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Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven pentameric sodium pump. Its ability to translocate cations other than protons and to create an electrochemical potential makes it an attractive optogenetic tool. Tailoring its ion pumping characteristics by mutations is therefore of great interest. In addition, understanding the functional and structural consequences of certain mutations helps to derive a functional mechanism of ion selectivity and transfer of KR2. Based on solid-state NMR spectroscopy, we report an extensive chemical shift resonance assignment of KR2 within lipid bilayers. This data set was then used to probe site-resolved allosteric effects of sodium binding, which revealed multiple responsive sites including the Schiff base nitrogen and the NDQ motif. Based on this data set, the consequences of the H180A mutation are probed. The mutant is silenced in the presence of sodium while in its absence, proton pumping is observed. Our data reveal specific long-range effects along the sodium transfer pathway. These experiments are complemented by time-resolved optical spectroscopy. Our data suggest a model in which sodium uptake by the mutant can still take place, while sodium release and backflow control are disturbed.

The project «Healthy claws - the foundation for the future» aims to establish a Swiss national claw health monitoring based on digital recordings by claw trimmers during claw trimming. To assess claw health on the participating farms, between-herd prevalence, within-herd prevalence and cow prevalence of all claw disorders based on the «ICAR Claw Health Atlas» were calculated during this study. Claw trimmers underwent an intensive training and examination in order to ensure data quality. To guarantee the representativity of the prevalences, only farm claw trimmings were considered, where ≥ 80 % of the cows in a herd were trimmed. The calculations were based on 7108 cows and 403 heifers from 238 farms, during the period from February 2020 to February 2021. At least one claw disorder was present in 99,2 % of the farms, with 49,6 % of the heifers and 77,7 % of the cows having at least one claw disorder. The high prevalence is seen as a result of all ICAR claw disorders being considered, whereas not all of them are painful and consequently not all of them cause lameness. The absence of lameness assessment limits the evaluation of existing herd problems. High between-herd and cow prevalences were observed for the following claw disorders: heel horn erosion (92,9 %/64,7 %), digital dermatitis (55,9 %/20,7 %), white line disease (81,5 %/17,7 %) and sole hemorrhage (66,4 %/11,6 %). Asymmetric claws, corkscrew claws, scissor claws, horn fissure, interdigital phlegmon, swelling of the coronet and/or bulb and toe necrosis had low prevalences. The proportion of cows treated with a hoof block (0,5 %) was comparatively small in regard of the cows suffering from ulcers (5,6 %) and white line abscesses (2,5 %). The median within-herd prevalence of digital dermatitis was 5,6 %, with a maximal within-herd prevalence of 87,5 %. Despite the contagious nature of digital dermatitis, no increase of between-herd and cow prevalence has been observed in the past ten years throughout Switzerland. Based on this data, the Swiss claw health situation can be monitored, compared over time and improved in the future.Im Projekt «Gesunde Klauen – das Fundament für die Zukunft» wird ein schweizweites Klauengesundheitsmonitoring auf Basis der digitalen Dokumentation der Klauenpflege durch Klauenpfleger umgesetzt. Um die Klauengesundheit der teilnehmenden Betriebe zu evaluieren, wurden in der vorliegenden Studie inter-Herden-, in­- tra-Herden- und Kuhprävalenzen aller Klauenveränderungen gemäss «ICAR Atlas der Klauengesundheit» berechnet. Für die Gewährleistung der Datenerhebungsqualität erfolgte eine intensive Ausbildung und Prüfung der Klauenpfleger. Um die Repräsentativität der Prävalenzen sicherzustellen, wurden nur Betriebsklauenpflegen berücksichtigt, bei denen ≥ 80 % der Kühe einer Herde gepflegt wurden. Die Berechnungen erfolgten anhand von 7108 Kühen und 403 Rindern in 238 Betrieben, über einen Zeitraum von Februar 2020 bis Februar 2021. Bei 99,2 % der Betriebe war mindestens eine Klauenveränderung vorhanden, wobei 49,6 % der Rinder und 77,7 % der Kühe eine Klauenveränderung aufwiesen. Die hohe Prävalenz ergibt sich aus der Berücksichtigung aller ICAR Klauenveränderungen, von welchen nicht alle schmerzhaft sind und folglich nicht alle Lahmheiten verursachen. Durch das Fehlen einer Lahmheitserhebung ist die Evaluierung von vorliegenden Bestandesproblemen eingeschränkt. Hohe inter-Herden- sowie Kuhprävalenzen zeigten sich für die Erkrankungen Ballenhornfäule (92,9 %/64,7 %), Dermatitis digitalis (55,9 %/20,7 %), Weisse-Linie-Erkrankung (81,5 %/17,7 %) und für Sohlenblutungen (66,4 %/11,6 %). Niedrige Prävalenzen wurden für Ungleiche Klauen, Rollklauen, Scherenklauen, Hornkluft/-spalt, sowie Zwischenklauenphlegmone, Schwellung des Kronsaums und/oder des Ballens und Sohlenspitzennekrose verzeichnet. Verhältnismässig gering war der Anteil mit Klotz behandelter Kühe (0,5 %) im Verhältnis zu Kühen mit Klauengeschwüren (5,6 %) und Weisse-Linie-Abszessen (2,5 %). Der Median der intra-Herdenprävalenz von Dermatitis digitalis lag bei 5,6 %, die maximale intra-Herdenprävalenz bei 87,5 %. Trotz der Kontagiösität von Dermatitis digitalis wurde in den letzten zehn Jahren schweizweit keine Steigerung der inter-Herden- und Kuhprävalenz beobachtet. Auf Basis dieser Daten kann die Schweizer Klauengesundheitssituation überwacht, über die Jahre verglichen und künftig verbessert werden.Le projet « Des onglons sains-de bon pied vers l’avenir » vise à établir une surveillance nationale suisse de la santé des onglons basée sur des enregistrements numériques effectués par des ongleurs pendant le parage des onglons. Pour évaluer la santé des onglons dans les exploitations participantes, la prévalence entre les troupeaux, la prévalence au sein du troupeau et la prévalence chez les vaches de tous les troubles des onglons sur la base de l’ « ICAR Claw Health Atlas » ont été calculées au cours de cette étude. Les ongleurs ont subi une formation et un examen intensifs afin de garantir la qualité des données. Afin de garantir la représentativité des prévalences, seuls ont été considérés les parages d’exploitations où ≥ 80% des vaches du troupeau ont été parées. Les calculs ont été basés sur 7108 vaches et 403 génisses de 238 élevages, au cours de la période de février 2020 à février 2021. Au moins une lésion des onglons était présente dans 99,2 % des élevages, avec 49,6 % des génisses et 77,7 % des vaches présentant au moins une lésion. La prévalence élevée est considérée comme le résultat de toutes les lésions selon ICAR, alors que toutes ne sont pas douloureuses et par conséquent ne provoquent pas toute de boiterie. L’absence d’évaluation de la boiterie limite l’évaluation des problèmes de troupeau existants. Des prévalences élevées entre les troupeaux et les vaches ont été observées pour les lésions des onglons suivantes : érosion de la corne du talon (92,9%/64,7%), dermatite digitale (55,9%/20,7%), maladie de la ligne blanche (81,5 %/17,7%) et hémorragie de la sole (66,4%/11,6%). Les onglons asymétriques, les onglons en tire-bouchon, les onglons en ciseaux, la fissure de la corne, le phlegmon interdigital, le gonflement de la couronne et/ou la nécrose de la pince avaient de faibles prévalences. La proportion de vaches traitées avec un sabot (0,5 %) était comparativement faible par rapport aux vaches souffrant d’ulcères (5,6%) et d’abcès de la ligne blanche (2,5%). La prévalence médiane intra-troupeau de dermatite digitale était de 5,6%, avec une prévalence intra-troupeau maximale de 87,5%. Malgré le caractère contagieux de la dermatite digitale, aucune augmentation de la prévalence entre troupeaux et vaches n’a été observée au cours des dix dernières années dans toute la Suisse. Sur la base de ces données, la situation sanitaire des onglons en Suisse peut être surveillée, comparée dans le temps et améliorée à l’avenir.Nel progetto «Zoccoli sani – un’ottima base per il futuro», si vuole implementare un monitoraggio della salute degli zoccoli in tutta la Svizzera basato sulla documentazione digitale della cura degli zoccoli da parte dei tagliatori degli zoccoli. Al fine di valutare la salute degli zoccoli delle aziende partecipanti, nel presente studio sono state calcolate le prevalenze tra le mandrie, all’interno delle mandrie e delle vacche di tutti i cambiamenti degli zoccoli secondo l’”Atlante ICAR delle lesioni podali del bovino”. Per garantire la qualità della raccolta dei dati, si è svolta un’intensa attività di formazione e di valutazione dei tagliatori di zoccoli. Per garantire la rappresentatività delle prevalenze, sono stati presi in considerazione per lo studio, solo i tagliatori per le aziende, che si sono occupati del ≥ 80% delle vacche di una mandria. I calcoli sono stati effettuati utilizzando 7108 mucche e 403 bovini provenienti da 238 aziende, in un periodo che si situa tra febbraio 2020 e febbraio 2021. Nel 99,2% delle aziende è stata individuata almeno una lesione agli zoccoli di cui il 49,6% dei bovini e il 77,7% delle mucche avevano una lesione agli zoccoli. L’alta prevalenza risulta dalla considerazione di tutte le lesioni dello zoccolo secondo ICAR, di cui non tutte sono dolorose e quindi non causano zoppia. La mancanza di un’indagine sulla zoppia limita la valutazione dei problemi esistenti nella mandria. Sono state riscontrate alte prevalenze tra mandrie e vacche per l’erosione del corno dei talloni (92,9%/64,7%), la dermatite digitale (55,9%/20,7%), la malattia della fessura della linea bianca (81,5%/17,7%) e il sanguinamento della punta (66,4%/11,6%). Basse prevalenze sono state registrate per gli unghioni irregolari, gli unghioni a cavatappi, gli unghioni a forbice, la fessura orizzontale del corno, la fessura verticale del corno, così come il flemmone interdigitale, la tumefazione della corona e/o del bulbo e la necrosi della punta. La percentuale di mucche trattate con blocco dello zoccolo (0,5%) è stata relativamente bassa rispetto agli animali affetti da ulcere degli unghioni (5,6%) e ascessi della linea bianca (2,5%). La prevalenza mediana intra-allevamento della dermatite digitale era del 5,6% e la massima prevalenza intra-allevamento era dell’ 87,5%. Nonostante la natura contagiosa della dermatite digitale, negli ultimi dieci anni in Svizzera non è stato osservato alcun aumento della prevalenza tra le mandrie e tra le vacche. Sulla base di questi dati, la situazione della salute degli zoccoli in Svizzera può essere monitorata, confrontata negli anni e migliorata in futuro.

