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Abstract Objective The present study was undertaken to examine the incidence and management of surgical site infection (SSI) in patients submitted to transapical transcatheter aortic valve implantation (TA-TAVI). Methods From April 2007 to December 2011, 154 patients underwent TA-TAVI with an Edwards Sapien bioprosthesis (ES) at the Institut Universitaire de Cardiologie et Pneumologie de Québec (IUCPQ) as part of a multidisciplinary program to prospectively evaluate percutaneous aortic valve implantation. Patient demographics, perioperative variables, and postoperative complications were recorded in a prospective registry. Results Five (3.2%) patients in the cohort presented with an SSI during the study period. The infections were all hospital-acquired (HAI) and were considered as organ/space SSI’s based on Center for Disease Control criteria (CDC). Within the first few weeks of the initial procedure, these patients presented with an abscess or chronic draining sinus in the left thoracotomy incision and were re-operated. The infection spread to the apex of the left ventricle in all cases where pledgeted mattress sutures could be seen during debridement. Patients received multiple antibiotic regimens without success until the wound was surgically debrided and covered with viable tissue. The greater omentum was used in three patients and the pectoralis major muscle in the other two. None of the patients died or had a recurrent infection. Three of the patients were infected with Staphylococcus epidermidis, one with Staphylococcus aureus, and one with Enterobacter cloacae. Patients with surgical site infections were significantly more obese with higher BMI (31.4±3.1 vs 26.2±4.4 p=0.0099) than the other patients in the cohort. Conclusions While TA-TAVI is a minimally invasive technique, SSIs, which are associated with obesity, remain a concern. Debridement and rib resection followed by wound coverage with the greater omentum and/or the pectoralis major muscle were used successfully in these patients.

TcdB is an intracellular bacterial toxin indispensable to Clostridioides difficile infections. The ability to use chondroitin sulfate proteoglycan 4 (CSPG4) as a primary cell surface receptor is evolutionarily conserved by the two major variants of TcdB. As CSPG4 does not typically undergo receptor-mediated endocytosis, we sought to identify environmental factors that stabilize interactions between TcdB and CSPG4 to promote cell binding and entry into the cytosol. Using a series of TcdB receptor-binding mutants and cell lines with various receptor expression profiles, we discovered that extracellular Ca 2+ promotes receptor-specific interactions with TcdB. Specifically, TcdB exhibits preferential binding to CSPG4 in the presence of Ca 2+ , with the absence of Ca 2+ resulting in CSPG4-independent cell surface interactions. Furthermore, Ca 2+ did not enhance TcdB binding to chondroitin sulfate (CS), the sole glycosaminoglycan of CSPG4. Instead, CS was found to impact the rate of cell entry by TcdB. Collectively, results from this study indicate that Ca 2+ enhances cell binding by TcdB and CS interactions contribute to subsequent steps in cell entry., Importance: Clostridioides difficile is a leading cause of antibiotic-associated gastrointestinal illness, and many disease pathologies are caused by the toxin TcdB. TcdB engages multiple cell surface receptors, with receptor tropisms differing among the variants of the toxin. Chondroitin sulfate proteoglycan 4 (CSPG4) is a critical receptor for multiple forms of TcdB, and insights into TcdB-CSPG4 interactions are applicable to many disease-causing strains of C. difficile . CSPG4 is modified by chondroitin sulfate (CS) and contains laminin-G repeats stabilized by Ca 2+ , yet the relative contributions of CS and Ca 2+ to TcdB cytotoxicity have not been determined. This study demonstrates distinct roles in TcdB cell binding and cell entry for Ca 2+ and CS, respectively. These effects are specific to CSPG4 and contribute to the activities of a prominent isoform of TcdB that utilizes this receptor. These findings advance an understanding of factors contributing to TcdB's mechanism of action and contribution to C. difficile disease., Competing Interests: The authors declare no conflict of interest.

