HoloMonitor i Nature x2 +1

 En kinesisk studie om benskörhet har publicerats i Nature,2/3 2024. Studien är tyvärr inlåst,men Mr google och studiens Supplementary information bekräftar användande av HoloMonitor.

The histone methyltransferase ASH1L protects against bone loss by inhibiting osteoclastogenesis

Published 02 March 2024

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3 forskare från skotska Dundee har idag 4/3 fått sina studier om bröstcancer publicerade i Nature.

The search for CDK4/6 inhibitor biomarkers has been hampered by inappropriate proliferation assays

Published: 04 March 2024

Abstract

CDK4/6 inhibitors are effective at treating advanced HR+ /HER2- breast cancer, however biomarkers that can predict response are urgently needed. We demonstrate here that previous large-scale screens designed to identify which tumour types or genotypes are most sensitive to CDK4/6 inhibitors have misrepresented the responsive cell lines because of a reliance on metabolic proliferation assays. CDK4/6-inhibited cells arrest in G1 but continue to grow in size, thereby producing more mitochondria. We show that this growth obscures the arrest using ATP-based proliferation assays but not if DNA-based assays are used instead. Furthermore, lymphoma lines, previously identified as the most sensitive, simply appear to respond the best using ATP-based assays because they fail to overgrow during the G1 arrest. Similarly, the CDK4/6 inhibitor abemaciclib appears to inhibit proliferation better than palbociclib because it also restricts cellular overgrowth through off-target effects. DepMap analysis of screening data using reliable assay types, demonstrates that palbociclib-sensitive cell types are also sensitive to Cyclin D1, CDK4 and CDK6 knockout/knockdown, whereas the palbociclib-resistant lines are sensitive to Cyclin E1, CDK2 and SKP2 knockout/knockdown. Potential biomarkers of palbociclib-sensitive cells are increased expression of CCND1 and RB1, and reduced expression of CCNE1 and CDKN2A. Probing DepMap with similar data from metabolic assays fails to reveal these associations. Together, this demonstrates why CDK4/6 inhibitors, and any other anti-cancer drugs that arrest the cell cycle but permit continued cell growth, must now be re-screened against a wide-range of cell types using an appropriate proliferation assay. This would help to better inform clinical trials and to identify much needed biomarkers of response.

Methods (urval)

Time lapse imaging

To characterise the arrest caused by palbociclib each cell line was plated at low density (15,000 per well) into an Ibidi μ-plate glass-bottomed 24 well plate. 
The following day cells were treated with drugs and then imaged using a Holomonitor M4 (Phase Holographic Imaging) at 37 °C with 5% CO2. 
Images were taken every 20 min for a total of 4 days. 
Image analysis was performed using the Holomonitor App Suite. 
For each condition, cells were selected at random and then followed by eye to record the length of time between the first and second mitosis (or the end of the movie).

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7 ungerska forskare har publicerat sin studie om den jobbiga bukspottskörtelcancern, där 5 årsöverlevnaden är låg (5-10%). Forskarna har studerat olika varianter av peptider (en molekyl bestående av aminosyror) i syfte att hitta en peptid att använda som bas i ett framtida cancerläkemedel.

Man tror sig ha nått det målet. Läs hela studien för mer info.

Characterisation of the cell and molecular biological effect of peptide-based daunorubicin conjugates developed for targeting pancreatic adenocarcinoma (PANC-1) cell line

Abstract

Pancreatic adenocarcinoma is one of the tumours with the worst prognosis, with a 5-year survival rate of 5–10%. Our aim was to find and optimise peptide-based drug conjugates with daunorubicin (Dau) as the cytotoxic antitumour agent. When conjugated with targeting peptides, the side effect profile and pharmacokinetics of Dau can be improved. The targeting peptide sequences (e.g. GSSEQLYL) we studied were originally selected by phage display. By Ala-scan technique, we identified that position 6 in the parental sequence (Dau=Aoa-LRRY-GSSEQLYL-NH2, ConjA) could be modified without the loss of antitumour activity (Dau=Aoa-LRRY-GSSEQAYL-NH2, Conj03: 14. 9% viability). Our results showed that the incorporation of p-chloro-phenylalanine (Dau=Aoa-LRRY-GSSEQF(pCl)YL-NH2, Conj16) further increased the antitumour potency (10−5 M: 9.7% viability) on pancreatic adenocarcinoma cells (PANC-1). We found that conjugates containing modified GSSEQLYL sequences could be internalised to PANC-1 cells and induce cellular senescence in the short term and subsequent apoptotic cell death. Furthermore, the cardiotoxic effect of Dau was markedly reduced in the form of peptide conjugates. In conclusion, Conj16 had the most effective antitumor activity on PANC-1 cells, which makes this conjugate promising for developing new targeted therapies without cardiotoxic effects.

Materials and Methods (urval)

2.7. Morphometric changes

To measure morphometric changes induced by the conjugates in comparison with Dau, a holographic transmission microscope (HoloMonitor M4; Phase Holographic Imaging AB, Lund, Sweden) was used, which uses a laser to produce pseudo-3D images of cells. 

This method enables real-time monitoring of cell morphological changes without any labelling. Cells were plated at a concentration of 3.5*105 cells/flask/4500 μL cell culture medium. 

After 24 hours of seeding, the cells were treated with the conjugates at 10 μM. 

We took images at 24, 48 and 72 hours after treatment. At least five images from different fields of view were taken for each culture dish. These images were analysed with the help of the microscope software (Hstudio M4, Phase Holographic Imaging AB, Lund, Sweden). For the evaluation, at least 25 cells per image (field of views) were identified by the minimum error histogram-based threshold algorithm of the in-built software. For morphometric analysis, the cells' average area, optical thickness and optical volume were used. 

The (J) holographic microscopy images of PANC-1 cells after treatment with Conj16 for 24, 48 and 72 hours.

Min kommentar

Återigen en publicering i det högt respekterade organet Nature.Denna gång handlar det om benskörhet och visar hur versatilt HoloMonitor är även utanför cancerområdet.Tillägg: 1 till Naturepublikation!

+1 är just inom detta förbaskade område,och den här gången studier om en skitjobbig cancer få överlever nån längre tid.De ungerska forskarna har i sin studie förhoppningsvis hittat embryot till ett läkemedel som tar kål på denna amöba. Värt att notera är att HoloMonitor använts i syfte att studera olika preparat och deras förmåga att ha ihjäl cancerceller. Läkemedelsframtagning med benäget användande av HoloMonitor.Börjar bli en hel del sådana studier.

                                     

                                        Mvh the99

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