Hursom,jag nås av information att en amerikansk doktorand i dagarna har disputerat med en avhandling som heter duga.Doktoranden Andrew Scott McNeal från San Fransisco har erövrat sin doktorstitel (och doktorshatt) med en ,minst sagt, gedigen studie kring hudcancer som andra forskare i gebitet kommer ha stor nytta av.
Jag tänker inte dissikera Andrews forskning närmare då jag är en enkel lekman med begränsade kunskaper inom biologi och kemi.Förvisso som hudcancerdrabbad är jag absolut intresserad av det Andrew har åstadkommit,men förmågan att förstå denna komplicerade forskning är av naturliga skäl begränsad.
Linkedin berättar att Andrew har varit inom forskarvärlden sen 2008,och de 5 senaste åren fått möjlighet att bedriva sin forskning hos ett universitet med den absolut bästa utrustningen.Japp,där ingår HoloMonitor.
Men om vi går till hans doktorsavhandling (135 sidor) betitlad:
Melanocytes, Melanocytic Nevi, and Progression to Melanoma
Abstract
Benign melanocytic nevi form when
melanocytes that acquire a BRAFV600E mutation undergo a period of rapid
proliferation followed by subsequent growth-arrest. Constitutive
activation of MAPK signaling downstream of BRAF drives the initial
proliferative phenotype. However, the factors that establish and
maintain growth-arrest in nevi remain elusive.
Here we investigate the contributions of several potential mediators of BRAFV600E-induced growth arrest in nevus melanocytes. These mediators include the gene products of the CDKN2A-CDKN2B genetic locus, nevus-enriched microRNAs MIR211-5p and MIR328-3p, as well as transcriptional networks associated with primary cilia expression.
p16INK4A, the cyclin dependent kinase inhibitor encoded by CDKN2A, has historically been associated with nevus formation. CDKN2B’s gene product, p15INK4B, remained under-studied in the context of nevus formation. Using primary melanocytes isolated directly from human nevi and naturally expressing the BRAFV600E-acitvating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAFV600E, we showed that p15INK4B expression is required for growth arrest in nevi and that loss promotes progression to melanoma. Investigating the role of the p16INK4A tumor suppressor in nevus formation and progression to melanoma, we found that loss of p16INK4A contributes to melanocyte hypermotility in vitro and increases invasive and metastatic behavior of melanoma cell lines in vivo. Using CRISPR-Cas9 to engineer a cellular model of melanoma initiation from primary human melanocytes, we determined that E2F1 upregulation as a consequence of CDKN2A loss drives these new phenotypes.
Additional acquired genetic alterations do not distinguish proliferating BRAFV600E melanocytes from their growth-arrested nevus counterparts, suggesting a role for regulatory elements. We investigated the role of microRNAs in the initiation and maintenance of nevus growth arrest. Using primary human melanocytes, melanocytic nevi, and adjacent melanoma, we show that MIR211-5p and MIR328-3p are enriched in nevi compared to normal melanocytes, then subsequently downregulated in adjacent melanoma. Both MIR211-5p and MIR328-3p proved necessary effectors of BRAFV600E-induced growth arrest in human melanocytes. We identified microRNA target networks which, when suppressed, phenocopy BRAFV600E-induced growth arrest and converge on inhibition of AURKB to block cell cycle progression in primary human melanocytes.
Similarly, expression of the primary cilium organelle has been shown to differentiate nevi from malignant melanoma. Investigations of primary cilia-associated gene expression in matched nevus and melanoma cases demonstrates that transcriptional changes in core primary cilia genes and related signaling molecules accompanies loss of the organelle during progression to melanoma.
Taken together, these data suggest that the BRAFV600E mutation leads to a cascade of intracellular changes that establish and maintain of growth arrest in human melanocytic nevi.
Och nej,jag har inte för avsikt att sätta mig in i abstractet närmare (kom igen,undertecknad är aningens bakis idag.OK?) mer än nämna att Andrew har utgått från en ofarlig leverfläck (nevus) på huden till utvecklande av en jobbig j-a hudcancer (malign melanom).Alltså har han följt ett skede som kan räknas i flera år,vilket är imponerande då forskare sällan har det tålamodet.
Men Andrew har med sina studier visat upp helt ny kunskap som kommer gagna hudcancerforskarna i förståelse för hur denna typ av cancer kan utvecklas från ett hyfsat ofarligt tillstånd (benign) till en dödlig variant (malign).
Andrew förtjänar definitivt sin doktorshatt med råge är undertecknads åsikt.
