340 research outputs found
UV-C Irradiation Reduces the Experimentally Induced Bacterial Load on the Surface of a Human Cadaver: An Additional Option for the Preservation of Cadavers in Anatomy
Safety is a major issue in the embalming procedures of human cadavers. Reduced application of formaldehyde is often recommended. The aim of this study was to investigate the potency of ultraviolet light (UV-C irradiation) on the bacterial load on the surface of a conserved human cadaver. To test UV-C irradiation, the cadaver was laid out in the dissection hall and, after preparation of the muscles, was covered with linen sheets moistened with water. Swabs of the surface and microbiological analysis revealed sporadic bacterial colonies. The surface area was then spiked with bacteria and irradiated by a UV lamp for 15 or 60âmin. Half of the area was covered by aluminum foil to serve as a control. After exposition, swabs were taken and analyzed. The exposition had reduced the number of colonies to one third (15âmin exposition) and to one tenth (60âmin exposition) of the control area. Thus, UV-C irradiation could be used in the preservation of cadavers without chemical pollution of the environment and without any risk for the employees. Clin. Anat. 32:113â116, 2019. © 2019 The Authors. Clinical Anatomy published by Wiley Periodicals LLC on behalf of American Association of Clinical Anatomists
Optimization of a two-plasmid system for the identification of promoters recognized by RNA polymerase containing Staphylococcus aureus alternative sigma factor ÏB
We optimized a previously established two-plasmid system for the identification of Staphylococcus aureus promoters that are recognized by the alternative transcription factor ÏB. The method allowed the identification of 18 S. aureusÏB-dependent promoters, 12 of which are reported here for the first time to be ÏB-dependent. S1-nuclease mapping of the respective transcriptional start points revealed that all the promoters contained sequences exhibiting high similarity to the consensus sequence of Bacillus subtilisÏB-dependent promoters. The promoters governed expression of genes encoding proteins proposed to be involved in various cellular functions, including the stress response genes and virulence-associated clfA gene for fibrinogen-binding clumping factor. Comparison of the nucleotide sequences upstream of the identified transcription start points identified a ÏB consensus promoter (GttTaa-N12-15-gGGTAt) that is highly homologous to that of ÏB of B. subtili
Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans
Pneumonia is a life-threatening disease often caused by infection with Streptococcus pneumo niae and Pseudomonas aeruginosa. Many of the mediators (e.g., TNF, IL-6R) and junction molecules
(e.g., E-cadherin) orchestrating inflammatory cell recruitment and loss of barrier integrity are prote olytically cleaved through a disintegrin and metalloproteinases (ADAMs). We could show by Western
blot, surface expression analysis and measurement of proteolytic activity in cell-based assays, that
ADAM10 in epithelial cells is upregulated and activated upon infection with Pseudomonas aeruginosa
and Exotoxin A (ExoA), but not upon infection with Streptococcus pneumoniae. Targeting ADAM10
by pharmacological inhibition or gene silencing, we demonstrated that this activation was critical
for cleavage of E-cadherin and modulated permeability and epithelial integrity. Stimulation with
heat-inactivated bacteria revealed that the activation was based on the toxin repertoire rather than
the interaction with the bacterial particle itself. Furthermore, calcium imaging experiments showed
that the ExoA action was based on the induction of calcium influx. Investigating the extracellular
vesicles and their proteolytic activity, we could show that Pseudomonas aeruginosa triggered exosomal
release of ADAM10 and proteolytic cleavage in trans. This newly described mechanism could consti tute an essential mechanism causing systemic inflammation in patients suffering from Pseudomonas
aeruginosa-induced pneumonia stimulating future translational studies
Role of Extracellular Vimentin in Cancer-Cell Functionality and Its Influence on Cell Monolayer Permeability Changes Induced by SARS-CoV-2 Receptor Binding Domain
The cytoskeletal protein vimentin is secreted under various physiological conditions.
Extracellular vimentin exists primarily in two forms: attached to the outer cell surface and secreted
into the extracellular space. While surface vimentin is involved in processes such as viral infections
and cancer progression, secreted vimentin modulates inflammation through reduction of neutrophil
infiltration, promotes bacterial elimination in activated macrophages, and supports axonal growth
in astrocytes through activation of the IGF-1 receptor. This receptor is overexpressed in cancer
cells, and its activation pathway has significant roles in general cellular functions. In this study,
we investigated the functional role of extracellular vimentin in non-tumorigenic (MCF-10a) and
cancer (MCF-7) cells through the evaluation of its effects on cell migration, proliferation, adhesion,
and monolayer permeability. Upon treatment with extracellular recombinant vimentin, MCF-7 cells
showed increased migration, proliferation, and adhesion, compared to MCF-10a cells. Further, MCF-7
monolayers showed reduced permeability, compared to MCF-10a monolayers. It has been shown
that the receptor binding domain of SARS-CoV-2 spike protein can alter bloodâbrain barrier integrity.
