Crystallographic Studies on Integral Membrane Light Harvesting Complexes From Photosynthetic Bacteria

The three dimensional crystal structure of the B800-820 light harvesting complex, an integral membrane pigment-protein complex, from the photosynthetic bacterium Rhodopseudomonas acidophila strain 7050, has been determined to a resolution of 2.8A. This thesis outlines the processes by which the structure was solved; gives an initial comparison between it and the crystal structure of the B800-850 LH complex from Rhodopseudomonas acidophila strain 100501; and suggests reasons for the observed spectroscopic differences between the two complexes. Additionally, biochemical investigations performed on a further three light harvesting complexes are described, and a novel spectroscopic method for monitoring the purity of the complexes is introduced

How do nurses facilitate shared decision making in HIV care?: an exploratory study of UK nurses knowledge, perspective and experience of facilitating shared decision making in clinical practice

Shared Decision Making (SDM) enables clinicians to promote self-management and to empower patients with long-term health conditions. Whilst it has been reported in other health conditions, there is little empirical research on the nature and practice of SDM in HIV care. This project aimed to explore current views and practices amongst UK HIV Nurses regarding SDM. A mixed methods study utilised focus groups and an online survey to identify opportunities, challenges and training needs. Nurses are supportive of SDM but face patient related, organisation and health system challenges to implement it, especially in supporting complex patients. SDM is an important aspect of nursing care for people living with HIV. Nurses need more training and resources to implement SDM effectively. In order to develop such training and resources to better understand SDM in relation to HIV care, there is a need for research on patient perspectives and experiences in this area

Substrate specificity and the effect of calcium on Trypanosomabrucei metacaspase 2

Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca<sup>2+</sup> on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has an unambiguous preference for basic amino acids at the P<sub>1</sub> position of peptide substrates and that this is most probably a result of hydrogen bonding from the P<sub>1</sub> residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P<sub>2</sub> and P<sub>3</sub>positions and for small residues at the prime side of a peptide substrate. Studies into the effects of Ca<sup>2+</sup> on the enzyme revealed the presence of two Ca<sup>2+</sup> binding sites and a reversible structural modification of the enzyme upon Ca<sup>2+</sup> binding. In addition, the concentration of Ca<sup>2+</sup> used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P<sub>2</sub> were examined, suggesting that Ca<sup>2+</sup>activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S2 binding pocket. Collectively, these data give new insights into the substrate specificity and Ca<sup>2+</sup> activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes and make attractive drug targets due to their absence in humans

A participatory approach to developing the HIV Nursing Research Strategy

As an illness that has changed dramatically over the past three decades, HIV has kept healthcare professionals working in the field engaged and challenged. From the caution and concerns of an emerging infection in the early 1980s providing palliative and end-of-life care, through the development of successful drug therapies, and finally to its identification as a long-term health condition, health professionals caring for people with HIV have had to adapt their knowledge and skills to meet their client group. Nurses have played an important role in the adaptation of care priorities and knowledge acquisition though the delivery of evidence-based clinical practice and research development when the evidence did not exist. This article outlines how the National HIV Nurses Association (NHIVNA) in the UK explored the research priorities for nurses in the changing environment of HIV nursing care and management in order to develop a progressive nursing research strategy

Hormonal control of gubernaculum development during testis descent: gubernaculum outgrowth in vitro requires both insulin-like factor and androgen

The gubernaculum connects the gonad to the inguinoscrotal region and is involved in testis descent. It rapidly develops in the male fetus, whereas development in the female fetus is lacking. Possible factors involved in gubernaculum development are androgens, anti-Mullerian hormone (AMH), and insulin-like factor (Insl3). Sexual dimorphism in gubernaculum development correlated with the mitotic activity of cells in the gubernacular bulbs from male and female fetuses. Androgen receptor expression was restricted to the mesenchymal core of the gubernacular bulb, whereas skeletal muscle was detected in its outer layer. In an organ culture system devised to further study gubernaculum development in vitro, morphology of gubernacular explants grown in the presence of testes was comparable with that of gubernacula developed in vivo. Testicular tissue or medium containing R1881, a synthetic androgen, had a growth stimulatory effect on gubernacular explants compared with ovarian tissue or basal medium only. Moreover, Amh-/-, Amh+/-, and Insl3+/- testes stimulated the growth of gubernacular explants to the same extent as control testes. Insl3-/- testes, however, did not produce such an activity. This study reveals an essential role for both androgen and Insl3 in the gubernaculum outgrowth during transabdominal testis descent

Ranking metabolite sets by their activity levels

Related metabolites can be grouped into sets in many ways, e.g., by their participation in series of chemical reactions (forming metabolic pathways), or based on fragmentation spectral similarities or shared chemical substructures. Understanding how such metabolite sets change in relation to experimental factors can be incredibly useful in the interpretation and understanding of complex metabolomics data sets. However, many of the available tools that are used to perform this analysis are not entirely suitable for the analysis of untargeted metabolomics measurements. Here, we present PALS (Pathway Activity Level Scoring), a Python library, command line tool, and Web application that performs the ranking of significantly changing metabolite sets over different experimental conditions. The main algorithm in PALS is based on the pathway level analysis of gene expression (PLAGE) factorisation method and is denoted as mPLAGE (PLAGE for metabolomics). As an example of an application, PALS is used to analyse metabolites grouped as metabolic pathways and by shared tandem mass spectrometry fragmentation patterns. A comparison of mPLAGE with two other commonly used methods (overrepresentation analysis (ORA) and gene set enrichment analysis (GSEA)) is also given and reveals that mPLAGE is more robust to missing features and noisy data than the alternatives. As further examples, PALS is also applied to human African trypanosomiasis, Rhamnaceae, and American Gut Project data. In addition, normalisation can have a significant impact on pathway analysis results, and PALS offers a framework to further investigate this. PALS is freely available from our project Web site