Lymph nodes and spleens are innervated by sympathetic nerve fibers that enter alongside arteries. Despite discovery of these nerve fibers nearly 40 years ago, the role of these nerves during response to infection remains poorly defined. We have found that chemical depletion of sympathetic nerve fibers compromises the ability of mice to develop protective immune memory to a Staphylococcus aureus infection. Innate control of the primary infection was not impacted by sympathectomy. Germinal center formation is also compromised in nerve-depleted animals; however, protective antibody responses are still generated. Interestingly, protective CD4+ T-cell memory fails to form in the absence of sympathetic nerves after S aureus infection., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The copackaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to posttranscriptionally regulate germline mRNAs. In Drosophila melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates containing multiple transcripts from the same gene. Nucleated by Oskar (Osk), homotypic clusters are generated through a stochastic seeding and self-recruitment process that requires the 3' untranslated region (UTR) of germ granule mRNAs. Interestingly, the 3' UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species and we hypothesized that this diversity influences homotypic clustering. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that clustering is a conserved process used to enrich germ granule mRNAs. However, we discovered germ granule phenotypes that included significant changes in the abundance of transcripts present in species' homotypic clusters, which also reflected diversity in the number of coalesced primordial germ cells within their embryonic gonads. By integrating biological data with computational modeling, we found that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels and/or homotypic clustering efficacy. Furthermore, we demonstrated how the nos 3' UTR from different species influences nos clustering, causing granules to have ∼70% less nos and increasing the presence of defective primordial germ cells. Our results highlight the impact that evolution has on germ granules, which should provide broader insight into processes that modify compositions and activities of other classes of biomolecular condensate., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Me31B/DDX6 is a DEAD-box family RNA helicase playing roles in post-transcriptional RNA regulation in different cell types and species. Despite the known motifs/domains of Me31B, the in vivo functions of the motifs remain unclear. Here, we used the Drosophila germline as a model and used CRISPR to mutate the key Me31B motifs/domains: helicase domain, N-terminal domain, C-terminal domain and FDF-binding motif. Then, we performed screening characterization on the mutants and report the effects of the mutations on the Drosophila germline, on processes such as fertility, oogenesis, embryo patterning, germline mRNA regulation and Me31B protein expression. The study indicates that the Me31B motifs contribute different functions to the protein and are needed for proper germline development, providing insights into the in vivo working mechanism of the helicase., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The signaling pathways and networks regulating expression of chondroitin sulfate proteoglycan 4 (CSPG4), a cancer-related protein that serves as a receptor for Clostridiodes difficile TcdB, are poorly defined. In this study, TcdB-resistant/CSPG4-negative HeLa cells were generated by exposure to increasing concentrations of the toxin. The cells that emerged (HeLa R5) lost expression of CSPG4 mRNA and were resistant to binding by TcdB. mRNA expression profiles paired with integrated pathway analysis correlated changes in the Hippo and estrogen signaling pathways with a CSPG4 decrease in HeLa R5 cells. Both signaling pathways altered CSPG4 expression when modulated chemically or through CRISPR-mediated deletion of key transcriptional regulators in the Hippo pathway. Based on the in vitro findings, we predicted and experimentally confirmed that a Hippo pathway inactivating drug (XMU-MP-1) provides protection from C. difficile disease in a mouse model. These results provide insights into key regulators of CSPG4 expression and identify a therapeutic for C. difficile disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Larabee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The packaging of specific mRNAs into ribonucleoprotein granules called germ granules is required for germline proliferation and maintenance. During Drosophila germ granule development, mRNAs such as nanos (nos) and polar granule component (pgc) localize to germ granules through a stochastic seeding and self-recruitment process that generates homotypic clusters: aggregates containing multiple copies of a specific transcript. Germ granules vary in mRNA composition with respect to the different transcripts that they contain and their quantity. However, what influences germ granule mRNA composition during development is unclear. To gain insight into how germ granule mRNA heterogeneity arises, we created a computational model that simulates granule development. Although the model includes known mechanisms that were converted into mathematical representations, additional unreported mechanisms proved to be essential for modeling germ granule formation. The model was validated by predicting defects caused by changes in mRNA and protein abundance. Broader application of the model was demonstrated by quantifying nos and pgc localization efficacies and the contribution that an element within the nos 3' untranslated region has on clustering. For the first time, a mathematical representation of Drosophila germ granule formation is described, offering quantitative insight into how mRNA compositions arise while providing a new tool for guiding future studies., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

An acid/alcohol-producing, Gram-stain-positive, obligately anaerobic, rod-shaped, non-motile, non-spore forming acetogen, designated as strain P21 T , was isolated from old hay after enrichment with CO as the substrate. Spores not observed even after prolonged incubation (30 days). Phylogenetic analysis of the 16S rRNA gene sequence of strain P21 T showed it was closely related to Clostridium carboxidivorans DSM 15243 T (97.9%), Clostridium scatologenes DSM 757 T (97.7 %) and Clostridium drakei DSM 12750 T (97.7 %). The genome is 5.6 Mb and the G+C content is 29.4 mol%. Average nucleotide identity between strain P21 T , C. carboxidivorans , C. scatologenes and C. drakei was 87.1, 86.4, 86.4 %, respectively. Strain P21 T grew on CO:CO 2 , H 2 :CO 2 , l-arabinose, ribose, xylose, fructose, galactose, glucose, lactose, mannose, cellobiose, sucrose, cellulose, starch, pyruvate, choline, glutamate, histidine, serine, threonine and casamino acids. End products of metabolism were acetate, butyrate, caproate, ethanol and hexanol. Dominant cellular fatty acids (>10 %) were C 16 : 0 (41.5 %), C 16 : 1  ω7 c /C 16 : 1  ω6 c (10.0 %), and a summed feature containing cyclo C 17 : 1 /C 18 : 0 (17.3 %). Based on the phenotypic, chemotaxonomic, phylogenetic and phylogenomic analyses, strain P21 T represents a new species in the genus Clostridium , for which the name Clostridium muellerianum sp. nov. is proposed. The type strain is P21 T (=DSM 111390 T =NCIMB 15261 T ).

