Search

Focuses on the biosynthesis of urdamycin A, the principal product of Streptomyces fradiae Tmu2717, an angucycline-type antibiotic and anticancer agent with C-glycosidically linked D-olivose. Bacterial strains, plasmids and culture conditions; Genetic manipulations; Library construction and screening; Chromosomal generation.

In this rubric we present various perspectives on theatre – historical and contemporary, intercultural and culture-specific, unexpectedly weird, unusually suspenseful, disturbedly gripping, fascinatingly enigmatic … The following extract is taken from Johann Wolfgang von Goethe’s tragedy FAUST (first published in 1808). A conversation between a theatre director, a writer (and a “merry person“) revolves around a fundamental question: to what extent should the audience’s desire for entertainment be the deciding factor when staging plays? Goethe worked on Faust nearly two centuries ago, but the text is still of high topicality nowadays, particularly in the face of increasing pressure on educational and cultural institutions. Indeed, the justification of performances is often intricately tied to measurable success (i.e. audience size). Apart from this we are looking at a downright pedagogical question: what plays should be staged, by which means, and to what end? MANAGER — DRAMATIC POET — MERRY-ANDREW MANAGER You two, who oft a helping hand Have lent, in need and tribulation. Come, let me know your expectation Of this, our enterprise, in German land! I wish the crowd to feel itself well treated, Especially since it lives and lets me live; The posts are set, the booth of boards completed.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation., Competing Interests: RD, AE, GC, PA, IY, BF, MT, IS No competing interests declared, SV, RL S.J.V.D. and R.M.L. are listed as inventors on a patent United States application: 17/505,561 for the use of chitinases to treat fibrotic lung disease. S.J.V.D., R.M.L., and J.S.F. are listed as inventors on a patent for mutant chitinases with enhanced expression and activity, JF S.J.V.D. and R.M.L. are listed as inventors on a patent for the use of chitinases to treat fibrotic lung disease. United States application: 17/505,561 S.J.V.D., R.M.L., and J.S.F. are listed as inventors on a patent for mutant chitinases with enhanced expression and activity, (© 2023, Díaz et al.)

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome 1-3 . How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Thyroid hormones are vital in metabolism, growth and development 1 . Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR) 2 . In patients with Graves' disease, autoantibodies that activate the TSHR pathologically increase thyroid hormone activity 3 . How autoantibodies mimic thyrotropin function remains unclear. Here we determined cryo-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. Thyrotropin selects an upright orientation of the extracellular domain owing to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody from a patient with Graves' disease selects a similar upright orientation of the extracellular domain. Reorientation of the extracellular domain transduces a conformational change in the seven-transmembrane-segment domain via a conserved hinge domain, a tethered peptide agonist and a phospholipid that binds within the seven-transmembrane-segment domain. Rotation of the TSHR extracellular domain relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to other G-protein-coupled receptors with large extracellular domains., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The opportunistic bacterium Pseudomonas aeruginosa can infect mucosal tissues of the human body. To persist at the mucosal barrier, this highly adaptable pathogen has evolved many strategies, including invasion of host cells. Here, we show that the P. aeruginosa lectin LecB binds and cross-links fucosylated receptors at the apical plasma membrane of epithelial cells. This triggers a signaling cascade via Src kinases and phosphoinositide 3-kinase (PI3K), leading to the formation of patches enriched with the basolateral marker phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) at the apical plasma membrane. This identifies LecB as a causative bacterial factor for activating this well-known host cell response that is elicited upon apical binding of P. aeruginosa. Downstream from PI3K, Rac1 is activated to cause actin rearrangement and the outgrowth of protrusions at the apical plasma membrane. LecB-triggered PI3K activation also results in aberrant recruitment of caveolin-1 to the apical domain. In addition, we reveal a positive feedback loop between PI3K activation and apical caveolin-1 recruitment, which provides a mechanistic explanation for the previously observed implication of caveolin-1 in P. aeruginosa host cell invasion. Interestingly, LecB treatment also reversibly removes primary cilia. To directly prove the role of LecB for bacterial uptake, we coated bacterium-sized beads with LecB, which drastically enhanced their endocytosis. Furthermore, LecB deletion and LecB inhibition with l-fucose diminished the invasion efficiency of P. aeruginosa bacteria. Taken together, the results of our study identify LecB as a missing link that can explain how PI3K signaling and caveolin-1 recruitment are triggered to facilitate invasion of epithelial cells from the apical side by P. aeruginosa. IMPORTANCE An intriguing feature of the bacterium P. aeruginosa is its ability to colonize highly diverse niches. P. aeruginosa can, besides forming biofilms, also enter and proliferate within epithelial host cells. Moreover, research during recent years has shown that P. aeruginosa possesses many different mechanisms to invade host cells. In this study, we identify LecB as a novel invasion factor. In particular, we show that LecB activates PI3K signaling, which is connected via a positive feedback loop to apical caveolin-1 recruitment and leads to actin rearrangement at the apical plasma membrane. This provides a unifying explanation for the previously reported implication of PI3K and caveolin-1 in host cell invasion by P. aeruginosa. In addition, our study adds a further function to the remarkable repertoire of the lectin LecB, which is all brought about by the capability of LecB to recognize fucosylated glycans on many different niche-specific host cell receptors.

