Single-Molecule Atomic Force Microscopy Reveals Clustering of the Yeast Plasma-Membrane Sensor Wsc1

Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains (“rafts”) is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of ∼200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches

Development of an HPLC-MS/MS Method for Chiral Separation and Quantitation of (R)- and (S)-Salbutamol and Their Sulfoconjugated Metabolites in Urine to Investigate Stereoselective Sulfonation

The aim of this study was to develop and optimize a chiral HPLC-MS/MS method for quantitative analysis of (R)-/(S)-salbutamol and (R)-/(S)-salbutamol-4′-O-sulfate in human urine to allow for bioanalytical quantitation of the targeted analytes and investigations of stereoselectivity in the sulfonation pathway of human phase Ⅱ metabolism. For analytical method development, a systematic screening of columns and mobile phases to develop a separation via enantiomerically selective high performance liquid chromatography was performed. Electrospray ionization settings were optimized via multiple-step screening and a full factorial design-of-experiment. Both approaches were performed matrix-assisted and the predicted values were compared. The full factorial design was superior in terms of prediction power and knowledge generation. Performing a longitudinal excretion study in one healthy volunteer allowed for the calculation of excretion rates for all four targeted analytes. For this proof-of-concept, either racemic salbutamol or enantiopure levosalbutamol was administered perorally or via inhalation, respectively. A strong preference for sulfonation of (R)-salbutamol for inhalation and peroral application was found in in vivo experiments. In previous studies phenol sulfotransferase 1A3 was described to be mainly responsible for salbutamol sulfonation in humans. Thus, in vitro and in silico investigations of the stereoselectivity of sulfotransferase 1A3 complemented the study and confirmed these findings

Biosynthesis of Salbutamol-4′-O-sulfate as Reference for Identification of Intake Routes and Enantiopure Salbutamol Administration by Achiral UHPLC-MS/MS

The aim of the study was a comprehensive and quantitative determination of salbutamol and its sulfoconjugated major metabolite in urine samples using achiral ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Therefore, salbutamol-4′-O-sulfate was biosynthesized as a reference using genetically modified fission yeast cells, and the product was subsequently characterized by NMR and HRMS. In competitive sports, salbutamol is classified as a prohibited drug; however, inhalation at therapeutic doses is permitted with a maximum allowance of 600 µg/8 h. In contrast, the enantiopure levosalbutamol is prohibited under any condition. For analytical discrimination, the amount of salbutamol and its main metabolite excreted in the urine was studied. As proof of concept, a longitudinal study in one healthy volunteer was performed in order to investigate excreted amounts and to study potential discrimination using achiral chromatography. Discrimination of administration of racemic salbutamol or the enantiopure levosalbutamol was not achieved by solely analyzing salbutamol as the parent compound. However, a distinction was possible by evaluation of the proportion of salbutamol-4′-O-sulfate in relation to salbutamol. Therefore, reference material of metabolites is of great importance in doping control, especially for threshold substances

Characterization of the essential role of Ynl152/Inn1 in cell division in Saccharomyces cerevisiae

The essential open reading frame YNL152w (now called INN1) of Saccharomyces cerevisiae was previously identified in a screen for negative regulators of cell integrity signaling. Subsequent studies and data from genome-wide functional analyses suggested, that the encoded protein plays a role in cell division. This was further addressed in the thesis presented here. Functional Inn1-GFP fusions were shown to co-localize with the contractile actomyosin ring component Myo1 during cytokinesis. Mutants depleted for Inn1 failed to form a primary septum, but did not affect the dynamics of the cytokinetic actin ring (CAR). This has been attributed to the inability to couple plasma membrane ingression (hence Inn1) to CAR constriction, a phenomenon also found by Sanchez-Diaz et al. (2008). Further investigations focused on the question of how Inn1 is recruited to the bud neck and identified the cytokinetic regulators Hof1 and Cyk3, which act in concert for this purpose. Each of them contains a SH3 domain, which interacts with the proline-rich carboxy-terminal part of Inn1. Localization studies and genetic analyses indicate that Inn1 acts downstream of Hof1 and Cyk3. Either the simultaneous repression of HOF1 and CYK3 gene expression or the deletion of their SH3 domains was lethal, with a concomitant failure to localize Inn1-GFP to the division site. Overproduction of either, Hof1 or Cyk3 perturbed the dynamics of Inn1-GFP distribution, which followed that of the overproduced proteins. This atypical CAR-independent localization of Inn1 supports a presumed role of Hof1 and Cyk3 in an alternative cytokinesis pathway to form a primary septum. Since INN1 is also a multicopy suppressor of a myo1 deletion, this further supports the notion that Inn1 may be required for plasma membrane ingression, also in CAR-independent cytokinesis. Preliminary data suggest, that the protein Vrp1 is responsible for the required removal of Inn1 from the bud neck after completion of cytokinesis. The essential amino-terminal C2 domain of Inn1 may mediate plasma membrane ingression by interaction with the membrane lipid phosphatidic acid, observed in biochemical studies. Alternatively, the C2 domain has been suggested to modulate chitin synthesis in the primary septum by modulating Chs2 activity (Nishihama et al., 2009)

Nutritional counseling in oncology - A questionnaire-based analysis to assess the subjective and medical counseling needs of patients with cancer at the Comprehensive Cancer Center of Würzburg University Hospital

Hintergrund Mangelernährung bleibt im klinischen Alltag noch oft unerkannt und wird häufig unterschätzt. Die durchgeführte Studie hatte das Ziel, die Häufigkeit eines Ernährungsrisikos sowie die Patientengruppen, welche am meisten von einer Ernährungsberatung profitieren würden, zu ermitteln. Methode Ambulant versorgte Patienten mit Tumorerkrankungen des Universitätsklinikums Würzburgs wurden mittels eines vom Ernährungsteam des Comprehensive Cancer Centers erstellten Fragebogens zwischen Mai 2017 und Januar 2018 befragt. Es wurden insbesondere Fragen zum Ernährungszustand und Ernährungsproblemen gestellt. Zudem wurde das Risiko für das Entstehen einer Mangelernährung mittels des validierten Screening-Fragebogens Malnutrition Universal Screening Tool (MUST) erfasst. Ergebnisse In der vorliegenden Studie wurden 311 Patienten befragt. Im MUST-Screening zeigte sich bei 16,4 % ein mittleres und bei 20,3 % ein hohes Risiko für eine Mangelernährung, wobei die Punktevergabe in 94,8 % der Fälle durch einen ungewollten Gewichtsverlust erfolgte. Insbesondere Patienten der Gastroenterologie sowie Patienten > 65 Jahre wiesen ein hohes Risiko auf. Es zeigte sich ein signifikanter Zusammenhang zwischen stattgehabter Chemotherapie und einem MUST-Score ≥ 2 (OR = 3,6). Als besondere Risikofaktoren ließen sich zudem Geschmackveränderungen, Schluckbeschwerden, Ekelempfinden und Appetitlosigkeit feststellen (OR = 2,3 – 3,2). Interesse am Thema „Ernährung bei Krebs“ zeigten vor allem junge, weibliche und normalgewichtige Patienten. Ein Gespräch mit dem behandelten Arzt hierzu fand nur bei 38 % aller Patienten statt. Schlussfolgerungen Jeder fünfte Patient unterlag einem hohen Ernährungsrisiko, nur ein Bruchteil wäre durch Erhebung des Body Mass Index aufgefallen. Ein valides Screeningverfahren mit aussagekräftigen Parametern sollte Einzug in den klinischen Alltag ambulant versorgter Krebspatienten finden und gemeinsam mit einer Ernährungsberatung standardisiert bei Diagnosestellung sowie in regelmäßigen Abständen im Verlauf stattfinden.Background Malnutrition still often remains unrecognized in clinical practice and is frequently underestimated. The aim of this study was to determine the frequency of nutritional risk and the patient groups that would benefit most from nutritional counseling. Methods Outpatients with tumor diseases at the University Hospital of Würzburg were surveyed using a questionnaire prepared by the Comprehensive Cancer Center's nutrition team between May 2017 and January 2018. In particular, questions were asked about nutritional status and nutritional problems. In addition, the risk of developing malnutrition was assessed using the validated screening questionnaire Malnutrition Universal Screening Tool (MUST). Results In the present study, 311 patients were interviewed. MUST screening showed 16.4% to be at moderate risk of malnutrition and 20.3% to be at high risk of malnutrition, with scoring by unintentional weight loss in 94.8% of cases. Gastroenterology patients and patients > 65 years of age were at particularly high risk. There was a significant association between chemotherapy given and a MUST score ≥ 2 (OR = 3.6). In addition, changes in taste, difficulty swallowing, disgust, and loss of appetite could be identified as particular risk factors (OR = 2.3 - 3.2). Interest in the topic of "nutrition in cancer" was mainly shown by young, female and normal-weight patients. A discussion with the treating physician on this topic took place in only 38% of all patients. Conclusions One in five patients was at high nutritional risk, only a fraction would have been detected by body mass index. A valid screening procedure with meaningful parameters should be introduced into the clinical routine of outpatient cancer patients and, together with nutritional counseling, should take place in a standardized manner at the time of diagnosis and at regular intervals during the course of treatment. Translated with www.DeepL.com/Translator (free version