Here, we report the genome sequence of Clostridium sp. strain P21, isolated from old hay from Stillwater, Oklahoma. This announcement describes the generation and annotation of the 5.6-Mb genomic sequence of strain P21, which will aid in studies targeting genes involved in the enhancement of acid-alcohol production., (Copyright © 2021 Doyle et al.)

Background: The E. coli pET system is the most widely used protein over-expression system worldwide. It relies on the assumption that all cells produce target protein and it is generally believed that integral membrane protein (IMP) over-expression is more toxic than their soluble counterparts., Results: Using GFP-tagged proteins, high level over-expression of either soluble or IMP targets results in > 99.9% cell loss with survival rate of only < 0.03%. Selective pressure generates three phenotypes: large green, large white and small colony variants. As a result, in overnight cultures, ~ 50% of the overall cell mass produces no protein. Genome sequencing of the phenotypes revealed genomic mutations that causes either the loss of T7 RNAP activity or its transcriptional downregulation. The over-expression process is bactericidal and is observed for both soluble and membrane proteins., Conclusions: We demonstrate that it is the act of high-level over-expression of exogenous proteins in E. coli that sets in motion a chain of events leading to > 99.9% cell death. These results redefine our understanding of protein over-production and link it to the adaptive survival response seen in the development of antimicrobial resistance.

Twelve isolates recovered from 10 cystic fibrosis/other patient types and a variety of clinical sources, were referred to Canada's National Microbiology Laboratory over 7 years. These were assignable to the genus Pseudoxanthomonas but were unidentifiable to species level. Patients included five males and five females from two geographically separated provinces, ranging in age from 2 months to 84 years. In contrast, most Pseudoxanthomonas species described to date have been derived from water, plants or contaminated soils. By 16S rRNA gene sequencing, the patient strains had ≥99.4 % similarity to each other but only 97.73-98.29 % to their closest relatives, Pseudoxanthomonas spadix or Pseudoxanthomonas helianthi . Bacteria were studied by whole genome sequencing using average nucleotide identity by Blastn, digital DNA-DNA hybridization, average amino acid identity, core genome and single nucleotide variant analyses, MALDI-TOF, biochemical and cellular fatty acid analyses, and by antimicrobial susceptibility testing. Bacterial structures were assessed using scanning and transmission electron microscopy. Strains were strict aerobes, yellowish-pigmented, oxidative, non-motile, Gram-stain-negative bacilli and generally unable to reduce nitrate. Strains were susceptible to most of the antibiotics tested; some resistance was observed towards carbapenems, several cephems and uniformly to nitrofurantoin. The single taxon group observed by 16S rRNA gene sequencing was supported by whole genome sequencing; genomes ranged in size from 4.36 to 4.73 Mb and had an average G+C content of 69.12 mol%. Based on this study we propose the name Pseudoxanthomonas winnipegensis sp. nov. for this cluster. Pseudoxanthomonas spadix DSM 18855 T , acquired for this study, was found to be non-motile phenotypically and by electron microscopy; we therefore propose the emendation of Pseudoxanthomonas spadix Young et al . 2007 to document that observation.

A Gram-stain-negative, microaerophilic, non-motile, rod-shaped bacterium strain designated PMP191F T , was isolated from a human peritoneal tumour. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the organism formed a lineage within the family Chitinophagaceae that was distinct from members of the genus Pseudoflavitalea (95.1-95.2 % sequence similarity) and Pseudobacter ginsenosidimutans (94.4 % sequence similarity). The average nucleotide identity values between strain PMP191F T and Pseudoflavitalea rhizosphaerae T16R-265 T and Pseudobacter ginsenosidimutans Gsoil 221 T was 68.9 and 62.3% respectively. The only respiratory quinone of strain PMP191F T was MK-7 and the major fatty acids were iso-C 15 : 0 , iso-C 15 : 1 G and summed feature 3 (C 16:1 ω7 c and/or C 16:1 ω6 c ). The polar lipids consisted of phosphatidylethanolamine and some unidentified amino and glycolipids. The G+C content of strain PMP191F T calculated from the genome sequence was 43.4 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, strain PMP191F T represents a novel species and genus for which the name Parapseudoflavitalea muciniphila gen. nov., sp. nov. is proposed. The type strain is PMP191F T (=DSM 104999 T =ATCC BAA-2857 T = CCUG 72691 T ). The phylogenetic analyses also revealed that Pseudobacter ginsenosidimutans shared over 98 % sequence similarly to members of the genus Pseudoflavitalea . However, the average nucleotide identity value between Pseudoflavitalea rhizosphaerae T16R-265 T , the type species of the genus and Pseudobacter ginsenosidimutans Gsoil 221 T was 86.8 %. Therefore, we also propose that Pseudobacter ginsenosidimutans be reclassified as Pseudoflavitalea ginsenosidimutans comb. nov.

