13.73 ± 1.61 M⊕. Because of our revised stellar parameters, the planetary radius we present is smaller than previously reported values. We also perform a second analysis of the transmission spectrum of the entire ensemble of transit observations to date, supporting the existence of an H_2-dominated atmosphere exhibiting a strong Rayleigh scattering slope