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| 2. |
- Drake, TM, et al.
(author)
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Surgical site infection after gastrointestinal surgery in children: an international, multicentre, prospective cohort study
- 2020
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In: BMJ global health. - : BMJ. - 2059-7908. ; 5:12
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Journal article (peer-reviewed)abstract
- Surgical site infection (SSI) is one of the most common healthcare-associated infections (HAIs). However, there is a lack of data available about SSI in children worldwide, especially from low-income and middle-income countries. This study aimed to estimate the incidence of SSI in children and associations between SSI and morbidity across human development settings.MethodsA multicentre, international, prospective, validated cohort study of children aged under 16 years undergoing clean-contaminated, contaminated or dirty gastrointestinal surgery. Any hospital in the world providing paediatric surgery was eligible to contribute data between January and July 2016. The primary outcome was the incidence of SSI by 30 days. Relationships between explanatory variables and SSI were examined using multilevel logistic regression. Countries were stratified into high development, middle development and low development groups using the United Nations Human Development Index (HDI).ResultsOf 1159 children across 181 hospitals in 51 countries, 523 (45·1%) children were from high HDI, 397 (34·2%) from middle HDI and 239 (20·6%) from low HDI countries. The 30-day SSI rate was 6.3% (33/523) in high HDI, 12·8% (51/397) in middle HDI and 24·7% (59/239) in low HDI countries. SSI was associated with higher incidence of 30-day mortality, intervention, organ-space infection and other HAIs, with the highest rates seen in low HDI countries. Median length of stay in patients who had an SSI was longer (7.0 days), compared with 3.0 days in patients who did not have an SSI. Use of laparoscopy was associated with significantly lower SSI rates, even after accounting for HDI.ConclusionThe odds of SSI in children is nearly four times greater in low HDI compared with high HDI countries. Policies to reduce SSI should be prioritised as part of the wider global agenda.
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| 3. |
- Nomura, Norimichi, et al.
(author)
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Structure and mechanism of the mammalian fructose transporter GLUT5
- 2015
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 526:7573, s. 397-
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Journal article (peer-reviewed)abstract
- The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward-and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a 'gated-pore' transport mechanism in such monosaccharide transporters.
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| 4. |
- Aziz-Qureshi, Abdul, et al.
(author)
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The MEMbrane Protein Single ShoT Amplification Recipe : MemStar
- 2017
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In: A Structure-Function Toolbox for Membrane Transporter and Channels. - San Diego : Elsevier. - 9780128123539 ; , s. 123-138
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Book chapter (peer-reviewed)abstract
- Here, we present a simple overexpression condition for high-throughput screening of membrane proteins in Escherichia coli. For the vast majority of bacterial membrane protein targets tested the MEMbrane protein Single shoT Amplification Recipe-MemStarleads to high production yields of target protein. The use of MemStar has facilitated structural studies of several transport proteins.
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| 5. |
- Lee, Chiara, et al.
(author)
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MemStar : A one-shot Escherichia coli-based approach for high-level bacterial membrane protein production
- 2014
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In: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 588:20, s. 3761-3769
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Journal article (peer-reviewed)abstract
- Optimising membrane protein production yields in Escherichia coli can be time- and resource-consuming. Here, we present a simple and effective Membrane protein Single shot amplification recipe: MemStar. This one-shot amplification recipe is based on the E. coli strain Lemo21(DE3), the PASM-5052 auto-induction medium and, contradictorily, an IPTG induction step. Using MemStar, production yields for most bacterial membrane proteins tested were improved to reach an average of 5 mg L-1 per OD600 unit, which is significantly higher than yields obtained with other common production strategies. With MemStar, we have been able to obtain new structural information for several transporters, including the sodium/proton antiporter NapA. (C) 2014 Federation of European Biochemical Societies.
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| 6. |
- Nji, Emmanuel, et al.
(author)
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Structural basis for the delivery of activated sialic acid into Golgi for sialyation
- 2019
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In: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 26:6, s. 415-423
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Journal article (peer-reviewed)abstract
- The decoration of secretory glycoproteins and glycolipids with sialic acid is critical to many physiological and pathological processes. Sialyation is dependent on a continuous supply of sialic acid into Golgi organelles in the form of CMP-sialic acid. Translocation of CMP-sialic acid into Golgi is carried out by the CMP-sialic acid transporter (CST). Mutations in human CST are linked to glycosylation disorders, and CST is important for glycopathway engineering, as it is critical for sialyation efficiency of therapeutic glycoproteins. The mechanism of how CMP-sialic acid is recognized and translocated across Golgi membranes in exchange for CMP is poorly understood. Here we have determined the crystal structure of a Zea mays CST in complex with CMP. We conclude that the specificity of CST for CMP-sialic acid is established by the recognition of the nucleotide CMP to such an extent that they are mechanistically capable of both passive and coupled antiporter activity.