Being able to visualize biology at the molecular level is essential for our understanding of the world. A structural biology approach reveals the molecular basis of disease processes and can guide the design of new drugs as well as aid in the optimization of existing medicines. However, due to the lack of a synchrotron light source, adequate infrastructure, skilled persons and incentives for scientists in addition to limited financial support, the majority of countries across the African continent do not conduct structural biology research. Nevertheless, with technological advances such as robotic protein crystallization and remote data collection capabilities offered by many synchrotron light sources, X-ray crystallography is now potentially accessible to Africa-based scientists. This leap in technology led to the establishment in 2017 of BioStruct-Africa, a non-profit organization (Swedish corporate ID: 802509-6689) whose core aim is capacity building for African students and researchers in the field of structural biology with a focus on prevalent diseases in the African continent. The team is mainly composed of, but not limited to, a group of structural biologists from the African diaspora. The members of BioStruct-Africa have taken up the mantle to serve as a catalyst in order to facilitate the information and technology transfer to those with the greatest desire and need within Africa. BioStruct-Africa achieves this by organizing workshops onsite at our partner universities and institutions based in Africa, followed by post-hoc online mentoring of participants to ensure sustainable capacity building. The workshops provide a theoretical background on protein crystallography, hands-on practical experience in protein crystallization, crystal harvesting and cryo-cooling, live remote data collection on a synchrotron beamline, but most importantly the links to drive further collaboration through research. Capacity building for Africa-based researchers in structural biology is crucial to win the fight against the neglected tropical diseases, e.g. ascariasis, hookworm, trichuriasis, lymphatic filariasis, active trachoma, loiasis, yellow fever, leprosy, rabies, sleeping sickness, onchocerciasis, schistosomiasis, etc., that constitute significant health, social and economic burdens to the continent. BioStruct-Africa aims to build local and national expertise that will have direct benefits for healthcare within the continent.

Caenorhabditis elegans is an informative model to study the neural basis of feeding. A useful paradigm is one in which adult nematodes feed on a bacterial lawn which has been pre-loaded with pharmacological agents and the effect on pharyngeal pumping rate scored. A crucial aspect of this assay is the availability of good quality bacteria to stimulate pumping to maximal levels. A potential confound is the possibility that the pharmacological agent impacts bacterial viability and indirectly influences feeding rate. Here, the actions of nicotine on pharyngeal pumping of C. elegans and on the Escherichia coli bacterial food source were investigated. Nicotine caused an immediate and concentration-dependent inhibition of C. elegans pharyngeal pumping, IC 50 4 mM (95% CI = 3.4 mM to 4.8 mM). At concentrations between 5 and 25 mM, nicotine also affected the growth and viability of E. coli lawns. To test whether this food depletion by nicotine caused the reduced pumping, we modified the experimental paradigm. We investigated pharyngeal pumping stimulated by 10 mM 5-HT, a food 'mimic', before testing if nicotine still inhibited this behaviour. The IC 50 for nicotine in these assays was 2.9 mM (95% CI = 3.1 mM to 5.1 mM) indicating the depletion of food lawn does not underpin the potency of nicotine at inhibiting feeding. These studies show that the inhibitory effect of nicotine on C. elegans pharyngeal pumping is mediated by a direct effect rather than by its poorly reported bactericidal actions.

Bacterial lipoproteins have been researched for decades due to their roles in a large number of biological functions. There were no structures of their main three membrane processing enzymes, until 2016 for Lgt and LspA, and then 2017 for Lnt with not one but three simultaneous, independent publications. We have analyzed the recent findings for this apolipoprotein N-acyltransferase Lnt, with comparisons between the novel structures, and with soluble nitrilases, to determine the significance of unique features in terms of substrate's recognition and binding mechanism influenced by exclusive residues, two transmembrane helices, and a flexible loop., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bacterial antibiotic resistance is rapidly becoming a major world health consideration. To combat antibiotics, microorganisms employ their pre-existing defence mechanisms that existed long before man's discovery of antibiotics. Bacteria utilise levels of protection that range from gene upregulation, mutations, adaptive resistance, and production of resistant phenotypes (persisters) to communal behaviour, as in swarming and the ultimate defence of a biofilm. A major part of all of these responses involves the use of antibiotic efflux transporters. At the single cell level, it is becoming apparent that the use of efflux pumps is the first line of defence against an antibiotic, as these pumps decrease the intracellular level of antibiotic while the cell activates the various other levels of protection. This frontline of defence involves a coordinated network of efflux transporters. In the future, inhibition of this efflux transporter network, as a target for novel antibiotic therapy, will require the isolation and then biochemical/biophysical characterisation of each pump against all known and new antibiotics. This depth of knowledge is required so that we can fully understand and tackle the mechanisms of developing antimicrobial resistance.

