Structure, stability and equilibrium (un)folding of flavodoxin

Flavodoxins function as low-potential one-electron carriers using a non-covalently bound FMN cofactor which can exist in three redox states. Flavodoxin structures are characterised by a five-stranded parallel-sheet (order2-1-3-4-5) surrounded by-helices at either side of the sheet. This topology is called the flavodoxin-like fold. In contrast to most folds, the flavodoxin-like fold is shared by many protein superfamilies which are sequentially and evolutionary unrelated.Studies on proteins with the flavodoxin-like fold can therefore be utilised to find answers to the so-called protein folding problem which can be captured by the following questions:What is the physical basis of the stability of the folded protein conformation?What processes or pathways determine which of the many possible conformations is the native folded conformation adopted by the protein?What are the rules governing the relation between the amino acid sequence and three-dimensional structure of a protein?Can the three-dimensional structure of a protein be predicted from its amino acid sequence?The research described in this thesis has been carried out to obtain a better understanding of the fundamental rules describing protein folding. This thesis focuses on the structure and stability of Azotobacter vinelandii (strain ATCC 478) flavodoxin II (henceforth designated flavodoxin) in its holo- and apoform (i.e. with and without cofactor FMN, respectively). The results obtained for this particular flavodoxin are compared with those for other flavodoxins, as well as with results obtained for sequentially unrelated proteins having a flavodoxin-like fold. The understanding of the equilibrium (un)folding of flavodoxin is a first step in the characterisation of the energy landscape determining the folding of this protein.A general introduction on protein folding, NMR spectroscopy and flavodoxins is presented in Chapter 1.To prevent dimerisation during structural and folding studies Cys69Ala and Cys69Ser mutants of wild-type flavodoxin were prepared. pH-Dependent semiquinone/hydroquinone redox potentials of wild-type, Cys69Ala and Cys69Ser flavodoxin were determined using cyclic voltammetry and confirmed by EPR-monitored redox titrations. No significant differences in redox properties of wild-type, Cys69Ala and Cys69Ser flavodoxin are observed. The pH dependence of the semiquinone/ hydroquinone redox potentials can be described using a model assuming two redox-linked protonation sites with a constant redox potential at high pH of -485 ± 4 mV. The electrochemical data which are presented in Chapter 2 show that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue do not alter the dielectric properties and structure of holoflavodoxin.In Chapter 3, a new doubly sensitivity-enhanced 3D 1H- 15N TOCSY-HSQC experiment is described and analysed using the product operator formalism . The overall gain in signal-to-noise ratio obtained using this doubly sensitivity-enhanced TOCSY-HSQC pulse sequence is, compared to the standard (non-enhanced) sequence, 2.49 or 1.89 for spectra obtained for 15N-labelled or 15N-/ 13C-labelled holoflavodoxin samples, respectively. The main factors leading to the signal-to-noise enhancement are the introduction of two enhanced coherence transfer sequences, the elimination of water presaturation and the inclusion of a water flip-back pulse. Incorporation of gradients for coherence pathway selection, however, leads to a reduction in signal intensity.The determination of the solution secondary structure of holoflavodoxin is described in Chapter 4. A five-stranded parallel-sheet (2-1-3-4-5) is surrounded by five-helices. The loops extending from the carboxy termini of strands1,3 and4 are involved in FMN binding. Hydrogen/deuterium exchange experiments suggest that (i) amide proton exchange within the core of holoflavodoxin occurs via the apoform of the molecule and that (ii) exchange of the N(3)H proton of FMN only occurs when the cofactor is free in solution. The solvent inaccessibility of the non-polar environment around N(3) could, at least in part, establish the low semiquinone /hydroquinone redox potential. The amide proton exchange rates do not suggest that holoflavodoxin is divided in two subdomains as has been found for the structurally, but not sequentially, homologous protein Che Y. The amide backbone protons of 65 residues and three indole side-chain protons exchange sufficiently slowly (k ex &lt; 10 -5s -1) to be able to perform hydrogen exchange pulse labelling experiments and to study the kinetics of flavodoxin folding in great structural detail.The structural characteristics of apoflavodoxin as determined by NMR spectroscopy are presented in Chapter 5. Apoflavodoxin has a stable, well-ordered core consisting of a five-stranded parallel-sheet surrounded by five-helices. Large parts of holo- and apoflavodoxin have identical conformations and similar internal dynamics. However, the flavin binding region in apoflavodoxin exhibits considerable conformational dynamics. Flexibility is a likely prerequisite to enable the flavin to enter the interior of the apoprotein. Hydrogen/deuterium exchange measurements suggest that the stable nucleus in apoflavodoxin at least comprises residues in strands1,3,4 and5a and in helices4 and5 in the C-terminal part of the protein. We propose that this is a general feature of flavodoxins. In contrast, the stable nucleus of the sequentially unrelated proteins cutinase and Che Y which share the flavodoxin-like fold is not found in their respective C-terminal parts. The amide proton exchange results show that the stable nucleus may be found in different parts of the flavodoxin-like topology. If folding of flavodoxin is initiated with the collapse of the stable nucleus, as has been found for several other proteins, the folding pathways of structurally homologous proteins seem to be unrelated as well.Chapter 6 reflects on the research described in this thesis and combines the NMR studies, as described in Chapters 4 and 5, with equilibrium (un)folding studies on apo- and holoflavodoxin using fluorescence and circular dischroism spectroscopy which have been performed by van Mierlo et al. The following picture for equilibrium (un)folding of flavodoxin arises: (i) holoflavodoxin has a compact stable fold consisting of a five-stranded parallel-sheet surrounded by five-helices; (ii) upon release of the FMN cofactor, apoflavodoxin is formed which has a stable core but a flexible FMN binding region; (iii) in the (un)folding pathway a relatively stable apoflavodoxin folding intermediate is found which is characterised by the loss of tertiary interactions without the complete loss of secondary structure; (iv) the unfolded state of flavodoxin presumably contains some residual structure of an aromatic cluster as a remnant of helix4. The results obtained on the equilibrium (un)folding of flavodoxin are discussed with respect to the implications for the kinetics of flavodoxin folding.</p

