28 research outputs found
Combining mass spectrometry and genetic labeling in mice to report TRP channel expression
Transient receptor potential (TRP) ion channels play important roles in fundamental biological processes throughout the body of humans and mice. TRP channel dysfunction manifests in different disease states, therefore, these channels may represent promising therapeutic targets in treating these conditions. Many TRP channels are expressed in several organs suggesting multiple functions and making it challenging to untangle the systemic pathophysiology of TRP dysfunction. Detailed characterization of the expression pattern of the individual TRP channels throughout the organism is thus essential to interpret data such as those derived from systemic phenotyping of global TRP knockout mice. Murine TRP channel reporter strains enable reliable labeling of TRP expression with a fluorescent marker. Here we present an optimized method to visualize primary TRP-expressing cells with single cell resolution throughout the entire organism. In parallel, we methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest. The TRP protein expression data are then correlated with the GFP reporter expression data. The combined methodological approach presented here can be adopted to generate expression data for other genes of interest and reporter mice.
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We present an optimized method to systemically characterize gene expression in fluorescent reporter mouse strains with a single cell resolution.
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We methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest in mice
Transient Receptor Potential Vanilloid 6 (TRPV6) Proteins Control the Extracellular Matrix Structure of the Placental Labyrinth
Calcium-selective transient receptor potential Vanilloid 6 (TRPV6) channels are expressed in
fetal labyrinth trophoblasts as part of the feto–maternal barrier, necessary for sufficient calcium supply,
embryo growth, and bone development during pregnancy. Recently, we have shown a less- compact
labyrinth morphology of Trpv6-deficient placentae, and reduced Ca2+ uptake of primary trophoblasts
upon functional deletion of TRPV6. Trpv6-/-
trophoblasts show a distinct calcium-dependent phenotype.
Deep proteomic profiling of wt and Trpv6-/- primary trophoblasts using label-free quantitative mass
spectrometry leads to the identification of 2778 proteins. Among those, a group of proteases,
including high-temperature requirement A serine peptidase 1 (HTRA1) and different granzymes
are more abundantly expressed in Trpv6-/-
trophoblast lysates, whereas the extracellular matrix
protein fibronectin and the fibronectin-domain-containing protein 3A (FND3A) were markedly
reduced. Trpv6-/- placenta lysates contain a higher intrinsic proteolytic activity increasing fibronectin
degradation. Our results show that the extracellular matrix formation of the placental labyrinth
depends on TRPV6; its deletion in trophoblasts correlates with the increased expression of proteases
controlling the extracellular matrix in the labyrinth during pregnancy
Mutations that affect the surface expression of TRPV6 are associated with the upregulation of serine proteases in the placenta of an infant
Recently, we reported a case of an infant with neonatal severe under-mineralizing skeletal dysplasia caused by mutations within both alleles of the TRPV6 gene. One mutation results in an in frame stop codon (R(510)stop) that leads to a truncated, nonfunctional TRPV6 channel, and the second in a point mutation (G(660)R) that, surprisingly, does not affect the Ca(2+) permeability of TRPV6. We mimicked the subunit composition of the unaffected heterozygous parent and child by coexpressing the TRPV6 G(660)R and R(510)stop mutants and combinations with wild type TRPV6. We show that both the G(660)R and R(510)stop mutant subunits are expressed and result in decreased calcium uptake, which is the result of the reduced abundancy of functional TRPV6 channels within the plasma membrane. We compared the proteomic profiles of a healthy placenta with that of the diseased infant and detected, exclusively in the latter two proteases, HTRA1 and cathepsin G. Our results implicate that the combination of the two mutant TRPV6 subunits, which are expressed in the placenta of the diseased child, is responsible for the decreased calcium uptake, which could explain the skeletal dysplasia. In addition, placental calcium deficiency also appears to be associated with an increase in the expression of proteases
Mutations That Affect the Surface Expression of TRPV6 Are Associated with the Upregulation of Serine Proteases in the Placenta of an Infant
Recently, we reported a case of an infant with neonatal severe under-mineralizing skeletal
dysplasia caused by mutations within both alleles of the TRPV6 gene. One mutation results in
an in frame stop codon (R510stop) that leads to a truncated, nonfunctional TRPV6 channel, and
the second in a point mutation (G660R) that, surprisingly, does not affect the Ca2+ permeability of
TRPV6. We mimicked the subunit composition of the unaffected heterozygous parent and child
by coexpressing the TRPV6 G660R and R510stop mutants and combinations with wild type TRPV6.