Background: Neonicotinoid insecticides are under review owing to emerging toxicity to non-target species. Interest has focused on biological pollinators while their effects on other organisms that are key contributors to the ecosystem remain largely unknown. To advance this, we have tested the effects of representatives of three major classes of neonicotinoids, thiacloprid, clothianidin and nitenpyram, on the free-living nematode Caenorhabditis elegans (C. elegans), as a representative of the Nematoda, an ecologically important phylum contributing to biomass., Results: Concentrations that are several-fold higher than those with effects against target species had limited impact on locomotor function. However, increased potency was observed in a mutant with a hyperpermeable cuticle, which shows that drug access limits the effects of the neonicotinoids in C. elegans. Thiacloprid was most potent (EC 50 714 μm). In addition, it selectively delayed larval development in wild-type worms at 1 mm., Conclusion: C. elegans is less susceptible to neonicotinoids than target species of pest insect. We discuss an approach in which this defined low sensitivity may be exploited by heterologous expression of insect nicotinic acetylcholine receptors from both pest and beneficial insects in transgenic C. elegans with increased cuticle permeability to provide a whole organism assay for species-dependent neonicotinoid effects. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

The widespread use of supplemental vitamin D has dramatically reduced the incidence of rickets. While generally considered a safe practice, there is potential for toxicity in patients with idiopathic infantile hypercalcemia (IIH). Inadequate 24-hydroxylase-enzyme activity renders these individuals unable to degrade active vitamin D, resulting in hypercalcemia due to increased intestinal calcium absorption, decreased renal calcium excretion, and increased osteoclastic bone activity. Clinicians should be aware that even therapeutic doses of vitamin D can prove harmful for patients with CYP24A1 mutations. Studies have also demonstrated a link between inadequate 24-hydroxylase activity and nephrocalcinosis, renal insufficiency, and calcium containing kidney stones, further emphasizing the importance of early recognition of this disease and judicious use of vitamin D. We present a case with an interesting diagnostic algorithm used to diagnose IIH when given an incomplete history and subsequently review the existing literature on the subject.

The fibroblast-populated three-dimensional (3-D) collagen matrix has been used to model matrix contraction, cell motility, and general fibroblast biology. MCPIP1 (monocyte chemotactic protein-induced protein 1) has been shown to regulate inflammation, angiogenesis, and cellular motility. In the present study, we demonstrated induction of MCPIP1 in human fibroblasts embedded in the stress-released 3-D collagen matrix, which occurred through activation of mitogen-activated protein kinases, phosphoinositide 3-kinase, and NF-κB. Furthermore, MCPIP1 induction was associated with inhibition of fibroblast migration out of the nested collagen matrix. MCPIP1 induction or ectopic expression also upregulated p53. RNA interference of p53 prevented the inhibition of migration produced by induction or ectopic expression of MCPIP1. Our findings suggest a new role for MCPIP1 as a molecular switch that regulates fibroblast migration in the nested collagen matrix model.

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid-polyamine-organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP from Pseudomonas aeruginosa was recombinantly expressed in Escherichia coli and purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space group P21, with unit-cell parameters a = 169.53, b = 169.53, c = 290.13 Å, γ = 120°.

The availability of fibroblasts that express green fluorescent protein (GFP) would be of interest for the monitoring of cell growth, migration, contraction, and other processes within the fibroblast-populated collagen matrix and other culture systems. A plasmid lentiviral vector-GFP (pLV-GFP) was utilized for gene delivery to produce primary human foreskin fibroblasts (HFFs) that stably express GFP. Cell morphology, cell migration, and collagen contraction were compared between nontransduced HFFs and transduced GFP-HFFs; no differences were observed. Immunocytochemical staining showed no differences in cell morphology between nontransduced and GFP-HFFs in both two-dimensional and three-dimensional culture systems. Furthermore, there was no significant difference in cellular population growth within the collagen matrix populated with nontransduced vs. GFP-HFFs. Within the limits of our assays, we conclude that transduction of GFP into HFFs did not alter the observed properties of HFFs compared with nontransduced fibroblasts. The GFP-HFFs may represent a new tool for the convenient monitoring of living primary fibroblast processes in two-dimensional or three-dimensional culture., (© 2014 by the Wound Healing Society.)

The transient receptor potential vanilloid 3 (TRPV3) cation channel is widely expressed in human tissues and has been shown to be activated by mild temperatures or chemical ligands. In spite of great progress in the TRP-channel characterization, very little is known about their structure and interactions with other proteins at the atomic level. This is mainly caused by difficulties in obtaining functionally active samples of high homogeneity. Here, we report on the high-level Escherichia coli expression of the human TRPV3 channel, for which no structural information has been reported to date. We selected a suitable detergent and buffer system using analytical size-exclusion chromatography and a thermal stability assay. We demonstrate that the recombinant purified protein contains high α-helical content and migrates as dimers and tetramers on native PAGE. Furthermore, the purified channel also retains its current inducing activity, as shown by electrophysiology experiments. The ability to produce the TRPV3 channel heterologously will aid future functional and structural studies., (© 2013 FEBS.)