Channelrhodopsin‐2 (ChR2) is a light‐gated cation channel and was used to lay the foundations of optogenetics. Its dark state X‐ray structure has been determined in 2017 for the wild‐type, which is the prototype for all other ChR variants. However, the mechanistic understanding of the channel function is still incomplete in terms of structural changes after photon absorption by the retinal chromophore and in the framework of functional models. Hence, detailed information needs to be collected on the dark state as well as on the different photointermediates. For ChR2 detailed knowledge on the chromophore configuration in the different states is still missing and a consensus has not been achieved. Using DNP‐enhanced solid‐state MAS NMR spectroscopy on proteoliposome samples, we unambiguously determined the chromophore configuration in the desensitized state, and we show that this state occurs towards the end of the photocycle., Channelrhodopsin‐2, a light driven cation channel with a retinal chromophore, undergoes a cyclic photoreaction with several photointermediates. Here, we analyze the chromophore configuration of the intermediates, which can be cryo‐trapped, in detail by DNP‐enhanced solid‐state NMR spectroscopy and reveal that the desensitized state is in a 13‐cis,15‐syn configuration. Our results also shed further light on the photocycle of the protein.

Microbial rhodopsins represent the most abundant phototrophic systems known today. A similar molecular architecture with seven transmembrane helices and a retinal cofactor linked to a lysine in helix 7 enables a wide range of functions including ion pumping, light-controlled ion channel gating, or sensing. Deciphering their molecular mechanisms therefore requires a combined consideration of structural, functional, and spectroscopic data in order to identify key factors determining their function. Important insight can be gained by solid-state NMR spectroscopy by which the large homo-oligomeric rhodopsin complexes can be studied directly within lipid bilayers. This chapter describes the methodological background and the necessary sample preparation requirements for the study of photointermediates, for the analysis of protonation states, H-bonding and chromophore conformations, for 3D structure determination, and for probing oligomer interfaces of microbial rhodopsins. The use of data extracted from these NMR experiments is discussed in the context of complementary biophysical methods.

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) deficiency (MIM 261680, EC 4.1.1.32, encoded by PCK1) is a rare disorder of gluconeogenesis presenting with recurrent hypoglycemia, hepatic dysfunction, and lactic acidosis. We report on a previously healthy 3-year-old boy who was initially admitted under the suspicion of a febrile seizure during an upper airway infection. Diagnostic workup revealed hypoglycemia as well as a cerebral edema and ruled out an infection. After a complicated course with difficult to treat symptomatic seizures, the child died on the 5th day of admission due to progressive cerebral edema. The metabolic screening showed elevated urinary lactate and Krebs cycle intermediates in line with a primary or secondary energy deficit. Due to the unclear and fatal course, trio exome sequencing was initiated postmortem (“molecular autopsy”) and revealed the diagnosis of cytosolic PEPCK deficiency based on the compound heterozygosity of a known pathogenic (c.925G > A, p.(Gly309Arg)) and a previously unreported (c.724G > A, p.(Gly242Arg)) variant in PCK1 (NM_002591.3). Sanger sequencing ruled out the disease and carrier status in three older brothers. Molecular autopsy was performed due to the unclear and fatal course. The diagnosis of a cytosolic PEPCK deficiency not only helped the family to deal with the grief, but especially took away the fear that the siblings could be affected by an unknown disease in the same manner. In addition, this case increases the genetic and phenotypic spectrum of cytosolic PEPCK deficiency.