We show that both the G660R and R510stop mutant subunits are expressed and result in decreased
calcium uptake, which is the result of the reduced abundancy of functional TRPV6 channels within
the plasma membrane. We compared the proteomic profiles of a healthy placenta with that of the
diseased infant and detected, exclusively in the latter two proteases, HTRA1 and cathepsin G. Our
results implicate that the combination of the two mutant TRPV6 subunits, which are expressed in the
placenta of the diseased child, is responsible for the decreased calcium uptake, which could explain
the skeletal dysplasia. In addition, placental calcium deficiency also appears to be associated with an
increase in the expression of proteases
The Evolution of Erythrocytes Becoming Red in Respect to Fluorescence
Very young red blood cells, namely reticulocytes, can be quite easily recognized and labeled by cluster of differentiation antibodies (CD71, transferrin receptor) or by staining remnant RNA with thiazol orange. In contrast, age specific erythrocyte labeling is more difficult in later periods of their life time. While erythrocytes contain band 4.1 protein, a molecular clock, so far it has not been possible to read this clock on individual cells. One concept to track erythrocytes during their life time is to mark them when they are young, either directly in vivo or ex vivo followed by a transfusion. Several methods like biotinylation, use of isotopes or fluorescent labeling have proved to be useful experimental approaches but also have several inherent disadvantages. Genetic engineering of mice provides additional options to express fluorescent proteins in erythrocytes. To allow co-staining with popular green fluorescent dyes like Fluo-4 or other fluorescein-based dyes, we bred a mouse line expressing a tandem red fluorescent protein (tdRFP). Within this Brief Research Report, we provide the initial characterisation of this mouse line and show application examples ranging from transfusion experiments and intravital microscopy to multicolour flow cytometry and confocal imaging. We provide a versatile new tool for erythrocyte research and discuss a range of experimental opportunities to study membrane processes and other aspects of erythrocyte development and aging with help of these animals
Implementing clinical trial data sharing requires training a new generation of biomedical researchers
Data sharing enhances the value of medical research and builds trust in clinical trials, but more biomedical researchers need to be trained in these approaches, which include meta-research, data science and ethical, legal and social issues
A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.
The COVID-19 pandemic has increased negative emotions and decreased positive emotions globally. Left unchecked, these emotional changes might have a wide array of adverse impacts. To reduce negative emotions and increase positive emotions, we tested the effectiveness of reappraisal, an emotion-regulation strategy that modifies how one thinks about a situation. Participants from 87 countries and regions (n = 21,644) were randomly assigned to one of two brief reappraisal interventions (reconstrual or repurposing) or one of two control conditions (active or passive). Results revealed that both reappraisal interventions (vesus both control conditions) consistently reduced negative emotions and increased positive emotions across different measures. Reconstrual and repurposing interventions had similar effects. Importantly, planned exploratory analyses indicated that reappraisal interventions did not reduce intentions to practice preventive health behaviours. The findings demonstrate the viability of creating scalable, low-cost interventions for use around the world
Improving the trustworthiness, usefulness, and ethics of biomedical research through an innovative and comprehensive institutional initiative.
The reproducibility crisis triggered worldwide initiatives to improve rigor, reproducibility, and transparency in biomedical research. There are many examples of scientists, journals, and funding agencies adopting responsible research practices. The QUEST (Quality-Ethics-Open Science-Translation) Center offers a unique opportunity to examine the role of institutions. The Berlin Institute of Health founded QUEST to increase the likelihood that research conducted at this large academic medical center would be trustworthy, useful for scientists and society, and ethical. QUEST researchers perform "science of science" studies to understand problems with standard practices and develop targeted solutions. The staff work with institutional leadership and local scientists to incentivize and support responsible practices in research, funding, and hiring. Some activities described in this paper focus on the institution, whereas others may benefit the national and international scientific community. Our experience, approaches, and recommendations will be informative for faculty leadership, administrators, and researchers interested in improving scientific practice
fiddle: A tool to combat publication bias by getting research out of the file drawer and into the scientific community
This preprint introduces a new open source tool (fiddle - file drawer data liberation effort; available at: http://s-quest.bihealth.org/fiddle/) that aims to help researchers to identify the most appropriate publication format for their dataset. Formats include data repositories, micropublications, preprints, data journals, publishing platforms and journals that are open to null results. Users can search for a publication format that meets their needs, compare and contrast formats, and find links to publishers