The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using (86)Rb(+) flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP(2)) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP(2) increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP(2), and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP(2) and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Many ion channels are modulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)), but studies examining the PIP(2) dependence of channel activity have been limited to cell expression systems, which present difficulties for controlling membrane composition. We have characterized the PIP(2) dependence of purified human Kir2.1 and Kir2.2 activity using (86)Rb(+) flux and patch clamp assays in liposomes of defined composition. We definitively show that these channels are directly activated by PIP(2) and that PIP(2) is absolutely required in the membrane for channel activity. The results provide the first quantitative description of the dependence of eukaryotic Kir channel function on PIP(2) levels in the membrane; Kir2.1 shows measureable activity in as little as 0.01% PIP(2), and open probability increases to ∼0.4 at 1% PIP(2). Activation of Kir2.1 by phosphatidylinositol phosphates is also highly selective for PIP(2); PI, PI(4)P, and PI(5)P do not activate channels, and PI(3,4,5)P(3) causes minimal activity. The PIP(2) dependence of eukaryotic Kir activity is almost exactly opposite that of KirBac1.1, which shows marked inhibition by PIP(2). This raises the interesting hypothesis that PIP(2) activation of eukaryotic channels reflects an evolutionary adaptation of the channel to the appearance of PIP(2) in the eukaryotic cell membrane.

The inward rectifier family of potassium (KCNJ) channels regulate vital cellular processes including cell volume, electrical excitability, and insulin secretion. Dysfunction of different isoforms have been linked to numerous diseases including Bartter's, Andersen-Tawil, Smith-Magenis Syndromes, Type II diabetes mellitus, and epilepsy, making them important targets for therapeutic intervention. Using a family-based approach, we succeeded in expressing 10 of 11 human KCNJ channels tested in Saccharomyces cerevisiae. GFP-fusion proteins showed that these channels traffic correctly to the plasma-membrane suggesting that the protein is functional. A 2-step purification process can be used to purify the KCNJ channels to >95% purity in a mono-dispersed form. After incorporation into liposomes, (86)Rb(+) flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

PDZ domains most commonly bind the C-terminus of their protein targets. Typically the C-terminal four residues of the protein target are considered as the binding motif, particularly the C-terminal residue (P0) and third-last residue (P-2) that form the major contacts with the PDZ domain's "binding groove". We solved crystal structures of seven human PDZ domains, including five of the seven PDLIM family members. The structures of GRASP, PDLIM2, PDLIM5, and PDLIM7 show a binding mode with only the C-terminal P0 residue bound in the binding groove. Importantly, in some cases, the P-2 residue formed interactions outside of the binding groove, providing insight into the influence of residues remote from the binding groove on selectivity. In the GRASP structure, we observed both canonical and noncanonical binding in the two molecules present in the asymmetric unit making a direct comparison of these binding modes possible. In addition, structures of the PDZ domains from PDLIM1 and PDLIM4 also presented here allow comparison with canonical binding for the PDLIM PDZ domain family. Although influenced by crystal packing arrangements, the structures nevertheless show that changes in the positions of PDZ domain side-chains and the alpha B helix allow noncanonical binding interactions. These interactions may be indicative of intermediate states between unbound and fully bound PDZ domain and target protein. The noncanonical "perpendicular" binding observed potentially represents the general form of a kinetic intermediate. Comparison with canonical binding suggests that the rearrangement during binding involves both the PDZ domain and its ligand.

Background: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Galpha-mediated GTP hydrolysis ("GTPase-accelerating proteins" or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Galpha GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras., Methodology/principal Findings: Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co-transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor-mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling., Conclusions/significance: In cells, RGS14 facilitates the formation of a selective Ras.GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras-binding domain architecture with RGS14.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules.

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein-ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.

PDZ domains are protein-protein interaction modules that generally bind to the C termini of their target proteins. The C-terminal four amino acids of a prospective binding partner of a PDZ domain are typically the determinants of binding specificity. In an effort to determine the structures of a number of PDZ domains we have included appropriate four residue extensions on the C termini of PDZ domain truncation mutants, designed for self-binding. Multiple truncations of each PDZ domain were generated. The four residue extensions, which represent known specificity sequences of the target PDZ domains and cover both class I and II motifs, form intermolecular contacts in the expected manner for the interactions of PDZ domains with protein C termini for both classes. We present the structures of eight unique PDZ domains crystallized using this approach and focus on four which provide information on selectivity (PICK1 and the third PDZ domain of DLG2), binding site flexibility (the third PDZ domain of MPDZ), and peptide-domain interactions (MPDZ 12th PDZ domain). Analysis of our results shows a clear improvement in the chances of obtaining PDZ domain crystals by using this approach compared to similar truncations of the PDZ domains without the C-terminal four residue extensions.

Centaurins are a family of proteins that contain GTPase-activating protein domains, with the gamma family members containing in addition a GTPase-like domain. Centaurins reside mainly in the nucleus and are known to activate phosphoinositide 3-kinase, a key regulator of cell proliferation, motility and vesicular trafficking. In the present study, using X-ray structural analysis, enzymatic assays and nucleotide-binding studies, we show that, for CENTG1 (centaurin gamma-1) the GTPase-like domain has broader trinucleotide specificity. Alterations within the G4 motif of CENTG1 from the highly conserved NKXD found in typical GTPases to TQDR result in the loss of specificity, a lower affinity for the nucleotides and higher turnover rates. These results indicate that the centaurins could be more accurately classified as NTPases and point to alternative mechanisms of cell signalling control.