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| 7. |
- Qureshi, Abdul Aziz, 1984-
(author)
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Establishing the mechanistic basis of sugar transport
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Sugar is a vital molecule required for cell viability and homeostasis. Sugar is important for metabolic energy, energy storage, signaling, structure and osmolyte regulation. Transport of sugar represents an important physiological process. Specific membrane transporter families have evolved to mediate the transport of sugar across biological membranes. In this thesis, we describe our work leading to a better mechanistic understanding of two sugar transporter families, namely glucose (GLUT) transporters and nucleotide-sugar (NST) transporters.Members of GLUT transporters, belonging to the Solute Carrier (SLC2) family, are involved in the uptake of various monosaccharides across the cellular membranes. Activity of different NSTs, belonging to the (SLC35) family, is crucial for the process of glycosylation by mediating the translocation of activated sugars from the cytoplasm into the lumen of either Golgi and/or ER organelles. GLUTs and NSTs families carry out transport processes fundamental to human physiology and pathophysiology. Despite the profound importance of GLUTs and NSTs in human health, comprehensive understanding of their architecture and mechanistic features with respect to determinants of substrate binding and allosteric coupling at the molecular level has remained elusive.In this thesis, we address key functional and structural properties of GLUT and NST mediated sugar transport. We combine crystal structures with robust binding and transport assays as well as computational approaches. The role of lipids in fine-tuning the activity of transporters is also exemplified by demonstrating the effect of lipid composition in the transport activity of GLUTs using in-vitro proteoliposome assays. Our work has not only enhanced the current understanding of GLUT and NST function, but also developed themes and methods that are likely relevant to many types of small molecule transporters.
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| 10. |
- Qureshi, Abdul Aziz, et al.
(author)
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The molecular basis for sugar import in malaria parasites
- 2020
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 578:7794, s. 321-325
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Journal article (peer-reviewed)abstract
- Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with d-glucose at a resolution of 3.6 Å. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 Å from d-glucose) are just as critical for transport as the residues that directly coordinate d-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics.
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| 11. |
- Stsiapanava, Alena, et al.
(author)
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Product formation controlled by substrate dynamics in leukotriene A4 hydrolase.
- 2014
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002. ; 1844:2, s. 439-446
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Journal article (peer-reviewed)abstract
- Leukotriene A4 hydrolase/aminopeptidase (LTA4H) (EC 3.3.2.6) is a bifunctional zinc metalloenzyme with both an epoxide hydrolase and an aminopeptidase activity. LTA4H from the African claw toad, Xenopus laevis (xlLTA4H) has been shown to, unlike the human enzyme, convert LTA4 to two enzymatic metabolites, LTB4 and another biologically active product Δ(6)-trans-Δ(8)-cis-LTB4 (5(S),12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid). In order to study the molecular aspect of the formation of this product we have characterized the structure and function of xlLTA4H. We solved the structure of xlLTA4H to a resolution of 2.3Å. It is a dimeric structure where each monomer has three domains with the active site in between the domains, similar as to the human structure. An important difference between the human and amphibian enzyme is the phenylalanine to tyrosine exchange at position 375. Our studies show that mutating F375 in xlLTA4H to tyrosine abolishes the formation of the LTB4 isomeric product Δ(6)-trans-Δ(8)-cis-LTB4. In an attempt to understand how one amino acid exchange leads to a new product profile as seen in the xlLTA4H, we performed a conformer analysis of the triene part of the substrate LTA4. Our results show that the Boltzmann distribution of substrate conformers correlates with the observed distribution of products. We suggest that the observed difference in product profile between the human and the xlLTA4H arises from different level of discrimination between substrate LTA4 conformers.
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| 12. |
- Suades, Albert, et al.
(author)
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Establishing mammalian GLUT kinetics and lipid composition influences in a reconstituted-liposome system
- 2023
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In: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Journal article (peer-reviewed)abstract
- Transport assays using purified glucose transporters (GLUTs) have proven to be difficult to implement, hampering deeper mechanistic insights. Here the authors have optimized a transport assay in liposomes that will provide insight to study other membrane transport proteins. Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals, and their dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUTs have proven to be difficult to implement, hampering deeper mechanistic insights. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids using a small number of synthetic lipids. We conclude that GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition to GLUT5. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar K-M,K- but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high K-M for D-glucose and yet a very slow turnover, which may have evolved to ensure uptake regulation by insulin-dependent trafficking. Overall, we outline a much-needed transport assay for measuring GLUT kinetics and our analysis implies that high-levels of free fatty acid in membranes, as found in those suffering from metabolic disorders, could directly impair glucose uptake.
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| 15. |
- Yen, Hsin-Yung, et al.
(author)
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Electrospray ionization of native membrane proteins proceeds via a charge equilibration step
- 2022
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In: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 12:16, s. 9671-9680
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Journal article (peer-reviewed)abstract
- Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion–ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.
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| 16. |
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