The metabolically engineered plant pathogen Ustilago maydis MB215 Delta cyp3 P(etef)ria1 has been cultivated to produce more than 80 g/L itaconate in 16 L scale pH and temperature controlled fermentation, in fed-batch mode with two successive feedings. The effect of pH as well as successive rounds of feeding has been quantified via elemental balances. Volumetric itaconic acid productivity gradually decreased with successive glucose feedings with increasing itaconic titers, with nearly constant product yield. Extracellular pH was decreased from 6 down to 3.5 and the fermentation was characterized in specific uptake, production, and growth rates. Notable is that the biomass composition changes significantly from growth phase to itaconic acid production phase, carbon content increases from 42% to around 62%. Despite the gradual decrease in itaconic acid levels with decreasing pH (nearly 50% decrease in itaconic acid at pH 3.5, compared to pH 6), significant itaconate production is still observed at pH 4 (around 63 g/L). Biomass yield remained nearly constant until pH 4. Taken together, these results strongly illustrate the potential of engineered Ustilago maydis in itaconate production at commercial levels.

Dynamic structural transitions within the seven‐transmembrane bundle represent the mechanism by which G‐protein‐coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist‐, and arrestin‐bound states of Y2R were prepared by cell‐free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were 13C‐labeled. NMR‐signal assignment was achieved by dynamic‐nuclear‐polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp2816.48 of the highly conserved SWLP motif and Trp3277.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved “cysteine lock”‐Trp11623.50 was identified., Structural transitions of crucial molecular switches of the activation of the human neuropeptide Y receptor type 2 are reported from the 13C‐tryptophan labeled molecule by solid‐state NMR spectroscopy in lipid membranes. The molecule shows high structural dynamics in the apo and agonist‐bound states, which is reduced by arrestin binding.

IntroductionThere is growing evidence that the health of young people with chronic health conditions deteriorates during the transfer from child-centred to adult-oriented health care. Risks include not only the deterioration of health status in general but also the occurrence of secondary diseases and adverse events. Transition programs have been implemented. However, there is a lack of evidence about whether they reduce these risks and which interventions should be principally included. Evidence-based guidelines for the transition of young people should be introduced. In this study we therefore aim to summarise actual evidence on somatic outcomes during the transition period.MethodsA systematic literature review was conducted. Two independent reviewers searched in electronic databases (Cochrane, Embase, PubMed, Web of Science) for intervention studies that aimed to improve transition. Last update of search was October 31st 2018. Grey literature was also searched. Studies were included if they examined participants aged 11 years or older suffering from a chronic health condition and evaluated interventions aimed to improve somatic outcomes after transition. Controlled trials or studies with a measurement before and after intervention were considered. The certainty of evidence was assessed using the GRADE approach. Additionally, each study was graded using a modified grading scale based on GRADE.Results28 studies met the inclusion criteria. Patients suffered from different chronic conditions such as type 1 diabetes, solid organ transplantation, inflammatory bowel disease or cystic fibrosis. Interventions had different components such as transition checklists, workshops, web-based interventions, transition plans, joint visits or transition coordinators. Outcomes included mortality and morbidity. They varied according to chronic condition. Thirteen studies showed beneficial effects in the intervention group or in post-intervention measurements. The certainty of evidence was very low.ConclusionA considerable number of studies evaluating transition interventions was identified. Transition interventions had some beneficial effects. Workshops, joint visits and longer or multidisciplinary appointments may be particularly effective components. Transition guidelines could be based on these results. However, due to the limitations of the included studies it is difficult to draw firm conclusions. More research is needed to further evaluate the effectiveness of transition interventions. It should address the deficits identified from prior studies, such as poor study design, short follow-up time or small sample sizes.

