Science this week

Stem cell research

Modeling Developmental and Tumorigenic Aspects of Trilateral Retinoblastoma via Human Embryonic Stem Cells (Avior et al. 2017, Stem Cell Research)

I love reading good papers reporting modelling of early-onset human diseases in human stem cells. There is one in Stem Cell Research that caught my attention this week, by Avior et al., modelling ablation of retinoblastoma protein (Rb) in human embryonic stem cells (hESCs). This is a well-known tumour suppressor and an inherited loss of Rb protein leads to retinoblastoma development, the most common primary intraocular pediatric cancer (95 % of cases diagnosed before the age of 5). In 6 % of cases, patients develop trilateral retinoblastoma characterised by neuroectodermal tumour occurence in additional to retinal tumours in the eye. Combined with results from conditional knockout mice, the Rb protein has been suggested to play a role in neural development. In the current study,  following CRISPR/Cas9 gene editing to knockout the Rb protein, modified hESCs are subjected to an array of assays to study the resulting phenotype. After observing normal neural stem cell generation in a 2D directed differentiation protocol, despite expectations of a phenotype compared to wildtype hESCs, the authors conclude that a more complex differentiation paradigm is needed. As a result, they move on to generate teratomas in immunocomprised mice and observe a substantial expansion of neural structures in Rb-null teratomas. This highlights the importance of using multiple methods to assess a phenotype reliable. I have come to the same conclusion in my studies as 2D differentiation protocols often rely on non-physiological concentrations of various drugs and peptides, whereby a phenotype may remain masked. This study also reveals mitochondrial dysfunction in Rb-null hESCs and establishes a framework for high-throughput testing of hundreds of FDA-approved chemotherapies in this human cell models.


High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells (D’Antonio et al. 2017, Stem Cell Research)

This paper made me aware of fluorescent cell barcoding as an efficient way to pool multiple stem cell samples together and save on antibodies during initial characterisation. To make pluripotency and multilineage analysis across multiple stem cell lines available, the authors provide an Excel file which users can use to generate their own scores and heatmaps using Ct values of a reference gene of interest and any chosen marker genes.

Another paper from the same lab: Aberrant DNA Methylation in Human iPSCs Associates with MYC-Binding Motifs in a Clone-Specific Manner Independent of Genetics (Panopoulus et al. 2017 Cell Stem Cell)

A lot of effort is invested into understanding how iPSCs differ from hESCs and whether somatic memory may limit their utility in disease modelling and as potential treatment strategies. It is not well understood whether aberrant methylation of CpG sites in iPSCs is mainly caused by genetic variability or are artefacts of the reprogramming factors. The current study reports the following: “Here we generated 22 iPSC clonal lines from six individuals (three pairs of older monozygotic twins). We profiled the 22 iPSC lines, at early (passages 5 [p5] and 9 [p9]) and late (passage 20 [p20]) passages, as well as fibroblasts (tissue of origin) using genome-wide methylation arrays and RNA sequencing (RNA- seq) data.We estimate aberrantmethylation of the iPSCs relative to ESCs, and we show that aberrant methylation affects gene expression and is enriched for CpGs associated with MYC and MYC-related protein motifs. We then identify genome-wide associations between CpG methylation variation and genetic background, clone, and passage, andwe showthat these associ- ationslikely result fromrelevant biological processes.Weexamine whether aberrant CpGs are enriched for CpGs associated with genetic and non-genetic effects, and we show that aberrant methylation preferentially occurs at CpGs showing clone-associ- ated effects and is less enriched at sites associated with genetic background. Our study shows that non-genetic regulatory mechanisms associated with clone-specific effects most strongly underlie iPSC aberrancy.”

My immediate reservations without having read the study in detail is the limited number of twin pairs and therefore cells examined as well as the fact that they are all female.

iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types (Panopoulus et al. 2017 Stem Cell Research)

An iPSC resource from the same lab  (very productive!). Will be useful for examining phenotypic diversity across individual induced pluripotent stem cell lines.  Pertinent to the same topic, a study in Cell Stem Cell examines the influence of genetic variation on gene expression in human iPSCs: Large-Scale Profiling Reveals the Influence of Genetic Variation on Gene Expression in Human Induced Pluripotent Stem Cells (DeBoever et al. 2017 Cell Stem Cell). Actually, this seems to be a theme in the current Cell Stem Cell issues and multiple other papers examine the same topic from different perspectives. For instance, Warren et al. provide proof-of-concept that large cohorts of human iPSCs can be used to performed GWAS studies in a dish, here in the context of metabolic disease.