A Parametric Study of Radiative Dipole Body Array Coil for 7 Tesla MRI

In this contribution we present numerical and experimental results of a parametric quantitative study of radiative dipole antennas in a phased array configuration for efficient body magnetic resonance imaging at 7T via parallel transmission. For magnetic resonance imaging (MRI) at ultrahigh fields (7T and higher) dipole antennas are commonly used in phased arrays, particularly for body imaging targets. This study reveals the effects of dipole positioning in the array (elevation of dipoles above the subject and inter-dipole spacing) on their mutual coupling, $B_1^{+}$ per $P_{acc}$ and $B_1^{+}$ per maximum local SAR efficiencies as well as the RF-shimming capability. The numerical and experimental results are obtained and compared for a homogeneous phantom as well as for a real human models confirmed by in-vivo experiments

Puberty Suppression in a Gender-Dysphoric Adolescent: A 22-Year Follow-Up

Puberty suppression by means of gonadotropin releasing hormone (GnRH) analogs is considered a diagnostic aid in gender dysphoric adolescents. However, there are also concerns about potential risks, such as poor outcome or post-surgical regret, adverse effects on metabolic and endocrine status, impaired increment of bone mass, and interference with brain development. This case report is on a 22-year follow-up of a female-to-male transsexual, treated with GnRH analogs at 13 years of age and considered eligible for androgen treatment at age 17, and who had gender reassignment surgery at 20 and 22 years of age. At follow-up, he indicated no regrets about his treatment. He was functioning well psychologically, intellectually, and socially; however, he experienced some feelings of sadness about choices he had made in a long-lasting intimate relationship. There were no clinical signs of a negative impact on brain development. He was physically in good health, and metabolic and endocrine parameters were within reference ranges. Bone mineral density was within the normal range for both sexes. His final height was short as compared to Dutch males; however, his body proportions were within normal range. This first report on long-term effects of puberty suppression suggests that negative side effects are limited and that it can be a useful additional tool in the diagnosis and treatment of gender dysphoric adolescents

Understanding contributors to racial and ethnic inequities in COVID-19 incidence and mortality rates