Increased antimicrobial use (AMU) and poor animal health are common problems on Swiss veal farms. The creation of incentive systems could contribute to reduce AMU and to implement supportive management measures. Voluntary government-funded programs could be an option. Therefore, the present study developed four hypothetical programs, which differed in their approach and funded measures. Depending on the program, the objective of reducing AMU should be achieved either by promotion of farm specific advice from the Swiss calf health service, benchmarking the antibiotic use, promotion of health measures on the source dairy (farm of birth) or by promoting veal calf fattening on the source dairy instead of selling calf to fattening operations. A survey using the contingent evaluation method evaluated the willingness of farm manager to participate in these programs. A total of 135 veal farm managers filled in the questionnaire, of which 131 were managers of family-run small scale veal fattening farms. In addition, 309 managers of dairy farms where no veal was produced participated in the study. These farms served as source dairies and sold excess calves for subsequent fattening mostly at the age of three to five weeks. Participation was moderate to good depending on the program. Approximately 30% of farm managers would participate in the program with farm specific advice from the Swiss calf health service and with benchmarking of AMU, 35% in the promotion of veal calf fattening on the source dairy, and 60% in health measures on the source dairy. Probability of participation in the program with farm specific advice and in health measures on source dairies can be influenced by the amount of the extra pay. The main reasons for which managers would not want to participate were having reservations about the need to improve animal health on the own farm, a lack of space and time, as well as concerns about increased administrative work. The study can be a starting point for the development of voluntary programs to reduce AMU on farms, in particular on source dairies and veal calf fattening farms. The results of this study may allow of estimation of the percentage of farm managers to participate in voluntary programs, thus addressing an optimal number of farms.Hoher Antibiotikaeinsatz und mangelnde Tiergesundheit sind häufige Problemstellungen auf Schweizer Kälbermastbetrieben. Zur Lösung könnte die Schaffung von Anreizsystemen beitragen, mittels derer die Reduktion des Antibiotikaeinsatzes und die Implementierung gezielter Managementmassnahmen gefördert würden. Dies könnte in Form freiwilliger, seitens der Behörden finanziell unterstützter Programme geschehen. Dafür wurden in der vorliegenden Studie vier hypothetische Programme erarbeitet, die sich in ihrer Vorgehensweise und den geförderten Massnahmen unterscheiden. Das Ziel «Antibiotikareduktion» soll je nach Programm entweder durch die Förderung betriebsspezifischer Beratung durch den Kälbergesundheitsdienst, ein Benchmarking des Antibiotikaeinsatzes, gesundheitsfördernde Massnahmen auf dem Geburtsbetrieb der Mastkälber oder durch die Förderung der Mast auf dem Geburtsbetrieb erreicht werden. Die Teilnahmebereitschaft an diesen Programmen seitens Betriebsleitender wurde mittels einer Umfrage mit der kontingenten Bewertungsmethode geprüft. Diese wurde von 135 Betriebsleitenden von Kälbermastbetrieben beantwortet, wovon 131 bäuerliche Kälbermast betreiben. Darüber hinaus nahmen 309 Betriebsleitende von Milchviehbetrieben teil, auf denen keine Kälber gemästet werden, welche aber als Geburtsbetriebe überzählige Kälber meist im Alter von drei bis fünf Wochen für die spätere Kälbermast verkaufen. Die Ergebnisse zeigen je nach Programm eine mässig bis gute Teilnahmebereitschaft. Am Programm mit betriebsspezifischer Beratung und am Programm mit Benchmarking des Antibiotikaeinsatzes würden rund 30% der Betriebsleitenden teilnehmen, am Programm für die Mast auf dem Geburtsbetrieb rund 35%, und bei Massnahmen auf dem Geburtsbetrieb rund 60%. Die Teilnahmewahrscheinlichkeit kann dabei beim Programm mit betriebsspezifischer Beratung und bei den Massnahmen auf dem Geburtsbetrieb durch die Höhe der Prämie beeinflusst werden. Die Hauptgründe für eine Nichtteilnahme sind Vorbehalte bezüglich der Notwendigkeit, Massnahmen am eigenen Betrieb zu ergreifen, mangelnder Platz und mangelnde Zeit sowie die Sorge vor erhöhtem administrativen Aufwand. Die Ergebnisse der Studie können als Ausgangspunkt für die Entwicklung freiwilliger Programme zur Antibiotikareduktion in der Kälbermast dienen, insbesondere für Geburtsbetriebe von Mastkälbern und Betriebe mit bäuerlicher Kälbermast. Mit den Schätzungen zur Teilnahmebereitschaft stehen im Voraus Informationen zur Verfügung, wie die Programme ausgestaltet werden müssten, um eine bestimmte Anzahl von Betrieben zu erreichen.L’utilisation élevée d’antibiotiques et la mauvaise santé des animaux sont des problèmes courants dans les élevages de veaux en Suisse. Une solution pourrait être la création de systèmes incitatifs visant à promouvoir la réduction de l’utilisation d’antibiotiques et la mise en place de mesures de gestion ciblées. Cela pourrait prendre la forme de programmes volontaires financés par le gouvernement. À cette fin, quatre programmes hypothétiques ont été développés dans la présente étude, lesquels diffèrent par leur approche et les mesures financées. Selon les programmes, l’objectif de «réduction des antibiotiques» doit être atteint soit par la promotion des conseils spécifiques à l’exploitation par le Service de santé des veaux, l’évaluation comparative de l’utilisation des antibiotiques, les mesures sanitaires dans les exploitations de naissance des veaux ou la promotion de l’engraissement sur l’exploitation de naissance. La volonté des exploitants de participer à ces programmes a été évaluée au moyen d’une enquête utilisant la méthode d’évaluation contingente. Cent trente-cinq responsables d’exploitations d’engraissement de veaux y ont répondu, dont 131 exploitations d’engraissement de veaux fermiers. De plus, 309 gestionnaires d’exploitations laitières qui n’engraissent aucun veau mais qui vendent pour l’engraissement des veaux excédentaires nés sur la ferme, principalement à l’âge de trois à cinq semaines, ont participé. Selon le programme, les résultats montrent une volonté modérée à bonne de participer. Environ 30% des exploitants participeraient au programme avec des conseils spécifiques à l’entreprise et au programme avec une évaluation comparative de l’utilisation des antibiotiques, environ 35% au programme d’engraissement à la ferme de naissance et environ 60% aux mesures à la ferme de naissance. La probabilité de participation au programme avec conseils spécifiques à l’entreprise et aux mesures sur la ferme d’origine peut être influencée par le montant de la prime. Les principales raisons de ne pas participer sont les réserves sur la nécessité de prendre des mesures dans la propre entreprise, le manque d’espace et de temps et les inquiétudes concernant l’augmentation des efforts administratifs. Les résultats de l’étude peuvent servir de base pour le développement de programmes volontaires de réduction de l’utilisation des antibiotiques chez les veaux d’engraissement, en particulier pour les fermes de naissance et les exploitations d’engraissement. Grâce aux estimations de la volonté de participer, des informations sont disponibles à l’avance sur la manière dont les programmes devraient être conçus pour atteindre un maximum d’exploitations.I problemi comuni nelle aziende da ingrasso di vitelli in Svizzera sono l’elevato uso di antibiotici e la cattiva salute degli animali. Per trovare delle soluzioni bisognerebbe creare un sistema di incentivi che favoriscano la diminuzione dell’uso di antibiotici e che implementino delle misure di gestione mirate. Questo potrebbe essere sotto forma di programmi su base volontaria sostenuti per la parte finanziaria dalle autorità. In questo studio sono stati creati quattro programmi ipotetici che si differenziano nella metodologia e nelle misure adottate. In relazione al programma, lo scopo della «riduzione di antibiotici» dovrebbe essere raggiunto o attraverso la promozione, da parte del servizio sanitario per i vitelli, di una consulenza specifica per l’azienda, o del monitoraggio dell’uso di antibiotici e delle misure per aumentare la salute nelle aziende di nascita dei vitelli da ingrasso oppure tramite la promozione dell’ingrasso nelle aziende di nascita. La disponibilità dei responsabili delle aziende di partecipare a questi programmi è stata valutata tramite un’inchiesta usando il metodo della valutazione contingente. Ben 135 responsabili delle aziende da ingrasso di vitelli hanno risposto, tra cui 131 effettuano l’ingrasso in fattoria. Hanno pure partecipato 309 responsabili di aziende per la produzione di latte, ma che non ingrassano animali, e che, come aziende di nascita, vendono i vitelli in eccesso di età tra le tre e le cinque settimane per il susseguente ingrasso. I risultati hanno mostrato che a seconda del programma vi è una disponibilità da moderata a buona. Circa il 30% dei responsabili delle aziende parteciperebbe al programma che provvede una consulenza specifica per l’azienda e al programma con il monitoraggio dell’uso di antibiotici, circa il 35% parteciperebbero al programma per l’ingrasso nell’azienda di nascita e circa il 60% per delle misure nelle aziende di nascita. La probabilità di partecipazione al programma con consulenza specifica per l’azienda e alle misure nell’azienda di nascita può essere influenzata dal livello del premio. Le ragioni principali della non partecipazione sono le riserve sulla necessità di prendere tali misure nella propria azienda, la mancanza di spazio e di tempo e le preoccupazioni per l’aumento del lavoro amministrativo. I risultati dello studio possono essere utilizzati come punti di riferimento per lo sviluppo di programmi a base volontaria per la diminuzione dell’uso di antibiotici nei vitelli da ingrasso, in particolare nelle aziende di nascita di vitelli da ingrasso e in aziende con vitelli da ingrasso. Grazie alle stime sulla disponibilità alla partecipazione si dispone già di informazioni su come i programmi dovrebbero essere concepiti alfine di raggiungere un definito numero di aziende.

Altering the properties of phospholipid membranes by light is an attractive option for the noninvasive manipulation of membrane proteins and cellular functions. Lipids with an azobenzene group within their acyl chains such as AzoPC are suitable tools for manipulating lipid order and dynamics through a light-induced trans-to-cis isomerization. However, the action of these photoswitchable lipids at the atomic level is still poorly understood. Here, liposomes containing AzoPC, POPE, and POPG have been characterized by solid-state NMR through chemical shift and dipolar CH order parameter measurements. Upon UV-light illumination, an efficient trans-to-cis conversion can be achieved resulting in a localized reduction of the CH order parameter within the bulk lipid acyl chains. This effect is even more pronounced in liposomes containing the integral membrane protein E. coli diacylglycerol kinase. The protein responds to the light-induced trans-to-cis isomerization by a site-specific increase in the molecular dynamics as observed by altered cross peak intensities in NCA spectra. This study represents a proof-of-concept demonstration for the use of photoswitchable lipids to modulate membrane properties by light for inducing dynamic changes within an embedded membrane protein.

Proteorhodopsin (PR) is a highly abundant, pentameric, light-driven proton pump. Proton transfer is linked to a canonical photocycle typical for microbial ion pumps. Although the PR monomer is able to undergo a full photocycle, the question arises whether the pentameric complex formed in the membrane via specific cross-protomer interactions plays a role in its functional mechanism. Here, we use dynamic nuclear polarization (DNP)-enhanced solid-state magic-angle spinning (MAS) NMR in combination with light-induced cryotrapping of photointermediates to address this topic. The highly conserved residue H75 is located at the protomer interface. We show that it switches from the (τ)- to the (π)-tautomer and changes its ring orientation in the M state. It couples to W34 across the oligomerization interface based on specific His/Trp ring orientations while stabilizing the pK(a) of the primary proton acceptor D97 within the same protomer. We further show that specific W34 mutations have a drastic effect on D97 and proton transfer mediated through H75. The residue H75 defines a cross-protomer Asp–His–Trp triad, which potentially serves as a pH-dependent regulator for proton transfer. Our data represent light-dependent, functionally relevant cross talk between protomers of a microbial rhodopsin homo-oligomer.

Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state. In addition, 50% of the KR2 13C and 15N resonances could be assigned by multidimensional high-field solid-state NMR experiments. Assigned residues include part of the NDQ motif as well as sodium binding sites. Based on these data, the structural effects of the H30A mutation, which seems to shift the ion selectivity of KR2 primarily to Na+, could be analysed. Our data show that it causes long-range effects within the retinal binding pocket and at the extracellular Na+ binding site, which can be explained by perturbations of interactions across the protomer interfaces within the KR2 complex. This study is complemented by data from time-resolved optical spectroscopy.

Based on Ernst-angle-type excitation and Acceleration by Sharing Adjacent Polarization (ASAP), a fast HSQC–TOCSY experiment is introduced. In the approach, the DIPSI–2 isotropic mixing period of the ASAP–HSQC is simply shifted, which provides a TOCSY period without additional application of rf-energy. The ASAP-HSQC-TOCSY allows the acquisition of a conventional 2D in about 30 s. Alternatively, it allows the acquisition of highly carbon-resolved spectra (several Hz digital resolution) on the order of minutes. An ASAP-HSQC-TOCSY-IPAP variant, finally, allows the sign-sensitive extraction of heteronuclear long-range coupling constants from a pair of highly resolved spectra in less than an hour. Pulse sequences, several example spectra, and a discussion of results are given.

Renin-angiotensin aldosterone system inhibitors are for a long time extensively used for the treatment of cardiovascular and renal diseases. AT1 receptor blockers (ARBs or sartans) act as antihypertensive drugs by blocking the octapeptide hormone Angiotensin II to stimulate AT1 receptors. The antihypertensive drug candesartan (CAN) is the active metabolite of candesartan cilexetil (Atacand, CC). Complexes of candesartan and candesartan cilexetil with 2-hydroxylpropyl-β-cyclodextrin (2-HP-β-CD) were characterized using high-resolution electrospray ionization mass spectrometry and solid state 13C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy. The 13C CP/MAS results showed broad peaks especially in the aromatic region, thus confirming the strong interactions between cyclodextrin and drugs. This experimental evidence was in accordance with molecular dynamics simulations and quantum mechanical calculations. The synthesized and characterized complexes were evaluated biologically in vitro. It was shown that as a result of CAN's complexation, CAN exerts higher antagonistic activity than CC. Therefore, a formulation of CC with 2-HP-β-CD is not indicated, while the formulation with CAN is promising and needs further investigation. This intriguing result is justified by the binding free energy calculations, which predicted efficient CC binding to 2-HP-β-CD, and thus, the molecule's availability for release and action on the target is diminished. In contrast, CAN binding was not favored, and this may allow easy release for the drug to exert its bioactivity.

Background Maternal immunological rejection of the semi-allogenic fetus is discussed as one of the significant factors involved in early pregnancy loss. An array of cytokines secreted by both maternal and fetal cells is involved in generating a delicate maternal immune tolerance. Interleukin 7 (IL-7) is discussed to play a key role in pro-inflammatory processes, but there is still limited insight into the pathophysiological input on placentation and embryonic development in early pregnancy loss. Patients and methods Cytokine level differences were identified with quantitative real-time PCR in placental tissue from spontaneous abortions (SA) (n=18), recurrent spontaneous abortions (RSA) (n=15), and healthy pregnancies (n = 15) at gestational weeks 7 to 14. Protein expression of IL-7 in the decidua was investigated by immunohistochemistry. IL-7 expressing cells were identified with double-immunofluorescence. Results Decidua of women with RSA expressed almost 51-times higher values of IL-7 in gene expression analysis. Immunohistochemistry identified a significant upregulation of IL-7 in the decidua of RSA specimens (p = 0.013) and in the decidua of women with SA (p = 0.004). Double-immunofluorescence confirmed decidual stroma cells as IL-7 expressing cells. Conclusion Significantly elevated IL-7 values in the decidua of spontaneous and recurrent miscarriages imply a crucial role of the cytokine in the signaling at the feto-maternal interface of the placenta. An overexpression of IL-7 could result in early pregnancy loss by inducing a pro-inflammatory environment. Proven to be valuable in other autoimmune diseases, targeting IL-7 signaling therapeutically may prove to be a very beneficial treatment option for RSA patients.