Hypertrophic pulmonary osteoarthropathy (HPOA) is a rare syndrome that causes clubbed fingers, periostitis, and synovial effusions. It can adversely impact a patient's quality of life. It occurs secondary to pulmonary disease - most commonly pulmonary malignancy. The most effective treatment for HPOA is to treat the underlying disease, usually through surgical resection, chemotherapy, or radiation. However, symptomatic treatments rather than definitive treatments (surgical, chemotherapy, or radiation) are more appropriate for the palliative care patient. Pamidronate is a promising medication for the treatment of HPOA for its safety and rapid onset of action. Further research is indicated to determine whether pamidronate is consistently effective., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s), 2022.)

There are numerous approaches and methodologies for assessing the identity and quantities of non-intentionally added substances (NIAS) in food contact materials (FCMs). They can give different results and it can be difficult to make meaningful comparisons. The initial approach was to attempt to prepare a prescriptive methodology but as this proved impossible; this paper develops guidelines that need to be taken into consideration when assessing NIAS. Different approaches to analysing NIAS in FCMs are reviewed and compared. The approaches for preparing the sample for analysis, recommended procedures for screening, identification, and quantification of NIAS as well as the reporting requirements are outlined. Different analytical equipment and procedures are compared. Limitations of today's capabilities are raised along with some research needs.

Objective: To examine the agreement between self- and informant-reported activities of daily living (ADL) deficits in Parkinson's Disease (PD) patients, and to examine factors influencing ADL ratings., Background: In PD, the loss of functional independence is an important outcome of disease progression. The valid assessment of ADL function in PD is essential, but it is unclear to what extent informants' and patients' perceptions of their daily functions concur, and how other factors may influence both ratings., Methods: Data of 150 PD patients who underwent cognitive and motor testing, as well as their informants were analyzed. The 10-item Functional Activities Questionnaire (FAQ), completed separately by patients (FAQ-S) and their informants (FAQ-I), assessed ADL function. Weighted κ statistics summarized level of agreement, and a discrepancy score (FAQ-I - FAQ-S) quantified agreement. Correlation analyses between FAQ total scores, patient and informant characteristics, and cognitive scores were conducted, with post hoc regressions to determine the associations between both FAQ scores and cognition, independent of patient characteristics., Results: The sample included 87 patients with normal cognition, 50 with mild cognitive impairment, and 13 with dementia. Overall, there was fair to moderate agreement between patients and informants on individual FAQ items (0.27 ≤ κ ≤ 0.61, p < 0.004), with greater discrepancies with increasing cognitive impairment. Patients' age, motor severity, non-motor burden, and depression also affected both ratings (0.27 ≤ r ≤ 0.50, p < 0.001), with motor severity showing the greatest influence on both ratings. Both the FAQ-I and FAQ-S were correlated with almost all cognitive domains. Post hoc regression analyses controlling for patient characteristics showed that the attention domain was a significant predictor of both the FAQ-S and FAQ-I scores, and memory was also a significant predictor of the FAQ-I score. Only 29.3% of patients agreed perfectly with informants on the FAQ total score, with informants most commonly rating ADL impairments as more severe than patients., Conclusions: Patient and informant ratings of ADL function using FAQ items showed moderate agreement, with only few items reaching substantial agreement. Ratings of both were associated with patient cognitive status, but also other characteristics. In addition to patient and informant reports, objective measures are needed to accurately classify ADL deficits in PD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Becker, Solbrig, Michaelis, Faust, Brockmann and Liepelt-Scarfone.)