The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein-protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners.

Detergent solubilization and purification of the E. coli heavy metal P-type ATPase ZntA yields an enzyme with reduced hydrolytic activity in vitro. Here, it is shown that the in vitro hydrolytic activity of detergent solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. The protein-lipid interaction of ZntA was characterized by enzyme-coupled ATPase assays and fluorescence spectroscopy. Among the most abundant naturally occurring phospholipids, only phosphatidyl-glycerol lipids (PG) enhance the in vitro enzymatic ATPase activity of ZntA. Re-lipidation of detergent purified ZntA with 1,2-dioleoylphosphatidyl-glycerol (DOPG) increases the ATPase activity four-fold compared to the purified state. All other E. coli phospholipids fail to activate the ATPase. Among the phosphatidyl-glycerol family, highest activity was observed for 1,2-dioleoyl-PG followed by 1,2-dimyristoyl-PG, 1,2-dipalmitoyl-PG and 1,2-distearoyl-PG. Increasing intrinsic Trp fluorescence quantum yield upon relipidation of ZntA was used to determine a pH maximum for lipid binding at pH 6.7. The pH dependence of the lipid binding was confirmed by pH-dependent ATPase assays showing maximum activity at pH 6.7. The biophysical characterization of detergent solubilized membrane proteins crucially relies on the conformational stability and functional integrity of the protein under investigation. The present study describes how the E. coli ZntA P-type ATPase can be stabilized and functionally activated in a detergent solubilized system.

The magnesium ion, Mg2+, is essential for myriad biochemical processes and remains the only major biological ion whose transport mechanisms remain unknown. The CorA family of magnesium transporters is the primary Mg2+ uptake system of most prokaryotes and a functional homologue of the eukaryotic mitochondrial magnesium transporter. Here we determine crystal structures of the full-length Thermotoga maritima CorA in an apparent closed state and its isolated cytoplasmic domain at 3.9 A and 1.85 A resolution, respectively. The transporter is a funnel-shaped homopentamer with two transmembrane helices per monomer. The channel is formed by an inner group of five helices and putatively gated by bulky hydrophobic residues. The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the transmembrane helices. The cytoplasmic neck of the pore is surrounded, on the outside of the funnel, by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane on the outside of the funnel and abut the ring of positive charge. An apparent Mg2+ ion was bound between monomers at a conserved site in the cytoplasmic domain, suggesting a mechanism to link gating of the pore to the intracellular concentration of Mg2+.

The bacterial K+ channel KcsA from Streptomyces lividans was analyzed by neutron and x-ray small-angle solution scattering. The C-terminally truncated version of KcsA, amenable to crystallographic studies, was compared with the full-length channel. Analyzing the scattering data in terms of radius of gyration reveals differences between both KcsA species of up to 13.2 A. Equally, the real-space distance distribution identifies a 40 to 50 A extension of full-length KcsA compared to its C-terminally truncated counterpart. We show that the x-ray and neutron scattering data are amenable for molecular shape reconstruction of full-length KcsA. The molecular envelopes calculated display an hourglass-shaped structure within the C-terminal intracellular domain. The C-terminus extends the membrane spanning region of KcsA by 54-70 A, with a central constriction 10-30 A wide. Solution scattering techniques were further employed to characterize the KcsA channel under acidic conditions favoring its open conformation. The full-length KcsA at pH 5.0 shows the characteristics of a dumbbell-shaped macromolecular structure, originating from dimerization of the tetrameric K+ channel. Since C-terminally truncated KcsA measured under the same low pH conditions remains tetrameric, oligomerization of full-length KcsA seems to proceed via structurally changed C-terminal domains. The determined maximum dimensions of the newly formed complex increase by 50-60%. Shape reconstruction of the pseudooctameric complex indicates the pH-induced conformational reorganization of the intracellular C-terminal domain.

Potassium channels allow the selective flow of K(+) ions across membranes. In response to external gating signals, the potassium channel can move reversibly through a series of structural conformations from a closed to an open state. 2D crystals of the inwardly rectifying K(+) channel KirBac3.1 from Magnetospirillum magnetotacticum have been captured in two distinct conformations, providing "snap shots" of the gating process. Analysis by electron cryomicroscopy of these KirBac3.1 crystals has resulted in reconstructed images in projection at 9 A resolution. Kir channels are tetramers of four subunits arranged as dimers of dimers. Each subunit has two transmembrane helices (inner and outer). In one crystal form, the pore is blocked; in the other crystal form, the pore appears open. Modeling based on the KirBac1.1 (closed) crystal structure shows that opening of the ion conduction pathway could be achieved by bending of the inner helices and significant movements of the outer helices.