The evaluation of new animal husbandry concepts includes, beyond the exploration of chosen parameters, that the question is addressed whether economic benefits for producers and ethical benefits for society result of the new system when compared to traditional systems. In a previous study, a novel management and housing concept for veal production («outdoor veal calf», OVC) was tested from October 2016 to July 2018. The OVC concept was associated with distinctly lower antimicrobial use while average daily weight gain was similar and calf welfare better; furthermore mortality was lower compared to conventional label fattening. The aim of the present study was to compare economics between the OVC concept on intervention farms (IF) and the IP-SUISSE label concept on control farms (CF) by calculating contribution margins (CM). The CM indicates the difference between the performance of a production branch (e. g. slaughter return) and the allocable costs for this production branch (e. g. feed costs). In the present study, two different approaches were used for CM calculation to analyze the available data in detail. No substantial differences in CM between the OVC concept and the IP-SUISSE concept were found in either approach. However, the specific CM varied between the approaches as different calculation criteria for quantities and prices were applied (approach «real» with accurate costs and returns from the OVC study and approach «standardized» with costs and returns based on the CM catalogue). The CM varied slightly for the CM per man-hour (IF «real»: 6.96 CHF/man-hour, «standardized»: 4.49 CHF/man-hour; CF: 7.90 CHF/man-hour and 8.08 CHF/man-hour, respectively) and considerably for the CM per fattening space and year (IF «real»: -380.36 CHF, «standardized»: -559.50 CHF; CF: -244.70 CHF and -318.80 CHF, -respectively). Producing veal with the OVC concept -allowed for achieving comparable CM/man-hour as in conventional label production. Thus, the farmers' economic interests should be warranted when implementing the OVC concept.Bei der Entwicklung neuer Tierhaltungssysteme wird versucht, diese im Hinblick auf ausgewählte Parameter zu testen und zu evaluieren, ob eine Umstellung zu einem wirtschaftlichen oder gesellschaftlichen Mehrwert gegenüber bisherigen Systemen führen würde. In einer vorhergehenden Studie wurde von Oktober 2016 bis Juli 2018 ein neuartiges System der Kälbermast (Freiluftkalb (FLK)-System) getestet. Im Vergleich zur konventionellen Labelmast waren mit dem FLK-System deutlich weniger Antibiotika bei gleicher Tagesmastleistung und verbessertem Tierwohl verbraucht worden; die Mortalität der Mastkälber war zudem tiefer. Das Ziel der aktuellen Studie war die vergleichende Wirtschaftlichkeitsanalyse der beiden Haltungssysteme (FLK-Betriebe, FLKB) und der IP-SUISSE-Labelmast (Vergleichsbetriebe, VB) mithilfe der Berechnung von Deckungsbeiträgen (DB). Der DB gibt die Differenz zwischen der Leistung eines Produktionszweiges (z. B. Schlachterlös) und den zuteilbaren Kosten dieses Produktionszweiges (z. B. Futterkosten) an. In der vorliegenden Studie wurden zwei Varianten des DB berechnet, um die vorhandenen Daten detailliert auszuwerten. Beide Varianten kamen zu dem Ergebnis, dass keine wesentlichen Unterschiede im DB zwischen dem neuen FLK-System und der IP-­SUISSE-Labelmast bestehen. Die spezifischen DB variierten jedoch zwischen den Varianten, was an unterschiedlichen Berechnungsgrundlagen für Mengen und Preise je Variante lag (Variante «real» mit genauen Leistungen und Kosten aus FLK-Studie sowie Variante «standardisiert» mit Leistungen und Preisen auf Basis des DB-Katalogs). Die DB variierten leicht für den DB pro Arbeitskraftstunde (Akh) (FLKB Variante «real»: 6.96 CHF/Akh, Variante «standardisiert»: 4.49; VB: 7.90 bzw. 8.08) und erheblich für den DB pro Mastplatz und Jahr (FLKB Variante «real»: –380.36 CHF, Variante «standardisiert»: –559.50; VB: –244.70 bzw. –318.80). Die Produktion von Kalbfleisch gemäss dem FLK-System erlaubt es, einen positiven DB/Akh in ähnlicher Höhe zu erwirtschaften, wie es in der konventionellen IP-SUISSE-­Labelmast möglich ist. Demnach werden die wirt­schaftlichen Interessen der Tierhaltenden im neuen ­Haltungssystem mit grosser Wahrscheinlichkeit nicht vernachlässigt.Lors du développement de nouveaux systèmes d’élevage, on s’efforce de les tester par rapport à des paramètres sélectionnés pour évaluer si un changement apporterait une valeur ajoutée économique ou sociétale par rapport aux systèmes déjà établis. Dans une étude précédente, un nouveau type de système d’engraissement des veaux (système «veau en plein air», VPA) a été testé d’octobre 2016 à juillet 2018. Par rapport à l’engraissement conventionnel labellisé, considérablement moins d’antibiotiques ont été utilisés avec ce système tout en conservant les mêmes performances de croissance quotidienne et en améliorant le bien-être des animaux; de plus, la mortalité des veaux d’engraissement était plus faible. Le but de la présente étude était de procéder à une analyse économique comparative des deux systèmes de détention, VPA et label IP-SUISSE (IPS), à l’aide du calcul des marges brutes (MB). La MB indique la différence entre les performances d’une branche de production (par exemple les revenus d’abattage) et les coûts imputables à cette branche de production (par exemple les coûts d’alimentation). Dans cette étude, deux variantes des marges brutes ont été calculées afin d’évaluer en détail les données disponibles. Les deux variantes sont arrivées à la conclusion qu’il n’y avait pas de différences majeures dans la MB entre le nouveau système VPA et le système du label IP-SUISSE. Cependant, la MB spécifique différait entre les variantes, en raison des bases de calcul différentes utilisées pour chaque variante pour les quantités et les prix (variante «réelle» aves des revenus et coûts concrets dérivés de l’étude VPA et variante «standardisée» avec des revenus et des prix basés sur le catalogue des MB). Les MB variaient légèrement en ce qui concerne la MB par heure de travail productive (VPA: variante «réelle»: 6.96 CHF/heure, «standardisée»: 4.49 CHF/heure; IPS: variante «réelle» 7.90 CHF/heure, «standardisée» 8.08 CHF/heure) et considérablement pour la MB par place d’engraissement et par année (VPA: variante «réelle»: –380.36 CHF, «standardisée»: –559.50 CHF; IPS: variante «réelle»: –244.70 CHF, «standardisée»: –318.80 CHF). La production de viande de veau selon le système VPA permet de générer une MB/heure de travail positive similaire à celle de l’engraissement conventionnel labellisé IP-SUISSE. En conséquence, les intérêts économiques des producteurs ne paraissent pas être prétérités dans ce nouveau système d’élevage.Durante lo sviluppo di nuovi sistemi di allevamento, si cerca di testarli in relazione a determinati parametri e di valutare se il passaggio a un nuovo sistema porterebbe a un valore economico o sociale aggiunto rispetto ai sistemi precedenti. In uno studio precedente, un nuovo sistema d’ingrasso dei vitelli (sistema di allevamento all’aperto) è stato testato tra ottobre 2016 e luglio 2018. Rispetto al sistema d’ingrasso convenzionale con marchio IP-SUISSE, nel sistema di allevamento all’aperto vengono consumati meno antibiotici, mantenendo le stesse prestazioni di ingrasso giornaliero e migliorando il benessere degli animali; anche la mortalità dei vitelli da ingrasso risultava inferiore. L’obiettivo del presente studio era di effettuare un’analisi economica comparativa dei due sistemi allevamento, all’aperto e convenzionale con marchio IP-SUISSE calcolando i margini di contribuzione (MdC). Il MdC indica la differenza tra leprestazioni di un ramo di produzione (per esempio i ricavi della macellazione) e i costi allocabili a questo ramo di produzione (per esempio, i costi dei mangimi). Nel presente studio, sono state calcolate due varianti del MdC per valutare in dettaglio i dati disponibili. valutare i dati disponibili. Con entrambe le varianti si è giunti alla conclusione che non ci sono differenze significative nel MdC tra il nuovo sistema di allevamento all’aperto e e quello convenzionale con marchio l’IP-­SUISSE. Tuttavia, il MdC specifico differiva tra le varianti, a causa delle diverse basi di calcolo per le quantità e i prezzi per variante (variante «reale» con costi e ricavi esatti dallo studio del sistema di allevamento all’aperto e variante «standardizzata» con costi e ricavi basati sul catalogo MdC). Il MdC varia leggermente per il MdC per ora di lavoro produttivo (variante «reale» del sistema di allevamento all’aperto: 6.96 CHF/ora, variante «standardizzata»: 4.49 CHF/ora; per il sistema convenzionale 7.90 CHF/ora e 8.08 CHF/ora rispettivamente) e notevolmente per il MdC per luogo d’ingrasso e anno (variante «reale» del sistema di allevamento all’aperto: –380.36 CHF, variante «standardizzata»: –559.50 CHF; per il sistema convenzionale –244.70 CHF e –318.80 CHF rispettivamente). La produzione di carne di vitello secondo il sistema di allevamento all’aperto permette di ottenere un MdC/ora di lavoro positivo di entità simile a quello ottenuto con il sistema convenzionale con marchio IP-SUISSE. Di conseguenza, è probabile che gli interessi economici degli allevatori non vengano trascurati con il nuovo sistema di allevamento.