Nursing care of the female patient with diagnosed amniotic fluid embolism during the Caesarean section in the spinal anesthesia — a case report

Wstęp. Zator płynem owodniowym jest powikłaniem rzadkim, ale poważnym, często ze skutkiem śmiertelnym dla matki. Patofizjologia zatorowości płynem owodniowym jest wieloczynnikowa i nadal słabo poznana. Cel pracy. Celem pracy było zdiagnozowanie problemów pielęgnacyjnych w opiece nad pacjentką z rozpoznaną zatorowością płucną płynem owodniowym w trakcie cięcia cesarskiego w znieczuleniu przewodowym oraz w czasie pobytu na oddziale intensywnej terapii. Materiał i metody. Do badań wykorzystano studium indywidualnego przypadku. Badanie miało charakter retrospektywny. Zastosowaną techniką badawczą był wywiad, analiza dokumentacji medycznej — historii choroby, karty znieczulenia, procesu pielęgnowania oraz wyników badań laboratoryjnych i obrazowych. Wyniki i wnioski. Wszystkie rozpoznane problemy zostały całościowo i pozytywnie rozwiązane, pozostała część problemów pielęgnacyjnych wymagała kontynuowania działań pielęgniarskich po przeniesieniu pacjentki z oddziału intensywnej terapii na kolejne etapy leczenia szpitalnego.Introduction. Amniotic fluid embolism is a rare, but serious complication, frequently leading to death of a delivering woman. The pathophysiology of the amniotic fluid embolism is multifactorial in origin and still remains poorly understood. Aim of the study. The purpose of the study was to nursing diagnoses for a female patient with amniotic fluid embolism during the caesarean section in the spinal anaesthesia and afterwards on the intensive care unit hospitalization. Materials and methods. The research is based on the professional clinical case report and has retrospective character. Data was collected on the basis of patient’s medical history and analysis of patient’s medical documentation — medical history, anaesthesia card, nursing process and laboratory tests results. Results and conclusions. All the diagnosed problems have been fully and successfully resolved. The remaining nursing problems had to be treated by nursing staff after moving the patient from the intensive care unit to next stages of hospitalization

The Small Yeast GTPase Rho5 and Its Dimeric GEF Dck1/Lmo1 Respond to Glucose Starvation

Rho5 is a small GTPase of Saccharomyces cerevisiae and a homolog of mammalian Rac1. The latter regulates glucose metabolism and actin cytoskeleton dynamics, and its misregulation causes cancer and a variety of other diseases. In yeast, Rho5 has been implicated in different signal transduction pathways, governing cell wall integrity and the responses to high medium osmolarity and oxidative stress. It has also been proposed to affect mitophagy and apoptosis. Here, we demonstrate that Rho5 rapidly relocates from the plasma membrane to mitochondria upon glucose starvation, mediated by its dimeric GDP/GTP exchange factor (GEF) Dck1/Lmo1. A function in response to glucose availability is also suggested by synthetic genetic phenotypes of a rho5 deletion with gpr1, gpa2, and sch9 null mutants. On the other hand, the role of mammalian Rac1 in regulating the action cytoskeleton does not seem to be strongly conserved in S. cerevisiae Rho5. We propose that Rho5 serves as a central hub in integrating various stress conditions, including a crosstalk with the cAMP/PKA (cyclic AMP activating protein kinase A) and Sch9 branches of glucose signaling pathways