BACKGROUND: Racial inequities in Coronavirus 2019 (COVID-19) have been reported over the course of the pandemic, with Black, Hispanic/Latinx, and Native American individuals suffering higher case rates and more fatalities than their White counterparts. METHODS: We used a unique statewide dataset of confirmed COVID-19 cases across Missouri, linked with historical statewide hospital data. We examined differences by race and ethnicity in raw population-based case and mortality rates. We used patient-level regression analyses to calculate the odds of mortality based on race and ethnicity, controlling for comorbidities and other risk factors. RESULTS: As of September 10, 2020 there were 73,635 confirmed COVID-19 cases in the State of Missouri. Among the 64,526 case records (87.7% of all cases) that merged with prior demographic and health care utilization data, 12,946 (20.1%) were Non-Hispanic (NH) Black, 44,550 (69.0%) were NH White, 3,822 (5.9%) were NH Other/Unknown race, and 3,208 (5.0%) were Hispanic. Raw cumulative case rates for NH Black individuals were 1,713 per 100,000 population, compared with 2,095 for NH Other/Unknown, 903 for NH White, and 1,218 for Hispanic. Cumulative COVID-19-related death rates for NH Black individuals were 58.3 per 100,000 population, compared with 38.9 for NH Other/Unknown, 19.4 for NH White, and 14.8 for Hispanic. In a model that included insurance source, history of a social determinant billing code in the patient\u27s claims, census block travel change, population density, Area Deprivation Index, and clinical comorbidities, NH Black race (OR 1.75, 1.51-2.04, p\u3c0.001) and NH Other/Unknown race (OR 1.83, 1.36-2.46, p\u3c0.001) remained strongly associated with mortality. CONCLUSIONS: In Missouri, COVID-19 case rates and mortality rates were markedly higher among NH Black and NH Other/Unknown race than among NH White residents, even after accounting for social and clinical risk, population density, and travel patterns during COVID-19

A systematic review of higher-risk myelodysplastic syndromes clinical trials to determine the benchmark of azacitidine and explore alternative endpoints for overall survival

The hypomethylating agent azacitidine can prolong overall survival (OS) in patients with higher risk-myelodysplastic syndromes (HR-MDS) compared to conventional regimens. However, outcomes differ largely between studies, making it challenging to determine the contribution of novel therapies added to azacitidine. Further, a discrepancy is seen between complete (CR) or partial (PR) response rates and OS improvement with azacitidine, making it challenging to rely on earlier endpoints than OS. We conducted a systematic literature search and study-level systematic review of 237 clinical studies to better understand outcomes for HR-MDS patients treated with azacitidine. Pooled marrow CR was 9% (N = 2654; 95% CI: 6-13 %), CR rate was 17 % (N = 6943; 95% CI: 15-20 %), and median OS (mOS) was 18.6 months (N = 2820; 95% CI: 15.3-21.9). A weak correlation to mOS was detected with CR rate (207 patient cohorts, Pearson\u27s r = 0.315; P \u3c 0.0005), and a much stronger correlation with median progression-free survival (mPFS) (r=0.88, P = 3 × 1

Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement

Transient structures in unfolded proteins are important in elucidating the molecular details of initiation of protein folding. Recently, native and non-native secondary structure have been discovered in unfolded A. vinelandii flavodoxin. These structured elements transiently interact and subsequently form the ordered core of an off-pathway folding intermediate, which is extensively formed during folding of this α–β parallel protein. Here, site-directed spin-labelling and paramagnetic relaxation enhancement are used to investigate long-range interactions in unfolded apoflavodoxin. For this purpose, glutamine-48, which resides in a non-native α-helix of unfolded apoflavodoxin, is replaced by cysteine. This replacement enables covalent attachment of nitroxide spin-labels MTSL and CMTSL. Substitution of Gln-48 by Cys-48 destabilises native apoflavodoxin and reduces flexibility of the ordered regions in unfolded apoflavodoxin in 3.4 M GuHCl, because of increased hydrophobic interactions in the unfolded protein. Here, we report that in the study of the conformational and dynamic properties of unfolded proteins interpretation of spin-label data can be complicated. The covalently attached spin-label to Cys-48 (or Cys-69 of wild-type apoflavodoxin) perturbs the unfolded protein, because hydrophobic interactions occur between the label and hydrophobic patches of unfolded apoflavodoxin. Concomitant hydrophobic free energy changes of the unfolded protein (and possibly of the off-pathway intermediate) reduce the stability of native spin-labelled protein against unfolding. In addition, attachment of MTSL or CMTSL to Cys-48 induces the presence of distinct states in unfolded apoflavodoxin. Despite these difficulties, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin

A perturbation approach for ultrafast calculation of RF field enhancements near medical implants in MRI