The neuropeptide substance P (SP) is important in pain and inflammation. SP activates the neurokinin-1 receptor (NK1R) to signal via G q and G s proteins. Neurokinin A also activates NK1R, but leads to selective G q signaling. How two stimuli yield distinct G protein signaling at the same G protein-coupled receptor remains unclear. We determined cryogenic-electron microscopy structures of active NK1R bound to SP or the G q -biased peptide SP6-11. Peptide interactions deep within NK1R are critical for receptor activation. Conversely, interactions between SP and NK1R extracellular loops are required for potent G s signaling but not G q signaling. Molecular dynamics simulations showed that these superficial contacts restrict SP flexibility. SP6-11, which lacks these interactions, is dynamic while bound to NK1R. Structural dynamics of NK1R agonists therefore depend on interactions with the receptor extracellular loops and regulate G protein signaling selectivity. Similar interactions between other neuropeptides and their cognate receptors may tune intracellular signaling., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity., Competing Interests: Declaration of interests B.W., W.-H.L., C.-W.H., Y.H., P.M.L.N., E.Z.X.N., H.C.T., C.Y.L., R.A.M., M.K.S., F.J.T., Y.Y.C.Y., and C.-I.W. are listed as inventors of a filed patent for all 27 monoclonal antibodies mentioned in this manuscript., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: there is a paradox between the development of strength-power abilities and the high volume of technical/tactical training in elite soccer players during the pre-season. This concurrent effect between aerobic and neuromuscular training regimes induce impairment in power performance., Methods: this study aimed to investigate the effect of an equalized program of strength-power training (4-5 sessions/week) and soccer training (4-6 sessions/week) in power and aerobic performance during 8-weeks of pre-season in elite women soccer players. Vertical jumps [squat jump (SJ); countermovement jump (CMJ)] and Yo-Yo intermittent recovery level 1 test (YOYO-R1) were assessed pre- and post pre-season. A paired sample t-test was used to compare differences between pre and post pre-season (Δ%). The level of significance was established at p ≤ 0.05., Results: the women soccer players improved the SJ (p<0.001; Δ%=12), CMJ (p<0.001; Δ%=8.5), and YOYO-R1 (p<0.001; Δ% =28.5). There was a body recomposition observed, lower body fat (p = 0.004; Δ%=15), higher fat free mass (p = 0.001; Δ%=5)., Conclusions: our results demonstrated that it is possible to develop aerobic and power abilities of elite women soccer players during pre-season using an equalized ratio of soccer training and strength-power training schedules.

Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Melanotransferrin (MTf) is an iron-binding member of the transferrin superfamily that can be membrane-anchored or secreted in serum. On cells, it can mediate transferrin-independent iron uptake and promote proliferation. In serum, it is a transcytotic iron transporter across the blood-brain barrier. MTf has been exploited as a drug delivery carrier to the brain and as an antibody-drug conjugate (ADC) target due to its oncogenic role in melanoma and its elevated expression in triple-negative breast cancer (TNBC). For treatment of TNBC, an MTf-targeting ADC completed a phase I clinical trial (NCT03316794). The structure of its murine, unconjugated Fab fragment (SC57.32) is revealed here in complex with MTf. The MTf N-lobe is in an active and iron-bound, closed conformation while the C-lobe is in an open conformation incompatible with iron binding. This combination of active and inactive domains displays a novel inter-domain arrangement in which the C2 subdomain angles away from the N-lobe. The C2 subdomain also contains the SC57.32 glyco-epitope, which comprises ten protein residues and two N-acetylglucosamines. Our report reveals novel features of MTf and provides a point of reference for MTf-targeting, structure-guided drug design.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration 1 . Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma 2 . Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models 3-5 , a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results-combined with signalling studies and molecular dynamics simulations-delineate the structural basis for PTCH1-SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.

Background: Internet- and mobile-based interventions are effective for the treatment of chronic pain. However, little is known about patients' willingness to engage with these types of interventions and how the uptake of such interventions can be improved., Objective: The aim of this study was to identify people's acceptance, uptake, and adherence (primary outcomes) with regard to an internet- and mobile-based intervention for chronic pain and the influence of an information video as an acceptance-facilitating intervention (AFI)., Methods: In this randomized controlled trial with a parallel design, we invited 489 individuals with chronic pain to participate in a Web-based survey assessing the acceptance of internet- and mobile-based interventions with the offer to receive an unguided internet- and mobile-based intervention for chronic pain after completion. Two versions of the Web-based survey (with and without AFI) were randomly sent to two groups: one with AFI (n=245) and one without AFI (n=244). Participants who completed the Web-based survey with or without AFI entered the intervention group or the control group, respectively. In the survey, the individuals' acceptance of pain interventions, measured with a 4-item scale (sum score ranging from 4 to 20), predictors of acceptance, sociodemographic and pain-related variables, and physical and emotional functioning were assessed. Uptake rates (log in to the intervention) and adherence (number of completed modules) to the intervention was assessed 4 months after intervention access. To examine which factors influence acceptance, uptake rate, and adherence in the internet- and mobile-based interventions, we conducted additional exploratory subgroup analyses., Results: In total, 57 (intervention group) and 58 (control group) participants in each group completed the survey and were included in the analyses. The groups did not differ with regard to acceptance, uptake rate, or adherence (P=.64, P=.56, P=.75, respectively). Most participants reported moderate (68/115, 59.1%) to high (36/115, 31.3%) acceptance, with 9.6% (11/115) showing low acceptance (intervention group: mean 13.91, SD 3.47; control group: mean 13.61, SD 3.50). Further, 67% (38/57, intervention group) and 62% (36/58, control group) had logged into the intervention. In both groups, an average of 1.04 (SD 1.51) and 1.14 (SD 1.90) modules were completed, respectively., Conclusions: The informational video was not effective with regard to acceptance, uptake rate, or adherence. Despite the high acceptance, the uptake rate was only moderate and adherence was remarkably low. This study shows that acceptance can be much higher in a sample participating in an internet- and mobile-based intervention efficacy trial than in the target population in routine health care settings. Thus, future research should focus not only on acceptance and uptake facilitating interventions but also on ways to influence adherence. Further research should be conducted within routine health care settings with more representative samples of the target population., Trial Registration: German Clinical Trial Registration DRKS00006183; http://www.drks.de/drks&#95;web/navigate.do ?navigationId=trial.HTML&TRIAL&#95;ID=DRKS00006183 (Archived by WebCite at http://www.webcitation.org/70ebHDhne)., (©Jiaxi Lin, Bianca Faust, David Daniel Ebert, Lena Krämer, Harald Baumeister. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 21.08.2018.)

The aim of this study was to compare the effects of two different mixed training programs (optimum power load [OPL] + resisted sprints [RS] and OPL + vertical/horizontal plyometrics [PL]) on neuromuscular performance of elite soccer players during a short-term training preseason. Eighteen male professional soccer players took part in this study. The athletes were pair-matched in two training groups: OPL + RS and OPL + PL. Unloaded and resisted sprinting speeds at 5-, 10-, 20-, and 30-m, change of direction (COD) speed, and performance in the squat jump (SJ), countermovement jump (CMJ), and horizontal jump (HJ) were assessed pre- and post- a 5-week training period. Magnitude based inference with the effect sizes were used for data analysis. A possible increase in the SJ and CMJ heights and a likely increase in the HJ distance were observed in the OPL + PL group. Meaningful improvements were observed in the COD speed test for both training groups comparing pre- and post-measures. In both unloaded and resisted sprints, meaningful decreases were observed in the sprinting times for all distances tested. This study shows that a mixed training approach which comprises exercises and workloads able to produce positive adaptations in different phases of sprinting can be a very effective strategy in professional soccer players. Moreover, the possibility of combining optimum power loads with resisted sprints and plyometrics emerges as a novel and suitable option for coaches and sport scientists, due to the applicability and efficiency of this strength-power training approach.

Objective: PeaceHealth Sacred Heart Medical Center at RiverBend is a 379 bed hospital; 15,060 surgical procedures were performed in 2014 with 254 being colorectal surgical cases. Using the ACS NSQIP program the hospital was identified as a high outlier for surgical site infection (SSI) in all cases and in colorectal procedures in our July 2012 semiannual report (SAR)., Methods: A best practice bundle to reduce SSI's and a colorectal enhanced recovery pathway were concurrently initiated, Results: After implementation of these best practice elements, our ACS NSQIP results showed a reduction in our colorectal SSI rate from 17.58% for 2011 to 5.11% (1st decile/low outlier) in the January 2015 SAR. Our SSI rate for all cases were reduced from 4.87% to 1.71% (1st decile/low outlier) during the same time period. A reduction in length of stay for our colorectal surgical patients was also noted., Conclusions: The ACS NSQIP program was essential to identify colorectal SSI as an area of concern and for providing data to drive quality improvement measures., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis. In particular, ABC subfamily B member 7 (ABCB7) and its homologues in yeast and plants are required for iron-sulfur (Fe-S) cluster biosynthesis outside of the mitochondria, whereas ABCB10 is involved in haem biosynthesis. They also play a role in preventing oxidative stress. Mutations in ABCB6 and ABCB7 have been linked to human disease. Recent crystal structures of yeast Atm1 and human ABCB10 have been key to identifying substrate-binding sites and transport mechanisms. Combined with in vitro and in vivo studies, progress is being made to find the physiological substrates of the different mitochondrial ABC transporters., (© 2015 Authors; published by Portland Press Limited.)