We have modeled the structure of KirBac1.1 in an open state using as a starting point the structure of KirBac1.1 in its closed conformation (Protein Data Bank 1P7B). To test the validity of the open-state model, molecular dynamics simulations in octane, a lipid bilayer mimetic, were carried out. Simulations of the closed conformer were used for comparison purposes. The total simulation time was approximately 138 ns. The initial open model was refined by using projection maps obtained from electron microscopy experiments on two-dimensional crystals of the inwardly rectifying K+ channel KirBac3.1 from Magentospirillum magnetotacticum captured in its open state (C. Vénien-Bryan, unpublished data). Significant movements of the outer helices take place in going from the closed to the open model in agreement with structural and biochemical data in potassium channels, which suggests that gating is accomplished by a conformational change that takes place in the transmembrane domain upon an external stimulus. The motion of the inner helices is mainly achieved by bending at conserved glycine residues that have been previously reported to act as molecular hinges. Overall, these simulations suggest that the open conformer is stable, providing a plausible all-atom model that will enable the study of potential gating mechanisms in more detail.

The prediction of transmembrane (TM) helices plays an important role in the study of membrane proteins, given the relatively small number (approximately 0.5% of the PDB) of high-resolution structures for such proteins. We used two datasets (one redundant and one non-redundant) of high-resolution structures of membrane proteins to evaluate and analyse TM helix prediction. The redundant (non-redundant) dataset contains structure of 434 (268) TM helices, from 112 (73) polypeptide chains. Of the 434 helices in the dataset, 20 may be classified as 'half-TM' as they are too short to span a lipid bilayer. We compared 13 TM helix prediction methods, evaluating each method using per segment, per residue and termini scores. Four methods consistently performed well: SPLIT4, TMHMM2, HMMTOP2 and TMAP. However, even the best methods were in error by, on average, about two turns of helix at the TM helix termini. The best and worst case predictions for individual proteins were analysed. In particular, the performance of the various methods and of a consensus prediction method, were compared for a number of proteins (e.g. SecY, ClC, KvAP) containing half-TM helices. The difficulties of predicting half-TM helices suggests that current prediction methods successfully embody the two-state model of membrane protein folding, but do not accommodate a third stage in which, e.g., short helices and re-entrant loops fold within a bundle of stable TM helices.

Objective: To study the NKX2-1 gene in two half-siblings with elevated thyroid-stimulating hormone (TSH) on state screen, prolonged neonatal respiratory distress despite term gestations, and persistent ataxia, dysarthria, and developmental delay., Study Design: We amplified and sequenced DNA samples from blood or buccal swab for subjects and their unaffected siblings., Results: The same mutation that prevents splicing together of exons 2 and 3 of the NKX2-1 gene was present in the affected siblings, their mother, and maternal grandmother but not in their unaffected siblings. The mutation was present in the heterozygous form, thus explaining the disease phenotype., Conclusions: Autosomal dominant transmission of mutations of NKX2-1 may cause congenital hypothyroidism, neonatal respiratory distress at term, and persistent neurologic findings such as ataxia, choreoathetosis, and dysarthria in families with affected subjects in multiple generations.

Potassium channels are signalling elements vital to vertebrate neurotransmission, and cardiac and renal function. Two inherent qualities equip them for their role in the interconversion of chemical and electrical messages: high selectivity for potassium ions and the ability to open (gate) on cue. The crystal structure of KcsA, published in 1998, explained much about potassium selectivity and high ion flux. The enormous diversity of potassium channels (some hundreds of genes in humans) may have hampered similar progress in understanding gating processes. The recent determination of several representative structures has provided us with a valuable reference for discriminating between features that are utilized in gating across the potassium channel genre and features that determine responsiveness to family-specific gating cues.

Water is probably the most important molecule in biology. It solvates molecules, all biochemical reactions occur in it and it is a major driving force in protein folding. Phospholipid membranes separate different water environments, but connections do exist between the different compartments. The integral membrane proteins (IMPs) form these connections. In the case of ions, IMPs form the passageways that regulate ion movement across the membrane. Structural information from three ion distinct channels are examined to see how these channels first select for and then control the movement of their target ions. This review focuses on how these channels select for target ions and control their movement while taking into account and using different properties of water. This includes the use of hydrophobic gates, mimicking the water environment, and controlling ions indirectly by controlling water.

Ion channels are generally multi-subunit complexes, with the ion conduction pathway formed at the subunit interface. In moving between the closed and open states, three structurally distinct channels, represented by the recently determined structures of a mechanosensitive, ligand-gated and K(+) selective channel, all move transmembrane helices away from the central ion conduction pathway. In all three cases, this results in the displacement of a hydrophobic gate from the ion conduction pathway, freeing ion movement. The channels achieve this by moving the transmembrane helices as rigid bodies using three major types of motion: MscL tilts its helices, the nicotinic ACh receptor rotates its helices, and KirBac1.1 bends its helices. In all cases, the gating motions are likely to take place rapidly. These large and fast movements provide a possible explanation for why the conduction pathways of a wide range of different ion channels are formed at the interface between subunits.