Introduction Whole-body cryotherapy (WBC) has shown to be beneficial in the treatment of fibromyalgia (FM). There is cumulative evidence that cytokines play a crucial role in FM. It’s unknown whether clinical effects of WBC can be demonstrated at the molecular level and how long the effects last. Methods We compared effects of serial WBC (6 sessions (− 130 °C in 6 weeks) in FM patients and healthy controls (HC). Primary outcome was the change in pain level (visual analogue scale 0–100 mm) after 6 sessions. Secondary outcomes were a change in disease activity (revised Fibromyalgia Impact Questionnaire) and pain after 3 sessions and 3 months after discontinued therapy and in cytokine levels (interleukin (IL-)1, IL-6, tumor necrosis factor α (TNF-α) and IL-10). The patients’ opinions on the satisfaction, effectiveness and significance of WBC were evaluated. Results Twenty-three FM patients and 30 HC were enrolled. WBC resulted in a significant reduction in pain and disease activity after 3 and 6 sessions. No clinical benefit could be measured 3 months after discontinued treatment. Overall, probands were satisfied with WBC and considered WBC to be important and effective. FM patients had significantly different levels of IL-1, IL-6, TNF-α and IL-10 at each reading point compared to HC. Levels of IL-1, IL-6 and IL-10 were significantly altered over time in FM patients. Compared to HC FM patients showed a significantly different response of IL1, − 6 and − 10 to WBC. Conclusion Serial WBC is a fast acting and effective treatment for FM. Proven effects of WBC may be explained by changes in cytokines.

Ustilago maydis, member of the Ustilaginaceae family, is a promising host for the production of several metabolites including itaconic acid. This dicarboxylate has great potential as a bio-based building block in the polymer industry, and is of special interest for pharmaceutical applications. Several itaconate overproducing Ustilago strains have been generated by metabolic and morphology engineering. This yielded stabilized unicellular morphology through fuz7 deletion, reduction of by-product formation through deletion of genes responsible for itaconate oxidation and (glyco)lipid production, and the overexpression of the regulator of the itaconate cluster ria1 and the mitochondrial tricarboxylate transporter encoded by mttA from Aspergillus terreus. In this study, itaconate production was further optimized by consolidating these different optimizations into one strain. The combined modifications resulted in itaconic acid production at theoretical maximal yield, which was achieved under biotechnologically relevant fed-batch fermentations with continuous feed.

Chronically ill young people must transition to the adult health care service after their 18th birthday. The transition from child centred paediatric care to the adult health care service is not simply limited to the change from familiar structures to something unknown, but includes the entire process of growing up, of individuals becoming independent from their parents and taking responsibility for their own disease management. Young people are at particular risk of losing the connection to medical care during this phase and the transition of young people with chronic conditions is associated with a high risk of declining adherence and worsening health status. Studies suggest that transition programs might be helpful, yet there is no evidence as to whether risks can be reduced or which intervention components are particularly conducive to better outcome. This study aimed to identify transition-specific interventions and evaluate their effect on the improvement of psychosocial parameters, such as health related quality of life and adherence of patients. A systematic literature review was conducted. Electronic databases (Cochrane, Embase, Pubmed, Web of Science) were searched by two independent reviewers for intervention studies aiming to improve transition. Grey literature was also searched. Studies were included if they evaluated transition-specific interventions aiming to improve psychosocial or adherence parameters of participants aged 12 years and older suffering from a chronic condition. Both controlled trials and studies with measurements before an after the intervention were included. The GRADE approach was used to assess the quality of evidence. The inclusion criteria was met by forty studies. Patients suffered from different chronic conditions, such as inflammatory bowel disease, type 1 diabetes or juvenile idiopathic arthritis. Transition interventions used several program components, such as transition coordinators, patient education programs or web-based interventions. Outcomes included quality of life, transition-specific knowledge, adherence and loss to follow up. Thirty-eight studies showed beneficial effects in the intervention group, respectively after intervention. The overall study quality was low. A large number of studies evaluating transition-specific interventions was included. Transition-specific interventions seem to have beneficial effects on psychosocial outcomes and adherence. The promotion of health literacy, appointment arrangement service and the use of technical elements (websites, SMS) seem to be particularly helpful in the transition process. As the patient population was diverse, the results can be transferred to other diseases. Even though the overall study quality was poor, it is possible to draw some conclusions. Future studies should aim to include large numbers of patients over extended periods of time in order to assess long-term outcomes.

Light‐induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid‐state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS‐NMR conditions. Uncaging of NPE‐ATP or of lipid substrate NPE‐DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real‐time 31P‐MAS NMR. This proof‐of‐concept illustrates that combining MAS‐NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers., Engineering light control: Light‐induced release of caged substrates to initiate enzymatic activity of the membrane enzyme diacylglycerol kinase. A first demonstration of in situ light control for biomolecular solid‐state NMR, highlighting its potential advantages to initiate reactions in membrane systems (see figure).

MsbA, a homodimeric ABC exporter, translocates its native substrate lipid A as well as a range of smaller, amphiphilic substrates across the membrane. Magic angle sample spinning (MAS) NMR, in combination with dynamic nuclear polarization (DNP) for signal enhancement, has been used to probe two specific sites in transmembrane helices 4 and 6 of full length MsbA embedded in lipid bilayers. Significant chemical shift changes in both sites were observed in the vanadate-trapped state compared to apo state MsbA. The reduced spectral line width indicates a more confined conformational space upon trapping. In the presence of substrates Hoechst 33342 and daunorubicin, further chemical shift changes and line shape alterations mainly in TM6 in the vanadate trapped state were detected. These data illustrate the conformational response of MsbA towards the presence of drugs during the catalytic cycle. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Isotope labeling enables the use of 13C-based metabolomics techniques with strongly improved resolution for a better identification of relevant metabolites and tracing of metabolic fluxes in cell and animal models, as required in fluxomics studies. However, even at high NMR-active isotope abundance, the acquisition of one-dimensional 13C and classical two-dimensional 1H,13C-HSQC experiments remains time consuming. With the aim to provide a shorter, more efficient alternative, herein we explored the ALSOFAST-HSQC experiment with its rapid acquisition scheme for the analysis of 13C-labeled metabolites in complex biological mixtures. As an initial step, the parameters of the pulse sequence were optimized to take into account the specific characteristics of the complex samples. We then applied the fast two-dimensional experiment to study the effect of different kinds of antioxidant gold nanoparticles on a HeLa cancer cell model grown on 13C glucose-enriched medium. As a result, 1H,13C-2D correlations could be obtained in a couple of seconds to few minutes, allowing a simple and reliable identification of various 13C-enriched metabolites and the determination of specific variations between the different sample groups. Thus, it was possible to monitor glucose metabolism in the cell model and study the antioxidant effect of the coated gold nanoparticles in detail. Finally, with an experiment time of only half an hour, highly resolved 1H,13C-HSQC spectra using the ALSOFAST-HSQC pulse sequence were acquired, revealing the isotope-position-patterns of the corresponding 13C-nuclei from carbon multiplets. Graphical abstract Fast NMR applied to metabolomics and fluxomics studies with gold nanoparticles.

Malaria blood stage parasites develop within red blood cells where they are contained in a vacuolar compartment known as the parasitophorous vacuole (PV). This compartment holds a key role in the interaction of the parasite with its host cell. However, the proteome of this compartment has so far not been comprehensively analysed. Here we used BioID in asexual blood stages of the most virulent human malaria parasite Plasmodium falciparum to identify new proteins of the PV. The resulting proteome contained many of the already known PV proteins and validation by GFP-knock-in of 10 previously in P. falciparum uncharacterised hits revealed 5 new PV proteins and two with a partial PV localisation. This included proteins peripherally attached to the inner face of the PV membrane as well as proteins anchored in the parasite plasma membrane that protrude into the PV. Using selectable targeted gene disruption we generated mutants for 2 of the 10 candidates. In contrast we could not select parasites with disruptions for another 3 candidates, strongly suggesting that they are important for parasite growth. Interestingly, one of these included the orthologue of UIS2, a protein previously proposed to regulate protein translation in the parasite cytoplasm but here shown to be an essential PV protein. This work extends the number of known PV proteins and provides a starting point for further functional analyses of this compartment.