Inhibin interferes with activin signaling at the level of the activin receptor complex in Chinese hamster ovary cells

To gain more insight in the mechanism of action of inhibin, we studied the effect of inhibin on activin signaling in Chinese hamster ovary cells. Inhibin specifically counteracted activin-induced expression of a plasminogen activator inhibitor 1 promoter element (3TP) and of the junB gene, but was ineffective when the responses were induced by transforming growth factor-beta. This indicates that inhibin acts only on the activin-specific part of these signaling cascades. Using a constitutively active activin type IB receptor we determined whether inhibin acted at the level of the activin-receptor complex or downstream of it. The mutant activin receptor stimulated the expression of the 3TP promoter in the absence of activin. This stimulation was insensitive to inhibin

Association between an AMH promoter polymorphism and serum AMH levels in PCOS patients

STUDY QUESTION: Do polymorphisms in the anti-Müllerian hormone (AMH) promoter have an effect on AMH levels in patients with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: We have identified a novel AMH promoter polymorphism rs10406324 that is associated with lower serum AMH levels and is suggested to play a role in the mechanism of regulation of AMH gene expression in women. WHAT IS KNOWN ALREADY: Follicle number is positively correlated with serum AMH levels, reflected by elevated AMH levels in women with PCOS. In addition, it is suggested that AMH production per follicle is higher in women with PCOS than in normo-ovulatory women, implying an altered regulation of AMH in PCOS. STUDY DESIGN, SIZE, DURATION: A discovery cohort of 655 PCOS women of Northern European ancestry and both an internal and external validation PCOS cohort (n = 458 and n = 321, respectively) were included in this study. Summary-level data of an AMH genome-wide association study meta-analysis including 7049 normo-ovulatory women was included as a control cohort. A genetic approach was taken through association analysis and in silico analysis of the associated variants in the AMH promoter. In vitro analysis was performed to investigate the functional mechanisms. PARTICIPANTS/MATERIALS, SETTING, METHODS: All common two-allelic single-nucleotide polymorphisms (SNPs) in the region Chr19:2 245 353–2 250 827 bp (Build 37) were selected for the analysis. Linear regression analyses were performed to determine the association between SNPs in the AMH promoter region and serum AMH levels. For the in silico analysis, the webtools ‘HaploReg’ v4.1 for ENCODE prediction weight matrices and ‘atSNP’ were used. In vitro analysis was performed using KK1 cells, a mouse granulosa cell line and COV434 cells, a human granulosa tumor cell line. Cells were transfected with the reference or the variant human AMH promoter reporter construct together with several transcription factors (TFs). Dual-Glo(®) Luciferase Assay was performed to measure the luciferase activity. MAIN RESULTS AND THE ROLE OF CHANCE: Polymorphism rs10406324 was significantly associated with serum AMH levels in all three PCOS cohorts. Carriers of the minor allele G had significantly lower log-transformed serum AMH levels compared to non-carriers (P = 8.58 × 10(−8), P = 1.35 × 10(−3) and P = 1.24 × 10(−3), respectively). This result was validated in a subsequent meta-analysis (P = 3.24 × 10(−12)). Interestingly, rs10406324 was not associated with follicle count, nor with other clinical traits. Also, in normo-ovulatory women, the minor allele of this variant was associated with lower serum AMH levels (P = 1.04 × 10(−5)). These findings suggest that polymorphism rs10406324 plays a role in the regulation of AMH expression, irrespective of clinical background. In silico analysis suggested a decreased binding affinity of the TFs steroidogenenic factor 1, estrogen-related receptor alpha and glucocorticoid receptor to the minor allele G variant, however in vitro analysis did not show a difference in promoter activity between the A and G allele. LIMITATIONS, REASONS FOR CAUTION: Functional analyses were performed in a mouse and a human granulosa cell line using an AMH promoter reporter construct. This may have limited assessment of the impact of the polymorphism on higher order chromatin structures. Human granulosa cells generated from induced pluripotent stem cells, combined with gene editing, may provide a method to elucidate the exact mechanism behind the decrease in serum AMH levels in carriers of the −210 G allele. We acknowledge that the lack of follicle number in the external validation and the control cohort is a limitation of the paper. Although we observed that the association between rs10406324 and AMH levels was independent of follicle number in our discovery and internal validation PCOS cohorts, we cannot fully rule out that the observed effects on serum AMH levels are, in part, caused by differences in follicle number. WIDER IMPLICATIONS OF THE FINDINGS: These results suggest that variations in serum AMH levels are not only caused by differences in follicle number but also by genetic factors. Therefore, the genetic context should be taken into consideration when assessing serum AMH levels in women. This may have clinical consequences when serum AMH levels are used as a marker for the polycystic ovarian morphology phenotype. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used. J.S.E.L. has received consultancy fees from the following companies: Ferring, Roche Diagnostics and Ansh Labs and has received travel reimbursement from Ferring. J.A.V. has received royalties from AMH assays, paid to the institute/lab with no personal financial gain. The other authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A

Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome.

Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7 Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well-ordered break in the polypeptide chain at K147, resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed K147 to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an auto-inhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts both Arg and Lys substrates and does not require Ca2+ for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms