53 research outputs found
Evidence for a Pathophysiological Role of Keratinocyte-Derived Type III Interferon (IFNλ) in Cutaneous Lupus Erythematosus
Type I IFNs (IFNα/β) have been shown to have a central role in the pathophysiology of lupus erythematosus (LE). The recently discovered type III IFNs (IFNλ1/IL29, IFNλ2/IL28a, IFNλ3/IL28b) share several functional similarities with type I IFNs, particularly in antiviral immunity. As IFNλs act primarily on epithelial cells, we investigated whether type III IFNs might also have a role in the pathogenesis of cutaneous LE (CLE). Our investigations demonstrate that IFNλ and the IFNλ receptor were strongly expressed in the epidermis of CLE skin lesions and related autoimmune diseases (lichen planus and dermatomyositis). Significantly enhanced IFNλ1 could be measured in the serum of CLE patients with active skin lesions. Functional analyses revealed that human keratinocytes are able to produce high levels of IFNλ1 but only low amounts of IFNα/β/γ in response to immunostimulatory nuclear acids, suggesting that IFNλ is a major IFN produced by these cells. Exposure of human keratinocytes to IFNλ1 induced the expression of several proinflammatory cytokines, including CXCL9 (CXC-motiv ligand 9), which drive the recruitment of immune cells and are associated with the formation of CLE skin lesions. Our results provide evidence for a role of type III IFNs in not only antiviral immunity but also autoimmune diseases of the skin
Desire to Receive More Pain Treatment: A Relevant Patient-Reported Outcome Measure to Assess Quality of Post-Operative Pain Management? Results From 79,996 Patients Enrolled in the Pain Registry QUIPS from 2016 to 2019
Acute postoperative pain is frequently evaluated by pain intensity scores. However, interpretation of the results is difficult and thresholds requiring treatment are notwell defined. Additional patientreported outcome measures (PROMs) might be helpful to better understand individual pain experience and quality of pain management after surgery.We used data from the QUIPS pain registry for a cross-sectional study in order to investigate associations between the desire to receive more pain treatment (D2RMPT) with pain intensity ratings and other PROMs. Responses from 79,996 patientswere analyzed, of whom 10.7% reported D2RMPT. A generalized estimating equation Poisson model showed that women had a lower risk ratio (RR) to answer this question with “yes” (RR: .92, P < .001). Factors that increased the risk most were “maximal pain intensity ≥ 6/10 on a numerical rating scale” (RR: 2.48, P < .001) and “any pain interference” (RR: 2.48, P < .001). The largest reduction in risk was observed if patients were “allowed to participate in pain treatment decisions” (RR: .41, P < .001) and if they felt that they “received sufficient treatment information” (RR: .58, P < .001). Our results indicate that the (easily assessed) question D2RMPT gives additional information to other PROMs like pain intensity. The small proportion of patients with D2RMPT (even for high pain scores) opens the discussion about clinicians’ understanding of over- und under-treatment and questions the exclusive use of pain intensity as quality indicator. Future studies need to investigatewhether asking about D2RMPT in clinical routine can improve postoperative pain outcome. Perspective: This article presents characteristics of the patient-reported outcome measure “Desire to receive more pain treatment.” This measure could be used to apply pain treatment in a more individualized way and lead to improved treatment strategies and quality
The “WWHow” Concept for Prospective Categorization of Post-operative Severity Assessment in Mice and Rats
The prospective severity assessment in animal experiments in the categories' non-recovery, mild, moderate, and severe is part of each approval process and serves to estimate the harm/benefit. Harms are essential for evaluating ethical justifiability, and on the other hand, they may represent confounders and effect modifiers within an experiment. Catalogs and guidelines provide a way to assess the experimental severity prospectively but are limited in adaptation due to their nature of representing particular examples without clear explanations of the assessment strategies. To provide more flexibility for current and future practices, we developed the modular Where-What-How (WWHow) concept, which applies findings from pre-clinical studies using surgical-induced pain models in mice and rats to provide a prospective severity assessment. The WWHow concept integrates intra-operative characteristics for predicting the maximum expected severity of surgical procedures. The assessed severity categorization is mainly congruent with examples in established catalogs; however, because the WWHow concept is based on anatomical location, detailed analysis of the tissue trauma and other intra-operative characteristics, it enables refinement actions, provides the basis for a fact-based dialogue with authority officials and other stakeholders, and helps to identify confounder factors of study findings
The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium
The uptake and processing of dietary lipids by the small intestine is a multistep process that involves several steps including vesicular and protein transport. The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. Here, we describe the essential role of ARFRP1 and its interaction with Rab2 in the assembly and lipidation of chylomicrons in the intestinal epithelium. Mice lacking Arfrp1 specifically in the intestine (Arfrp1vil−/−) exhibit an early post-natal growth retardation with reduced plasma triacylglycerol and free fatty acid concentrations. Arfrp1vil−/− enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triacylglycerol content. In addition, the release of apolipoprotein A-I (ApoA-I) was dramatically decreased, and ApoA-I accumulated in the Arfrp1vil−/− epithelium, where it predominantly co-localized with Rab2. The release of chylomicrons from Caco-2 was markedly reduced after the suppression of Rab2, ARL1 and Golgin-245. Thus, the GTPase ARFRP1 and its downstream proteins are required for the lipidation of chylomicrons and the assembly of ApoA-I to these particles in the Golgi of intestinal epithelial cells
Estimating global mortality from potentially foodborne diseases: an analysis using vital registration data
<p>Abstract</p> <p>Background</p> <p>Foodborne diseases (FBD) comprise a large part of the global mortality burden, yet the true extent of their impact remains unknown. The present study utilizes multiple regression with the first attempt to use nonhealth variables to predict potentially FBD mortality at the country level.</p> <p>Methods</p> <p>Vital registration (VR) data were used to build a multiple regression model incorporating nonhealth variables in addition to traditionally used health indicators. This model was subsequently used to predict FBD mortality rates for all countries of the World Health Organization classifications AmrA, AmrB, EurA, and EurB.</p> <p>Results</p> <p>Statistical modeling strongly supported the inclusion of nonhealth variables in a multiple regression model as predictors of potentially FBD mortality. Six variables were included in the final model: <it>percent irrigated land, average calorie supply from animal products, meat production in metric tons, adult literacy rate, adult HIV/AIDS prevalence</it>, and <it>percent of deaths under age 5 caused by diarrheal disease</it>. Interestingly, nonhealth variables were not only more robust predictors of mortality than health variables but also remained significant when adding additional health variables into the analysis. Mortality rate predictions from our model ranged from 0.26 deaths per 100,000 (Netherlands) to 15.65 deaths per 100,000 (Honduras). Reported mortality rates of potentially FBD from VR data lie within the 95% prediction interval for the majority of countries (37/39) where comparison was possible.</p> <p>Conclusions</p> <p>Nonhealth variables appear to be strong predictors of potentially FBD mortality at the country level and may be a powerful tool in the effort to estimate the global mortality burden of FBD.</p> <p>Disclaimer</p> <p>The views expressed in this document are solely those of the authors and do not represent the views of the World Health Organization.</p
Accessing the Anisotropic Nonthermal Phonon Populations in Black Phosphorus
We combine ultrafast electron diffuse scattering experiments and first-principles calculations of the coupled electron-phonon dynamics to provide a detailed momentum-resolved picture of lattice thermalization in black phosphorus. The measurements reveal the emergence of highly anisotropic nonthermal phonon populations persisting for several picoseconds after exciting the electrons with a light pulse. Ultrafast dynamics simulations based on the time-dependent Boltzmann formalism are supplemented by calculations of the structure factor, defining an approach to reproduce the experimental signatures of nonequilibrium structural dynamics. The combination of experiments and theory enables us to identify highly anisotropic electron-phonon scattering processes as the primary driving force of the nonequilibrium lattice dynamics in black phosphorus. Our approach paves the way toward unravelling and controlling microscopic energy flows in two-dimensional materials and van der Waals heterostructures, and may be extended to other nonequilibrium phenomena involving coupled electron-phonon dynamics such as superconductivity, phase transitions, or polaron physics
Observing the Evolution of the Universe
How did the universe evolve? The fine angular scale (l>1000) temperature and
polarization anisotropies in the CMB are a Rosetta stone for understanding the
evolution of the universe. Through detailed measurements one may address
everything from the physics of the birth of the universe to the history of star
formation and the process by which galaxies formed. One may in addition track
the evolution of the dark energy and discover the net neutrino mass.
We are at the dawn of a new era in which hundreds of square degrees of sky
can be mapped with arcminute resolution and sensitivities measured in
microKelvin. Acquiring these data requires the use of special purpose
telescopes such as the Atacama Cosmology Telescope (ACT), located in Chile, and
the South Pole Telescope (SPT). These new telescopes are outfitted with a new
generation of custom mm-wave kilo-pixel arrays. Additional instruments are in
the planning stages.Comment: Science White Paper submitted to the US Astro2010 Decadal Survey.
Full list of 177 author available at http://cmbpol.uchicago.ed
Child maltreatment victimization by type in relation to criminal recidivism in juvenile offenders
The Calcineurin Inhibitor-Sparing (CIS) Trial - individualised calcineurin-inhibitor treatment by immunomonitoring in renal allograft recipients: protocol for a randomised controlled trial
Regulated intramembrane proteolysis of transferrin receptor 1
INHALTSVERZEICHNIS Abkürzungen
...........................................................................................................
6 1
Zusammenfassung...................................................................................................
9 Summary
...............................................................................................................
11 2 Einleitung
..............................................................................................................
13 2.1 Biologische Bedeutung von Eisen
................................................................. 13 2.1.1
Eisenaufnahme in den Organismus
............................................................ 13 2.1.2
Zellulärer Eisenstoffwechsel
...................................................................... 14
2.1.3 Regulation des systemischen Eisenstoffwechsels
...................................... 14 2.2 Rolle des Transferrinrezeptors
...................................................................... 15
2.2.1 Transferrinrezeptor 1
.................................................................................
15 2.2.2 Shedding des Transferrinrezeptors
............................................................ 16 2.3 Regulierte
intramembranäre Proteolyse
........................................................ 16 2.4 Zielsetzung
....................................................................................................
19 3 Material
.................................................................................................................
21 3.1 Allgemeine Hinweise
....................................................................................
21 3.2 Geräte
............................................................................................................
21 3.2.1 Zell- und Bakterienkultur
........................................................................... 21
3.2.2 Zentrifugen
.................................................................................................
21 3.2.3 Elektrophorese und Westernblot
................................................................ 22 3.2.4
ELISA
........................................................................................................
22 3.2.5 Sonstige Geräte
..........................................................................................
22 3.3 Verbrauchsmaterial
........................................................................................
22 3.4 Zell- und Bakterienkultur
..............................................................................
23 3.5 Chemikalien
...................................................................................................
23 3.6 Antikörper
.....................................................................................................
24 3.6.1 Primärantikörper
........................................................................................
24 3.6.2 Sekundärantikörper
....................................................................................
25 3.7 Proteaseinhibitoren
........................................................................................
25 3.8 Kits
................................................................................................................
25 3.9 Oligonukleotide
.............................................................................................
26 4 Methoden
...............................................................................................................
26 4.1 Zellbiologische Methoden
.............................................................................
26 4.1.1 Kultivierung von HEK293 Zellen
............................................................. 26 4.1.2
Einfrieren und Auftauen von Zellen
.......................................................... 27 4.1.3
Transfektion von HEK293-Zellen
............................................................. 27 4.1.4
Immunfluoreszenz von HEK293-Zellen
................................................... 27 4.2 Proteinbiochemische
Methoden ....................................................................
28 4.2.1 Lyse von Zellen
.........................................................................................
28 4.2.2 Konzentrationsbestimmung von Proteinen
................................................ 29 4.2.3 Gelelektrophorese
......................................................................................
29 4.2.4 Westernblot und Immunodetektion
........................................................... 31 4.2.5 Stripping
....................................................................................................
32 4.2.6 Immunpräzipitation
...................................................................................
32 4.2.7 Membranlysatpräparation
.......................................................................... 33
4.2.8 MALDI-TOF-MS
......................................................................................
33 4.2.9 Antikörperaufreinigung
.............................................................................
34 4.2.10 Enzymgekoppelter Immunadsorptionstest
............................................. 35 4.3 Molekularbiologische
Methoden ...................................................................
36 4.3.1 Agarosegelelektrophorese
......................................................................... 36
4.3.2 Restriktionsverdau
.....................................................................................
37 4.3.3 Isolierung von DNA aus Agarosegelen
..................................................... 37 4.3.4 Ligation von
DNA-Fragmenten
................................................................. 37 4.3.5
Transformation von Plasmid-DNA
............................................................ 37 4.3.6
Präparation von Plasmid-DNA
.................................................................. 38 4.3.7
Polymerasekettenreaktion
.......................................................................... 39
4.3.8 DNA-Sequenzierung
..................................................................................
39 5 Ergebnisse
.............................................................................................................
40 5.1 Charakterisierung des Flag-TfR1-NTF-V5
................................................... 40 5.2 Identifizierung des
TfR1-C Peptides und der Spalt-stelle ............................. 41 5.3
Antikörperaufreinigung
.................................................................................
44 5.4 Etablierung eines Sandwich-ELISA
.............................................................. 53 5.5 Bindung
des TfR1-C Peptids an Serum Albumin .........................................
55 5.6 Identifizierung der Protease
........................................................................... 58
5.7 Lokalisierung von TfR1-NTF und den verschiedenen SPPLs
...................... 66 6 Diskussion
.............................................................................................................
69 6.1 Entdeckung des TfR1-C-Peptid
..................................................................... 69 6.2
Entwicklung von Nachweismethoden
........................................................... 72 6.3
Intramembranäre Proteolyse von TfR1-NTF durch SPPL2a und SPPL2b ... 75 7
Literaturverzeichnis
..............................................................................................
80 8 Anhang
..................................................................................................................
89 Publikationsverzeichnis
........................................................................................
89 Lebenslauf
.............................................................................................................
90 Danksagung
..........................................................................................................
91Der Transferrinrezeptor 1 (TfR1) ist ein homodimeres Typ II
Transmembranprotein und vermittelt die Eisenaufnahme über die Bindung des
eisenbeladenen Transferrin in die Zelle. Die transferrinbindende
extrazelluläre Domäne wird von einem als a disintegrin and metalloproteinase
(ADAM) bezeichneten Enzym im juxtamembranen Teil des TfR1 C-terminal von
Arginin 100 geschnitten. Der dabei entstehende lösliche Transferrinrezeptor
(sTfR) dient als diagnostischer Marker für erythropoetische Aktivität,
besonders um zwischen einer Eisenmangelanämie und einer inflammatorischen
Anämie zu unterscheiden, wobei die biologische Funktion des sTfR1 unverstanden
ist. Der Verbleib des durch die Proteolyse der Ektodomäne entstandenen
membranständigen N-terminalen Fragments (NTF) des TfR1 ist unbekannt. Für Typ
I Transmembranproteine wie dem Notch- oder dem ErbB4-Rezeptor konnte eine
weitere intramembranäre Proteolyse zur Proteindegradierung oder für weitere
Signaltransduktionen nachgewiesen werden. Dies gelang für Typ II
Transmembranproteine erst im Jahr 2002 nach der Entdeckung der
Signalpeptidpeptidase ähnlichen-Proteasen (SPPL) für die Substrate
Tumornekrosefaktoralpha (TNFα), FasLigand und british dementia Protein (Bri2).
Im Laufe dieser Arbeit konnte mit Hilfe eines N-terminal Flag-getaggten und
C-terminal V5-getaggten TfR1-NTF zum ersten Mal gezeigt werden, dass das
TfR1-NTF intramembranär gespalten wird. Das dabei extrazellulär entstehende
C-Peptid des TfR1 (TfR1-Cp) wurde im Medium als oxidiertes Monomer, d. h. mit
einer intramolekularen Disulfidbrücke zwischen dem Cystein 89 und 98
nachgewiesen. Zudem konnte die Sequenz des TfR1-Cp mit Hilfe der MALDI-TOF-
TOF-MS-Analyse bestimmt werden. Die intramembranäre Proteolyse erfolgte
demnach zwischen dem Glycin 84 und dem Tyrosin 85. Weiterhin wurden
polyklonale Antikörper gegen TfR1-Cp in Kaninchen generiert und aus dem
Kaninchenserum aufgereinigt. Diese Antikörper wurden für die Etablierung eines
Sandwich-ELISA zum Nachweis des TfR1-Cp im Serum verwendet. Es konnte eine
hohe Spezifität des Sandwich-ELISA mit Hilfe eines kompetitiven ELISA, bei dem
biotinyliertes TfR1-Cp mit verschiedenen Mengen ungelabeltem TfR1 versetzt
wurde, gezeigt werden. Weiterhin wurde mit Hilfe des ELISA humanes Albumin als
Interaktionspartner des TfR1-Cp identifiziert. In dieser Arbeit konnte zudem
gezeigt werden, dass die endogene Protease für die intramembranäre Proteolyse
des TfR1-NTF ein Mitglied der GxGD-Aspartylproteasen, speziell der SPP/SPPL-
Familie ist. Weitere Experimente mit überexprimierten SPPLs oder ihren
katalytisch inaktiven Mutanten zeigten bei SPPL2a und SPPL2b eine erhöhte
Freisetzung des TfR1-Cp. Während bei der katalytisch inaktiven SPPL2b D/A
überhaupt kein TfR1-Cp detektiert werden konnte, zeigte die Überexpression der
katalytisch inaktiven SPPL2a D/A eine verringerte jedoch nicht vollständig
unterdrückte Freisetzung des TfR1-Cp gegenüber Kontrollzellen. Zudem konnte
eine Kolokalisierung der SPPL2b mit dem TfR1-NTF nachgewiesen werden, dies
gelang nicht für die SPPL2a. Dies weist auf die SPPL2b als Hauptprotease des
TfR1-NTF hin, während die SPPL2a die am Prozess unwesentlichere Protease zu
sein scheint. Ferner gelang es in einer Kooperation mit Frau Fluhrer (DZNE,
LMU, München) die intrazelluläre Domäne des TfR1 (TfR1-ICD) in Membranlysaten
von mit SPPL2b überexprimierten Zellen nachzuweisen. Die physiologische
Bedeutung der intramembranären Proteolyse des TfR1-NTF und der entstehenden
Fragmente TfR1-Cp und TfR1-ICD ist ungeklärt. Die intramembranäre Proteolyse
könnte dem Abbau des TfR1 oder weiteren Regulationsmechanismen der Zelle und
des Körpers dienen. Während die TfR1-ICD in der Zelle als zellulärer Regulator
für einen Eisenmangel fungieren könnte, könnte das TfR1-Cp als systemischer
Regulator für eine Freisetzung von Eisen aus retikuloendothelialen Makrophagen
oder den Enterozyten des Darms durch eine mögliche Stabilisierung des
Eisenexportproteins Ferroportin wirken. Dies ist rein spekulativ und müsste
noch bewiesen werden.The transferrin receptor-1 (TfR1) is a homodimeric type II transmembrane
protein and mediates the cellular iron uptake via binding of ferri-
transferrin. The extracellular transferrin binding domain is cleaved by a
disintegrin and metallo- (ADAM) protease C-terminal of arginin-100 within the
juxtamembrane region of the ectodomain. The thereby generated soluble TfR1
(sTfR1) is used as a diagnostic marker for erythropoietic activity, in
particular to discriminate between iron deficiency anemia and anemia of