Patients with medical implants often are deprived of magnetic resonance imaging examination because of safety risks. One specific risk is the enhancement of the radiofrequency fields around the medical implant potentially resulting in significant tissue heating and damage. The assessment of this enhancement is a computationally demanding task, with simulations taking hours or days to converge. Conventionally the source of the radiofrequency fields, patient anatomy, and the medical implant are simulated concurrently. To alleviate the computational burden, we reformulate a fast simulation method that views the medical implant as a small perturbation of the simulation domain without the medical implant and calculates the radiofrequency fields associated with this perturbation. Previously, this method required an extensive offline stage where the result is intractable for large simulation domains. Currently, this offline stage is no longer required and the method is completely online. The proposed method results in comparable radiofrequency fields but is orders of magnitude faster compared to standard simulation technique; the finite-difference time-domain, the finite-sums, and the finite element methods. This acceleration could enable patient-specific and potentially online radiofrequency safety assessment

Longitudinal Outcomes of Gender Identity in Children (LOGIC): study protocol for a retrospective analysis of the characteristics and outcomes of children referred to specialist gender services in the UK and the Netherlands

INTRODUCTION: Specialist gender services for children and young people (CYP) worldwide have experienced a significant increase in referrals in recent years. As rates of referrals increase, it is important to understand the characteristics and profile of CYP attending these services in order to inform treatment pathways and to ensure optimal outcomes. METHODS AND ANALYSIS: A retrospective observational study of clinical health records from specialist gender services for CYP in the UK and the Netherlands. The retrospective analysis will examine routinely collected clinical and outcome measures data including demographic, clinical, gender identity-related and healthcare resource use information. Data will be reported for each service and also compared between services. This study forms part of a wider programme of research investigating outcomes of gender identity in children (the Longitudinal Outcomes of Gender Identity in Children study). ETHICS AND DISSEMINATION: The proposed study has been approved by the Health Research Authority and London-Hampstead Research Ethics Committee as application 19/LO/0181. The study findings will be published in peer-reviewed journals and presented at both conferences and stakeholder events

Proportions of bird damage in tree fruits are higher in low-fruit-abundance contexts

Frugivorous birds impose significant costs on tree fruit growers through direct consumption of fruit and grower efforts to manage birds.We documented factors that influenced tree fruit bird damage from 2012 through 2014 with a coordinated field study in Michigan, New York, and Washington. For sweet cherries, percent bird damage was higher in 2012 compared to 2013 and 2014, in Michigan and New York compared toWashington, and in blocks with more edges adjacent to non-sweet cherry land-cover types. These patterns appeared to be associated with fruit abundance patterns; 2012 was a particularly lowyield year for tree fruits in Michigan and New York and percent bird damage was high. In addition, percent bird damage to sweet and tart cherries in Michigan was higher in landscapes with low to moderate forest cover compared to higher forest cover landscapes. \u27Honeycrisp\u27 apple blocks under utility wires were marginally more likely to have greater bird damage compared to blocks without wires. We recommend growers prepare bird management plans that consider the spatial distribution of fruit and non-fruit areas of the farm. Growers should generally expect to invest more in bird management in low-yield years, in blocks isolated from other blocks of the same crop, and in blocks where trees can provide entry to the crop for frugivorous birds

Simulation-based evaluation of SAR and flip angle homogeneity for five transmit head arrays at 14 T

INTRODUCTION: Various research sites are pursuing 14 T MRI systems. However, both local SAR and RF transmit field inhomogeneity will increase. The aim of this simulation study is to investigate the trade-offs between peak local SAR and flip angle uniformity for five transmit coil array designs at 14 T in comparison to 7 T. METHODS: Investigated coil array designs are: 8 dipole antennas (8D), 16 dipole antennas (16D), 8 loop coils (8D), 16 loop coils (16L), 8 dipoles/8 loop coils (8D8L) and for reference 8 dipoles at 7 T. Both RF shimming and k T-points were investigated by plotting L-curves of peak SAR levels vs flip angle homogeneity. RESULTS: For RF shimming, the 16L array performs best. For k T-points, superior flip angle homogeneity is achieved at the expense of more power deposition, and the dipole arrays outperform the loop coil arrays. DISCUSSION AND CONCLUSION: For most arrays and regular imaging, the constraint on head SAR is reached before constraints on peak local SAR are violated. Furthermore, the different drive vectors in k T-points alleviate strong peaks in local SAR. Flip angle inhomogeneity can be alleviated by k T-points at the expense of larger power deposition. For k T-points, the dipole arrays seem to outperform loop coil arrays