An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells (Guo et al. 2017 Protein & Cell)

A variation on a system that has been introduced in the past – the ability to activate genes from their endogenous locus via a Dox-inducible expression of dCas9 fused to a transcriptional activation domain.

Other bits and pieces that I have not read in detail, but that caught my interest – mostly metabolism-related

Mitochondrial Patch Clamp of Beige Adipocytes Reveals UCP1-Positive and UCP1-Negative Cells Both Exhibiting Futile Creatine Cycling (Bertholet et al. 2017 Cell Metabolism) – up until now all beige adipocytes were assumes to function like brown adipocytes, utilising UCP1 dissipate energy as heat; this paper suggests that there is a subset of beige adipocytes in mice, in epididymal fat in particular, that are UCP-negative yet perform thermogenesis by engaging in futile creatine cycling. Quite biophysical, by the way! If of interest, there is a Preview commentary on this article as well: Now UCP(rotein), Now You Don’t: UCP1 Is Not Mandatory for Thermogenesis (Szabo and Zoratti 2017, Cell Metabolism).

Another paradigm-shaking paper in Cell Metabolism modifies our understanding of the incretin effect mediated by GLP1. I have actually only read the Preview by Habener and Stanojevic (Pancreas and Not Gut Mediates the GLP-1-Induced Glucoincretin Effect) and it is always best to have a look at the actual paper and its data instead, so acknowledging the absence of appropriate assessment on my own, it might still be worth a read for those interested in this field!

Surprising results are the theme in Cell Metabolism this week; another one: FGF21 Regulates Metabolism Through Adipose-Dependent and -Independent Mechanisms by BonDurant et al., providing evidence that adiponectin is dispensable for the metabolic effects of FGF21.

A paper in PNAS by Steptoe and Wardle (Life skills, wealth, health, and wellbeing in later life) looks at the importance of 5 key life skills – conscientiousness, emotional stability, determination, control, and optimism – in later life (cross-sectionally and longitudinally), concluding that (after adjustment of multiple confounding factors) these life skills are associated with wealth, income, subjective wellbeing, less depression, low social isolation and loneliness, more close relationships, better self-rated health, fewer chronic diseases and impaired activities of daily living, faster walking speed, and favorable objective biomarkers (concentration of high-density lipoprotein cholesterol, vitamin D and C-reactive protein, and less central obesity). They were also associated with greater psychological well-being and less loneliness, and a lower incidence of new chronic disease and physical impairment over a 4-y period. I like this study – a good reason to practice mindfulness throughout life!






The weekly science round up – 5 April

Actually, some of  the papers already came out last week, but only just got round to having a proper look at them.


Lysosomal cholesterol activates mTORC1 via an SLC38A9–Niemann-Pick C1 signaling complex (Castellano et al.)

Copied from the final bit of the paper: “This work identifies dedicated machinery that couples cholesterol trafficking through the lyso- some to regulation of cellular growth signaling. LDL-derived cholesterol affects mTORC1 through the combined action of a positive regulator, SLC38A9, and a negative regulator, NPC1 (Fig. 4G). SLC38A9 conveys increases in lysosomal cholesterol through its conserved CARC and CRAC motifs, leading to lysosomal recruitment and activation of mTORC1. In contrast, NPC1 associates with the mTORC1 scaffolding complex and translates cholesterol depletion into mTORC1 inhibition.”


Structural insights into adiponectin receptors suggest ceramidase activity (Vasiliauskaité-Brooks et al.)

Discovery that ADIPORs (1+2) have ceramidase activity that can be enhanced upon adiponectin stimulation. Overall, this enzymatic activity is low so future studies are required to understand whether it bears relevance to the function of adiponectin in vivo and its link to metabolic improvements.


The cold-induced lipokine 12,13-diHOME promotes fatty acid transport into brown adipose tissue (Lynes et al.) 