The Na(+)-coupled betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.

Understanding the role of specific bilayer components in controlling the function of G-protein coupled receptors (GPCRs) will be a key factor in the development of novel pharmaceuticals. Cholesterol-dependence in particular has become an area of keen interest with respect to GPCR function; not least since the 2.6Å crystal structure of the β2 adrenergic receptor revealed a putative cholesterol binding motif conserved throughout class-A GPCRs. Furthermore, experimental evidence for cholesterol-dependent GPCR function has been demonstrated in a limited number of cases. This modulation of receptor function has been attributed to both direct interactions between cholesterol and receptor, and indirect effects caused by the influence of cholesterol on bilayer order and lateral pressure. Despite the widespread occurrence of cholesterol binding motifs, available experimental data on the functional involvement of cholesterol on GPCRs are currently limited to a small number of receptors. Here we investigate the role of cholesterol in the function of the neurotensin receptor 1 (NTS1) a class-A GPCR. Specifically we show how cholesterol, and the analogue cholesteryl hemisuccinate, influence activity, stability, and oligomerisation of both purified and reconstituted NTS1. The results caution against using such motifs as indicators of cholesterol-dependent GPCR activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: The pharmacokinetics, bioavailability, and tolerability of dalfampridine extended-release tablets in healthy adults under fed and fasted conditions were evaluated., Methods: The study participants (n = 30) were randomly assigned to receive one 10-mg dalfampridine tablet in a fasted condition (no food for 10-12 hours) or a fed condition (after a high-fat meal); after a seven-day washout period, participants received the same dalfampridine dosage under the converse condition. The endpoints were the maximum plasma drug concentration (C(max)) and areas under the plasma-concentration curve (AUC) for 24-hour exposure (AUC(0-24)) and total exposure (AUC(0-∞)). A 90% two-sided confidence interval (CI) within predefined limits for the fed:fasted ratio of the geometric mean values was used as the standard for determining the absence of a significant food effect., Results: Among the participants who received both treatments (n = 28), food intake was associated with a 23% increase in the log-transformed geometric mean C(max) of dalfampridine (p ≤ 0.10) but no significant change in mean AUC values. Eight (26.7%) of the study participants reported a total of 13 adverse events (AEs), of which only dizziness and upper abdominal pain occurred in more than one participant; all AEs were of mild-to-moderate severity., Conclusion: When a single 10-mg dose of dalfampridine was given to healthy volunteers after a high-fat meal, a significant increase in C(max) was observed. However, overall differences in dalfampridine pharmacokinetics when the drug was administered to participants under fasting and fed conditions did not exceed predefined limits, indicating that the extended-release formulation may be taken without regard to meals.

We have studied a number of effects that can give rise to errors in small-angle measurement systems when they are used to calibrate artifacts such as optical polygons. Of these sources of uncertainty, the most difficult to quantify are errors associated with the measurement of imperfect, non-flat faces of the artifact, causing the instrument to misinterpret the average orientation of the surface. In an attempt to shed some light on these errors, we have compared autocollimator measurements to angle measurements made with a Fizeau phase-shifting interferometer. These two instruments have very different operating principles and implement different definitions of the orientation of a surface, but (surprisingly) we have not yet seen any clear differences between results obtained with the autocollimator and with the interferometer. The interferometer is in some respects an attractive alternative to an autocollimator for small-angle measurement; it implements an unambiguous and robust definition of surface orientation in terms of the tilt of a best-fit plane, and it is easier to quantify likely errors of the interferometer measurements than to evaluate autocollimator uncertainty.

Background: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2x D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis., Results: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines., Conclusions: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.

From a cosmid library of Streptomyces cyanogenus S136, DNA fragments encompassing approximately 35 kb of the presumed landomycin biosynthetic gene cluster were identified and sequenced, revealing 32 open reading frames most of which could be assigned through data base comparison.