Figure 2.1:BRAFV600E-Driven Melanocyte Growth Arrest.
(A) Clinical image of a benign nevus used in this study.
Till Materials and Methods.Och som vanligt klipper jag enbart in HoloMonitor-relaterad info.
Live Quantitative Phase Imaging
Digital holographic cytometry was performed using HoloMonitor M4 imaging cytometers (Phase Holographic Imaging, Lund, Sweden) and analyzed for cell proliferation, volume and death using HStudio (v2.6.3) as previously described in Hejna et al. 2017 (Hejna et al., 2017).
For normal human melanocyte proliferati on experiments, cells were seeded into 6-well culture plates (Sarstedt, 83.3920) at 100,000 cells per well and either live-imaged in a standard mammalian cell incubator or serially imaged at different days as indicated.
For dose response curves, 60,000 501Mel cells or 150,000 HCIMel019 cells were plated per well. The next day, media was replaced with media containing indicated concentrations of barasertib (AurK-B inhibitor; AZD1152-HQPA | AZD2811) obtained from Selleckchem.com (Catalog No. A1147 –5 mg) and cells were imaged for 48-72 hours.Live quantitative phase imaging coupled with fluorescent imaging was conducted using the Livecyte platform (Phasefocus, Sheffield, UK).
Holographic imaging and microscopy
Holographic imaging was conducted as previously described 34. Briefly, imaging was conducted with a HoloMonitor M4 imaging cytometer with high precision automated stage (Phase Holographic Imaging, Lund, Sweden) installed in a standard tissue culture incubator.
Cells were plated in 6-well standard tissue culture plates, plated at 10,000 – 100,000 cells per well depending on cell type and experiment and covered with a Hololid (PHI).
For motility analyses, holograms were generated every hour for indicated time and analyzed using the Hstudio software package (PHI). For growth arrest analyses, cells were classified based upon quantitative optical metrics as previously described 34, where cells were defined as growth arrested if they stopped dividing while adopting a morphology indistinguishable from NHMs treated with CDK4/6 inhibitor 62. Fluorescent images were captured with an EVOS FL inverted microscope (ThermoFisher), colored images were captured with a Lieca Dmi1 light microscope, and high quality scans of clinical specimens were scanned with a ScanScope XT scanner (Aperio).
Figure 2.9: CDKN2A loss promotes melanocyte motility and invasion(A)Schematic of experimental set-up.
For each experiment, NHMs are derived from donated tissue and engineered for CDKN2A loss as in Figure 1. After isolation, CDKN2A null and wild-type sibling cells are monitored via digital holographic cytometry for 72 hr. The rate of cell division, motility, morphology, growth arrest and detachment are quantified. Representative holographic phase shift images (bottom left) show colored comet tails tracking cells.(B)Single cell motility analysis of CDKN2A null NHMs compared to wild-typesiblings. Box and whisker plot represents mean, 10th, 25th,75th and 90th percentiles of at least fifty cells each from three CRISPR reactions of three preparations of NHMs.(C)Quantification as in (B) but excluding cells that divided during the 72 hr time period surrounding 24 hr of analysis.(D and E)Time-lapsed holographic phase shift images (D) and quantification (E) of NHMs migrating into scratched region. Yellow line indicates scratch limit at first time point. Error bars represent standard deviation of the mean of three CRISPR reactions of three preparations of NHMs.(F)Quantification of transwell invasion assays where CDKN2A null NHMs and wild-type siblings were required to migrate through high-density basement membrane extract. Box and whisker plots represent mean, 25th and 75th percentiles, minimum and maximum values of five CRISPR reactions of three preparations of NHMs.(G)Single cell motility analysis of CDKN2A null NHMs transduced with and sorted for the indicated vectors. Box and whisker plot represents mean, 10th, 25th, 75th and 90th percentiles of at least fifty cells each from three transductions.Asterisks indicate p value of * <0.05 to ***** <0.000005 from unpaired t-test. N.S. indicates no significant difference.
Min kommentar
Som hudcancerdrabbad är det naturligtvis lätt att beskriva Andrews forskning i hyllande ordalag.
Och förmodligen kommer den mottas i samma tonläge bland hans forskarkollegor.
Jag menar,läs hans forskningsrapport och fatta hur väl underbyggd den är på 185 !! tidigare forskningsrapporter. Hundraåttiofem genomgånga studier för att vidimera resultaten Andrew har lyckats nå.
Så ja,undertecknad hyllar hans forskning högt.
Det Andrew åstadkommit är troligtvis helt ny kunskap hans kollegor kommer ha stor nytta av i sina egna studier.
