2 nya forskningsrapporter

Vår HoloMonitorfrälste forskare från Ungern, Robert Horvath, har tillsammans m kollegor fått sina senaste studier publicerade i det vetenskapliga organet European Journal of Cell Biology.

 

Studien (som är alldeles för komplicerad för undertecknad att förstå sig på i detalj) handlar i stora drag om hur och vilka mekanismer bakom cancerceller fås att utveckla dessa cancerceller till metastaser. 

En mycket värdefull forskning som kommer leda till bättre kunskap och förståelse för världens alla cancerforskare i deras arbete att finna bot för dessa vandrande förb-ade amöbor.

Robert har använt funktionen single cell observation i sitt HoloMonitorinstrument för att nå dessa nya insikter.

Cell-substratum and Cell-cell Adhesion Forces and Single-cell Mechanical Properties in Mono- and Multilayer Assemblies From Robotic Fluidic Force Microscopy

Highlights

•The direct measurement of cellular forces in a precise and high-throughput manner is still challenging.
•We first employ the robotic fluidic force microscopy (robotic FluidFM) to study cellular forces in mono- and multilayered cell assemblies.
•This novel technology allowed us to study cellular forces and single-cell biomechanical properties with an unprecedented throughput and resolution.

Abstract

The epithelium covers, protects, and actively regulates various formations and cavities of the human body. During embryonic development the assembly of the epithelium is crucial to the organoid formation, and the invasion of the epithelium is an essential step in cancer metastasis. Live cell mechanical properties and associated forces presumably play an important role in these biological processes. However, the direct measurement of cellular forces in a precise and high-throughput manner is still challenging. We studied the cellular adhesion maturation of epithelial Vero monolayers by measuring single-cell force-spectra with high-throughput fluidic force microscopy (robotic FluidFM). Vero cells were grown on gelatin-covered plates in different seeding concentrations, and cell detachment forces were recorded from the single-cell state, through clustered island formation, to their complete assembly into a sparse and then into a tight monolayer. A methodology was proposed to separate cell-substratum and cell-cell adhesion force and energy (work of adhesion) contributions based on the recorded force-distance curves. For comparison, cancerous HeLa cells were also measured in the same settings. During Vero monolayer formation, a significantly strengthening adhesive tendency was found, showing the development of cell-cell contacts. Interestingly, this type of step-by-step maturation was absent in HeLa cells. The attachment of cancerous HeLa cells to the assembled epithelial monolayers was also measured, proposing a new high-throughput method to investigate the biomechanics of cancer cell invasion. We found that HeLa cells adhere significantly stronger to the tight Vero monolayer than cells of the same origin. Moreover, the mechanical characteristics of Vero monolayers upon cancerous HeLa cell influence were recorded and analyzed. All these results provide insight into the qualitative assessment of cell-substratum and cell-cell mechanical contacts in mono- and multilayered assemblies and demonstrate the robustness and speed of the robotic FluidFM technology to reveal biomechanical properties of live cell assemblies with statistical significances.

Materials and Methods (urval)

4.5. Digital holographic microscopy

The digital holographic cytometer, Holomonitor M4 (Phase Holographic Imaging PHI AB, Lund, Sweden) was placed inside a humidified incubator with 37°C and 5% CO₂. Vero cell spreading and growth was monitored for 24 hours, during which they established a confluent monolayer on a gelatin coated 35 mm glass bottom dish (VWR). Gelatin coating was performed as described above, and for the recording the dish was filled with 3 ml of completed cell culture medium. Prior to the recording 200 µl cell suspension (~6×10⁵ cells) were added to the 35 mm gelatin-coated dish, which was covered with the HoloLid (Phase Holographic Imaging PHI AB, Lund, Sweden). The set-up of the Holomonitor and the HoloLid are determined by the manufacturer, and all guidelines were followed according to the manual.

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Sen om inte Roberts studier var tillräckligt komplicerade att förstå sig på kommer här en studie som är helt omöjlig att bena ut detaljerna i. 

Ett bamseforskningsprojekt involverande 26 forskare från 8 länder, under ledning av våra vänner från Norge Arnesen Lab.

Cellforskning på allra högsta nivå som visar på koppling mellan ett specifikt protein och manlig sterilitet samt förkortad livslängd. Utöver det vågar jag mig inte på att kommentera innehållet.

Jo,möjligtvis att forskarna som i Roberts studie här ovan använt sig av single cell observation funktionen i sitt (sina?) HoloMonitorinstrument.

Studien är såpass ny så den är under granskning för att sen publiceras i förmodligen tämligen respekterade organ. Jag utesluter inte Nature.

N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

SUMMARY

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.

In Brief Varland, Silva et al. define that a major cellular role of N-terminal acetylation is shielding proteins from proteasomal degradation by specific ubiquitin ligases. The human N-terminal acetyltransferase NatC protects the neddylation regulator UBE2M from degradation, while overexpression of Drosophila UBE2M/UbcE2M rescues the longevity and motility defects of NatC deletion.

Methods (urval)

Holographic live cell imaging

HAP1 cells were seeded in a 24-well plate (16,000 cells/well) in 2.5 mL filtrated IMDM medium and incubated for 20-30 min at RT to ensure cell attachment. 

Cells were monitored for 48 h in a HoloMonitor M4 live cell imaging system (Phase Holographic Imaging PHI AB, Lund, Sweden) acquiring holographic images every 10 min at 20× magnification. Each cell line was imaged from three different wells with one randomly chosen field of view per well (n = 3). Images were analyzed using HoloMonitor App Suite version 3.5.0.214. 

Single cells were automatically identified using auto minimum error with background threshold set to 120-140 and minimum object size set to 15-25. For analysis, images were manually examined to ensure correct cell number, size and morphology was identified, and out-of-focus images were removed. 

Cell proliferation analysis is based on cell count from images taken every 4^th h in the timeframe 0-42 h. Growth curve show mean ± SEM of three independent experiments. Cell proliferation was quantified by comparing relative increase in cell numbers at 42 h. 

Data are shown as mean ± SEM of three independent experiments pooled together and are expressed relative to WT (n=9). Significance was determined using one-way ANOVA with Dunnett’s correction.

Min kommentar

Oavsett våra vänner garanternas ihärdiga aktiesäljeri fortsätter HoloMonitor att leverera delaktighet i högst avancerad forskning. Som Roberts resultat i ny kunskap om bakomliggande faktorer varför cancerceller metastaserar. Till ny kunskap om manlig fertilitet och livslängd. 

HoloMonitor är ett förbaskat versatilt och användbart instrument för många typer av forskning.

Måtte forskningsvärlden lista ut det snart.

                                            Mvh the99

 

Ps. Håll ögonen öppna den närmsta tiden,dvs kolla bloggen. Undertecknad jobbar för att gå i mål med insamlingen. Går allt i lås kommer pengarna verkligen göra nytta. Ds.

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