Previously we introduced two novel NMR experiments for small molecules, the so-called ASAP-HSQC and ALSOFAST-HSQC (Schulze-Sunninghausen et al., 2014), which allow the detection of heteronuclear one-bond correlations in less than 30s at natural abundance. We propose an improved symmetrized pulse scheme of the basic experiment to minimize artifact intensities and the combination with non-uniform sampling to enable the acquisition of conventional HSQC spectra in as short as a couple of seconds and extremely 13C-resolved spectra in less than ten minutes. Based on steady state investigations, a first estimate to relative achievable signal intensities with respect to conventional, ASAP-, and ALSOFAST-HSQC experiments is given. In addition, we describe several extensions to the basic pulse schemes, like a multiplicity-edited version, a revised symmetrized CLIP-ASAP-HSQC, an ASAP-/ALSOFAST-HSQC sequence with broadband BIRD-based 1H,1H decoupling, and a symmetrized sequence optimized for water suppression. Finally, RF-power considerations with respect to the high duty cycle of the experiments are given.

The interactions of irbesartan (IRB) and irbesartan-2-hydroxypropyl-β-cyclodextrin (HP-β-CD) complex with dipalmitoyl phosphatidylcholine (DPPC) bilayers have been explored utilizing an array of biophysical techniques ranging from differential scanning calorimetry (DSC), small angle X-ray scattering (SAXS), ESI mass spectrometry (ESI-MS) and solid state nuclear magnetic resonance (ssNMR). Molecular dynamics (MD) calculations have been also conducted to complement the experimental results. Irbesartan was found to be embedded in the lipid membrane core and to affect the phase transition properties of the DPPC bilayers. SAXS studies revealed that irbesartan alone does not display perfect solvation since some coexisting irbesartan crystallites are present. In its complexed form IRB gets fully solvated in the membranes showing that encapsulation of IRB in HP-β-CD may have beneficial effects in the ADME properties of this drug. MD experiments revealed the topological and orientational integration of irbesartan into the phospholipid bilayer being placed at about 1nm from the membrane centre.

Renin Angiotensin System (RAS) plays a key role in the pathophysiology of the cardiovascular and renal system. Within this system stimulation of the G-protein coupled receptor (GPCR) AT1 by the peptide angiotensin II (AII) has a central role. Molecules have been discovered which either block the formation of AII or compete with the activation of the AT1 receptor. Angiotensin Receptor Blockers (ARBs) or sartans are the first synthetic drugs of the latter class. Up to now the question has not been answered how the drug molecules reach the receptor site. Are these molecules incorporated in the lipid bilayers core before they freely diffuse to reach the active site or do they enter at the receptor binding site directly from the water phase? Both mechanisms are possible and may have a synergistic action. Various biophysical techniques, such as calorimetric, spectroscopic and X-ray scattering methods can provide valuable information on the dynamic and thermodynamic changes caused when drugs are incorporated into the lipid bilayers. Molecular Dynamics (MD) simulations are a valuable tool to acquire atomic level information on the molecular basis of the interaction between the drugs and the RAS system. The purpose of this chapter is to review the contribution of the various biophysical techniques and MD on drug-membrane interactions on the RAS system. Studies described in the manuscript are mainly referred to drugs acting on the GPCR AT1 receptor, however it is envisaged that the discussed concepts of drug interaction can apply also to other GPCRs.

Amorphous formulation technologies to improve oral absorption of poorly soluble active pharmaceutical ingredients (APIs) have become increasingly prevalent. Currently, polymer-based amorphous formulations manufactured by spray drying, hot melt extrusion (HME), or co-precipitation are most common. However, these technologies have challenges in terms of the successful stabilization of poor glass former compounds in the amorphous form. An alternative approach is mesoporous silica, which stabilizes APIs in non-crystalline form via molecular adsorption inside nano-scale pores. In line with these considerations, two poor glass formers, haloperidol and carbamazepine, were formulated as polymer-based solid dispersion via HME and with mesoporous silica, and their stability was compared under accelerated conditions. Changes were monitored over three months with respect to solid-state form and dissolution. The results were supported by solid-state nuclear magnetic resonance spectroscopy (SS-NMR) and scanning electron microscopy (SEM). It was demonstrated that mesoporous silica was more successful than HME in the stabilization of the selected poor glass formers. While both drugs remained non-crystalline during the study using mesoporous silica, polymer-based HME formulations showed recrystallization after one week. Thus, mesoporous silica represents an attractive technology to extend the formulation toolbox to poorly soluble poor glass formers.

Background: The diagnosis of low-grade infections of endoprostheses is challenging. There are still no unified guidelines for standardised diagnostic approaches, recommendations are categorised into major and minor criteria. Additional histopathological samples might sustain the diagnosis. However, ambulatory preoperative biopsy collection is not widespread. Method: 102 patients with hip or knee endoprosthesis and suspected periprosthetic joint infection (PJI) were examined by arthrocentesis with microbiological sample and histopathological punch biopsy. The data were retrospectively analysed for diagnosis concordance. Results: Preoperative microbiology compared to intraoperative results was positive in 51.9% (sensitivity 51.9%, specificity 97.3%). In comparison of preoperative biopsy to intraoperative diagnostic results 51.9% cases were positive (sensitivity 51.9%, specificity 100.0%). The combination of preoperative biopsy and microbiology in comparison to intraoperative results was positive in 70.4% of the cases (sensitivity 70.4%, specificity 97.3%). Conclusion: The diagnosis of PJI is complex. One single method to reliably detect an infection is currently not available. With the present method histopathological samples might be obtained quickly, easily and safely for the preoperative detection of PJI. A combination of microbiological and histopathological sampling increases the sensitivity up to 18.5% to detect periprosthetic infection.

Mesoporous silica has emerged as an enabling formulation for poorly soluble active pharmaceutical ingredients (APIs). Unlike other formulations, mesoporous silica typically does not inhibit precipitation of supersaturated API therefore, a suitable precipitation inhibitor (PI) should be added to increase absorption from the gastrointestinal (GI) tract. However, there is limited research about optimal processes for combining PIs with silica formulations. Typically, the PI is added by simply blending the API-loaded silica mechanically with the selected PI. This has the drawback of an additional blending step and may also not be optimal with regard to release of drug and PI. By contrast, loading PI simultaneously with the API onto mesoporous silica, i.e. co-incorporation, is attractive from both a performance and practical perspective. The aim of this study was to demonstrate the utility of a co-incorporation approach for combining PIs with silica formulations, and to develop a mechanistic rationale for improvement of the performance of silica formulations using the co-incorporation approach. The results indicate that co-incorporating HPMCAS with glibenclamide onto silica significantly improved the extent and duration of drug supersaturation in single-medium and transfer dissolution experiments. Extensive spectroscopic characterization of the formulation revealed that the improved performance was related to the formation of drug-polymer interactions already in the solid state; the immobilization of API-loaded silica on HPMCAS plates, which prevents premature release and precipitation of API; and drug-polymer proximity on disintegration of the formulation, allowing for rapid onset of precipitation inhibition. The data suggests that co-incorporating the PI with the API is appealing for silica formulations from both a practical and formulation performance perspective.

Isotope labeling enables the use of