Surface vimentin also acts as a co-receptor between the SARS-CoV-2 spike protein and the cell-surface
angiotensin-converting enzyme 2 receptor. Therefore, we also investigated the permeability of
MCF-10a and MCF-7 monolayers upon treatment with extracellular recombinant vimentin, and
its modulation of the SARS-CoV-2 receptor binding domain. These findings show that binding of
extracellular recombinant vimentin to the cell surface enhances the permeability of both MCF-10a
and MCF-7 monolayers. However, with SARS-CoV-2 receptor binding domain addition, this effect is
lost with MCF-7 monolayers, as the extracellular vimentin binds directly to the viral domain. This
defines an influence of extracellular vimentin in SARS-CoV-2 infections
T cell stiffness is enhanced upon formation of immunological synapse
T cells are activated by target cells via an intimate contact, termed immunological synapse (IS). Cellular mechanical properties, especially stiffness, are essential to regulate cell functions. However, T cell stiffness at a subcellular level at the IS still remains largely elusive. In this work, we established an atomic force microscopy (AFM)-based elasticity mapping method on whole T cells to obtain an overview of the stiffness with a resolution of ~60 nm. Using primary human CD4+ T cells, we show that when T cells form IS with stimulating antibody-coated surfaces, the lamellipodia are stiffer than the cell body. Upon IS formation, T cell stiffness is enhanced both at the lamellipodia and on the cell body. Chelation of intracellular Ca2+ abolishes IS-induced stiffening at the lamellipodia but has no influence on cell-body-stiffening, suggesting different regulatory mechanisms of IS-induced stiffening at the lamellipodia and the cell body
Characteristics of a New Carbonaceous Chondrite, Metal-Rich-Lithology Found in the Carbonaceous Chondrite Breccia Aguas Zarcas
The Aguas Zarcas meteorite fell in Costa Rica on 23 April 2019 at 21:07 local time, with a total mass of about 27 kg. Hundreds of fusion-crusted stones ranging from 0.1 to 1868 g were recovered (The Meteoritical Bulletin). The meteorite was classified as a CM chondrite, but some lithlogies show a different texture to that of CM. In this study, we investigated the petrography, mineral-ogy, chemistry, and isotopic composition of an unusual Metal-rich-lithology from this fresh fall
Characterization of the Elasticity of CD4+ T Cells: An Approach Based on Peak Force Quantitative Nanomechanical Mapping
CD4+ T cells are essential players in orchestrating the specific immune response against intracellular pathogens, and
in inhibiting tumor development in an early stage. The activation of T cells is triggered by engagement of T cell
receptors (TCRs). Here, CD3 and CD28 molecules are key factors, (co)stimulating signaling pathways essential for
activation and proliferation of CD4+ T cells. T cell activation induces the formation of a tight mechanical bond
between T cell and target cell, the so-called immunological synapse (IS). Due to this, mechanical cell properties,
including stiffness, play a significant role in modulating cell functions. In the past, many approaches were made to
investigate mechanical properties of immune cells, including micropipette aspiration, microplate-based rheometry,
techniques based on deformation during cytometry, or the use of optical tweezers. However, the stiffness of T
lymphocytes at a subcellular level at the IS still remains largely elusive.
With this protocol, we introduce a method based on atomic force microscopy (AFM), to investigate the local
cellular stiffness of T cells on functionalized glass/Polydimethylsiloxan (PDMS) surfaces, which mimicks focal
stimulation of target cells inducing IS formation by T cells. By applying the peak force nanomechanical mapping
(QNM) technique, cellular surface structures and the local stiffness are determined simultaneously, with a
resolution of approximately 60 nm. This protocol can be easily adapted to investigate the mechanical impact of
numerous factors influencing IS formation and T cell activation
The Low-Molecular Weight Protein Arginine Phosphatase PtpB Affects Nuclease Production, Cell Wall Integrity, and Uptake Rates of Staphylococcus aureus by Polymorphonuclear Leukocytes
The epidemiological success of Staphylococcus aureus as a versatile pathogen in mammals
is largely attributed to its virulence factor repertoire and the sophisticated regulatory network
controlling this virulon. Here we demonstrate that the low-molecular-weight protein arginine
phosphatase PtpB contributes to this regulatory network by affecting the growth phase-dependent
transcription of the virulence factor encoding genes/operons aur, nuc, and psmα, and that of the
small regulatory RNA RNAIII. Inactivation of ptpB in S. aureus SA564 also significantly decreased the
capacity of the mutant to degrade extracellular DNA, to hydrolyze proteins in the extracellular milieu,
and to withstand Triton X-100 induced autolysis. SA564 âptpB mutant cells were additionally ingested
faster by polymorphonuclear leukocytes in a whole blood phagocytosis assay, suggesting that PtpB
contributes by several ways positively to the ability of S. aureus to evade host innate immunity
Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity
DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of â131mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsb
A detailed guideline for the fabrication of single bacterial probes used for atomic force spectroscopy
The atomic force microscope (AFM) evolved as a standard device in modern microbiological research. However, its capability as a sophisticated force sensor is not used to its full capacity. The AFM turns into a unique tool for quantitative adhesion research in bacteriology by using âbacterial probesâ. Thereby, bacterial probes are AFM cantilevers that provide a single bacterium or a cluster of bacteria as the contact-forming object. We present a step-by-step protocol for preparing bacterial probes, performing force spectroscopy experiments and processing force spectroscopy data. Additionally, we provide a general insight into the field of bacterial cell force spectroscopy
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