inflammation, however, the physiological function of sTfR1 is unclear. The
fate of the remaining N-terminal fragment of the TfR1 (TfR1-NTF) is unknown so
far. For type I transmembrane proteins such as Notch or ErbB4 receptor, an
intramembrane proteolysis for further protein degradation or for subsequent
signaling is known. For type II transmembrane proteins, this was the case
after the discovery of new proteins in 2002 referred as signalpeptide
peptidase-like proteases (SPPL) that cleave substrates such as tumornecrosis
factor-α (TNF-α), Fas ligand and the british dementia protein (Bri2). In this
work the intramembrane proteolysis of TfR1-NTF was detected for the first time
by using an N-terminal Flag tag and a C-terminal V5 tag fused to the TfR1-NTF.
The generated extracellular C-terminal peptide of TfR1 (TfR1-Cp) was detected
as an oxidized monomer, with an intramolecular disulfide bridge between
cystein-89 and cystein-98. Furthermore, the sequence of the peptide was
determined by a fragmentation analysis via MALDI-TOF-TOF-MS, which revealed
that intramembrane proteolysis occurs between glycin-84 and tyrosin-85.
Furthermore, polyclonal antibodies against the TfR1-Cp were generated in
rabbit and purified to be used for a sandwich-ELISA that was established to
determine the TfR1-Cp amount in human serum. There was a high specificity
detected by using a competitive assay with biotinylated TfR1-Cp and different
amounts of unlabelled TfR1-Cp. In addition, human serum albumin was identified
as an interaction partner of TfR1-Cp by using the sandwich-ELISA. Moreover,
the endogenous protease for the intramembrane proteolysis of the TfR1-NTF was
identified in this work. It belongs to the GxGD-aspartyl proteases, especially
to the SPP/SPPL-family. Further experiments with overexpressed SPPLs and their
catalytically inactive mutants showed an increased release of TfR1-Cp from
cells overexpressing SPPL2a and SPPL2b. By using the catalytically inactive
mutant SPPL2b D/A, absolutely no TfR1-Cp was detectable, whereas with the
catalytically inactive SPPL2a D/A a reduced but not completely abolished
TfR1-Cp signal was observed in contrast to the control cells. Furthermore, a
colocalisation of TfR1-NTF with SPPL2b but not with SPPL2a was detected in the
plasma membrane using confocal fluorescence microscopy. This indicates that
the SPPL2b is the major protease responsible for TfR1-NTF cleavage and the
SPPL2a only the minor one. In a cooperation with Regina Fluhrer (DZNE, LMU,
Munich) it was possible to detect the intracellular domain of the TfR1
(TfR1-ICD) in membrane lysates obtained from cells with co-expression of
SPPL2b. The physiologic relevance of the intramembrane proteolysis of the
TfR1-NTF and of the released fragments TfR1-ICD and TfR1-Cp is unknown. The
intramembrane proteolysis could be useful for the denaturation of the TfR1 or
for further regulation within the cell or in body. The TfR1-ICD might be
valuable as a cellular regulator for iron deficiency whereas the TfR1-Cp could
act as a systemic iron regulator for the iron release from reticuloendothelial
macrophages or from the enterocytes in the intestine by stabilizing the iron
exporter Ferroportin, but this is speculative and has to be proven
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