Och som bloggen brukar avsluta : utan PHI´s excellenta teknik i form av HoloMonitor skulle forskarna i dagsläget inte ha denna kunskap.
Ps. Mycket av Andrews forskning går att härleda till PHI`s HoloMonitor-frälste forskare Robert Judson-Torres.
Robert som undertecknad tror kommer bli en framtida Nobelpriskandidat.
Han är fortfarande ganska ung,men med den intensitet han bedriver sin hudcancerforskning är det en lågoddsare att han till slut kommer knäcka Malign Melanom problematiken.
Mvh (en ovanligt törstig) the99
Here we investigate the contributions of several potential mediators of BRAFV600E-induced growth arrest in nevus melanocytes. These mediators include the gene products of the CDKN2A-CDKN2B genetic locus, nevus-enriched microRNAs MIR211-5p and MIR328-3p, as well as transcriptional networks associated with primary cilia expression.
p16INK4A, the cyclin dependent kinase inhibitor encoded by CDKN2A, has historically been associated with nevus formation. CDKN2B’s gene product, p15INK4B, remained under-studied in the context of nevus formation. Using primary melanocytes isolated directly from human nevi and naturally expressing the BRAFV600E-acitvating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAFV600E, we showed that p15INK4B expression is required for growth arrest in nevi and that loss promotes progression to melanoma. Investigating the role of the p16INK4A tumor suppressor in nevus formation and progression to melanoma, we found that loss of p16INK4A contributes to melanocyte hypermotility in vitro and increases invasive and metastatic behavior of melanoma cell lines in vivo. Using CRISPR-Cas9 to engineer a cellular model of melanoma initiation from primary human melanocytes, we determined that E2F1 upregulation as a consequence of CDKN2A loss drives these new phenotypes.
Additional acquired genetic alterations do not distinguish proliferating BRAFV600E melanocytes from their growth-arrested nevus counterparts, suggesting a role for regulatory elements. We investigated the role of microRNAs in the initiation and maintenance of nevus growth arrest. Using primary human melanocytes, melanocytic nevi, and adjacent melanoma, we show that MIR211-5p and MIR328-3p are enriched in nevi compared to normal melanocytes, then subsequently downregulated in adjacent melanoma. Both MIR211-5p and MIR328-3p proved necessary effectors of BRAFV600E-induced growth arrest in human melanocytes. We identified microRNA target networks which, when suppressed, phenocopy BRAFV600E-induced growth arrest and converge on inhibition of AURKB to block cell cycle progression in primary human melanocytes.
Similarly, expression of the primary cilium organelle has been shown to differentiate nevi from malignant melanoma. Investigations of primary cilia-associated gene expression in matched nevus and melanoma cases demonstrates that transcriptional changes in core primary cilia genes and related signaling molecules accompanies loss of the organelle during progression to melanoma.
Taken together, these data suggest that the BRAFV600E mutation leads to a cascade of intracellular changes that establish and maintain of growth arrest in human melanocytic nevi.
Och nej,jag har inte för avsikt att sätta mig in i abstractet närmare (kom igen,undertecknad är aningens bakis idag.OK?) mer än nämna att Andrew har utgått från en ofarlig leverfläck (nevus) på huden till utvecklande av en jobbig j-a hudcancer (malign melanom).Alltså har han följt ett skede som kan räknas i flera år,vilket är imponerande då forskare sällan har det tålamodet.
Men Andrew har med sina studier visat upp helt ny kunskap som kommer gagna hudcancerforskarna i förståelse för hur denna typ av cancer kan utvecklas från ett hyfsat ofarligt tillstånd (benign) till en dödlig variant (malign).
Andrew förtjänar definitivt sin doktorshatt med råge är undertecknads åsikt.
Figure 2.1:BRAFV600E-Driven Melanocyte Growth Arrest.
(A) Clinical image of a benign nevus used in this study.
Till Materials and Methods.Och som vanligt klipper jag enbart in HoloMonitor-relaterad info.
Live Quantitative Phase Imaging
Digital holographic cytometry was performed using HoloMonitor M4 imaging cytometers (Phase Holographic Imaging, Lund, Sweden) and analyzed for cell proliferation, volume and death using HStudio (v2.6.3) as previously described in Hejna et al. 2017 (Hejna et al., 2017).
For normal human melanocyte proliferati on experiments, cells were seeded into 6-well culture plates (Sarstedt, 83.3920) at 100,000 cells per well and either live-imaged in a standard mammalian cell incubator or serially imaged at different days as indicated.
For dose response curves, 60,000 501Mel cells or 150,000 HCIMel019 cells were plated per well. The next day, media was replaced with media containing indicated concentrations of barasertib (AurK-B inhibitor; AZD1152-HQPA | AZD2811) obtained from Selleckchem.com (Catalog No. A1147 –5 mg) and cells were imaged for 48-72 hours.Live quantitative phase imaging coupled with fluorescent imaging was conducted using the Livecyte platform (Phasefocus, Sheffield, UK).
Holographic imaging and microscopy
Holographic imaging was conducted as previously described 34. Briefly, imaging was conducted with a HoloMonitor M4 imaging cytometer with high precision automated stage (Phase Holographic Imaging, Lund, Sweden) installed in a standard tissue culture incubator.
Cells were plated in 6-well standard tissue culture plates, plated at 10,000 – 100,000 cells per well depending on cell type and experiment and covered with a Hololid (PHI).
For motility analyses, holograms were generated every hour for indicated time and analyzed using the Hstudio software package (PHI). For growth arrest analyses, cells were classified based upon quantitative optical metrics as previously described 34, where cells were defined as growth arrested if they stopped dividing while adopting a morphology indistinguishable from NHMs treated with CDK4/6 inhibitor 62. Fluorescent images were captured with an EVOS FL inverted microscope (ThermoFisher), colored images were captured with a Lieca Dmi1 light microscope, and high quality scans of clinical specimens were scanned with a ScanScope XT scanner (Aperio).
Figure 2.9: CDKN2A loss promotes melanocyte motility and invasion(A)Schematic of experimental set-up.
For each experiment, NHMs are derived from donated tissue and engineered for CDKN2A loss as in Figure 1. After isolation, CDKN2A null and wild-type sibling cells are monitored via digital holographic cytometry for 72 hr. The rate of cell division, motility, morphology, growth arrest and detachment are quantified. Representative holographic phase shift images (bottom left) show colored comet tails tracking cells.(B)Single cell motility analysis of CDKN2A null NHMs compared to wild-typesiblings. Box and whisker plot represents mean, 10th, 25th,75th and 90th percentiles of at least fifty cells each from three CRISPR reactions of three preparations of NHMs.(C)Quantification as in (B) but excluding cells that divided during the 72 hr time period surrounding 24 hr of analysis.(D and E)Time-lapsed holographic phase shift images (D) and quantification (E) of NHMs migrating into scratched region. Yellow line indicates scratch limit at first time point. Error bars represent standard deviation of the mean of three CRISPR reactions of three preparations of NHMs.(F)Quantification of transwell invasion assays where CDKN2A null NHMs and wild-type siblings were required to migrate through high-density basement membrane extract. Box and whisker plots represent mean, 25th and 75th percentiles, minimum and maximum values of five CRISPR reactions of three preparations of NHMs.(G)Single cell motility analysis of CDKN2A null NHMs transduced with and sorted for the indicated vectors. Box and whisker plot represents mean, 10th, 25th, 75th and 90th percentiles of at least fifty cells each from three transductions.Asterisks indicate p value of * <0.05 to ***** <0.000005 from unpaired t-test. N.S. indicates no significant difference.
Min kommentar
Som hudcancerdrabbad är det naturligtvis lätt att beskriva Andrews forskning i hyllande ordalag.
Och förmodligen kommer den mottas i samma tonläge bland hans forskarkollegor.
Jag menar,läs hans forskningsrapport och fatta hur väl underbyggd den är på 185 !! tidigare forskningsrapporter. Hundraåttiofem genomgånga studier för att vidimera resultaten Andrew har lyckats nå.
Så ja,undertecknad hyllar hans forskning högt.
Det Andrew åstadkommit är troligtvis helt ny kunskap hans kollegor kommer ha stor nytta av i sina egna studier.
Och som bloggen brukar avsluta : utan PHI´s excellenta teknik i form av HoloMonitor skulle forskarna i dagsläget inte ha denna kunskap.
Ps. Mycket av Andrews forskning går att härleda till PHI`s HoloMonitor-frälste forskare Robert Judson-Torres.
Robert som undertecknad tror kommer bli en framtida Nobelpriskandidat.
Han är fortfarande ganska ung,men med den intensitet han bedriver sin hudcancerforskning är det en lågoddsare att han till slut kommer knäcka Malign Melanom problematiken.
Mvh (en ovanligt törstig) the99
Tack! Låter mycket flrhoppningsfullt för alla parter; drabbade, forskningen och PHI.
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