Cold-induced activation of brown adipose tissue (BAT) is known to result in increased energy expenditure, and it is believed that BAT energy expenditure can be harnessed therapeutically in metabolic disorders. Using global lipodmics, this study identifies a BAT-specific lipokine 12,13-diHOME which is released in response to cold exposure and activates BAT fuel utilisation. Consistent with a decrease in BAT mass with obesity, 12,13-diHOME levels were negatively correlated with obesity and other phenotypes characteristic of the metabolic syndrome. It is impressive that this lipokine is first identified in human participants exposed to cold, followed by mechanistic studies in mouse models, where the effects of 12,13-diHOME on free fatty acid uptake are comparable to noradrenaline-mediated BAT activation. Overall, an elegant study advancing the field with a new candidate for pharmacologic BAT activation.


Nature Biotechnology

Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells (Weinberg et al.)

My brain gets mushy when I try to read this in detail at 10 pm in the evening, but just quite cool to see the engineering of one of the most sophisticated computational biological circuits to date. This done by combining orthogonal recombinases and various DNA sequences that control transcription. Again, I think an electrical engineer would understand this paper better than me, but still impressive…

Synergistic drug combinations for cancer identified in a crisPr screen for pairwise genetic interactions (Han et al.)

In this paper, a global CRISPR screen is performed to identify combinatorial interactions between cancer drug targets. 490,000 sgRNAs are used to screen for synthetic lethal drug target pairs in K562 leukemia cells. The approach is interesting and applicable to other areas. Worth noting the technical aspects of the screen.

A similar paper has also been published in Nature Methods: Combinatorial CRISPR–Cas9 screens for de novomapping of genetic interactions (Shen et al.)

Nature Protocols

From the Zhang lab on CRISPR screens: Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening (Joung et al.)


Nature Genetics

Estimating the selective effects of heterozygous protein-truncating variants from human exome data (Cassa et al.)

Not the best person to understand this, but a study that looks at protein truncating variatns in ExAC if they are predicted to be consequential and estimates gene-based fitness effects and individual gene fitness cost in heterozygotes (only using data from individuals with non-Mendelian disorders). Basically useful to look at novel genes that might have important functions in development as mutations would be highly selected against. The mTOR and MAPK pathways are in the list of pathways with genes that seem to have a high gene fitness cost (unsurprising).

Nature Chemical Biology

Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase (Stanford et al.)

Worth a read – actually quite metabolic and signalling-relevant. Here is the abstract:

“Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.”

JCI Insight

Insulin’s direct hepatic effect explains the inhibition of glucose production caused by insulin secretion (Edgerton et al.)

Given the redundancy in insulin’s ability to control hepatic glucose production (direct and indirect mechanisms), this study set out to explore the contextual importance of some of these mechanisms.

An interesting excerpt from the introduction of the paper, discussing the physiological difference between peripheral insulin infusion and direct infusion into the portal vein: “It is apparent, therefore, that when insulin is infused into a peripheral vein its direct effects on the liver are underemphasized, while its indirect effects are exaggerated. Thus, the route of insulin delivery can have a major impact on both the overall response of the liver and the mechanisms by which that response is achieved. Indeed, over a range of insulin doses, peripheral insulin infusion was not as effective at suppressing HGP as compared with direct infusion into the portal vein.”

Based on this information, “the purpose of this study was to assess whether insulin’s acute indirect effects on HGP are additive to, redundant to, or synergistic with its direct hepatic effects, in the context of a physiologic increase in portal vein insulin level in a large animal model. The impact of eliminating each of insulin’s indirect effects, either alone or in combination, was determined. We focused on insulin’s ability to lower FFAs, activate brain insulin signaling, and reduce glucagon secretion since recent studies have concluded that suppression of lipolysis is the major mechanism by which insulin suppresses HGP (11), that increased brain insulin action is required for the rapid suppression of HGP (17), and because a fall in plasma gluca- gon is potentially a powerful contributor to insulin’s ability to inhibit HGP (15).”

They perform portal insulin infusion (in mice) and simulate a 6-fold increase in insulin secretion, but manage to maintain the physiologic insulin gradient between liver and the rest of the body.

This paper is very interesting given the multiple demonstrations of a key role of insulin’s indirect effect on HGP via modulation of FFA release from adipose tissue; in contrast, this study seems to imply that FFA don’t play a role in HGP suppression under what is described as more physiologic conditions. Similarly, a fall in glucagon secretion is not necessary for insulin direct ability to suppress HGP. The same is true when insulin’s brain action is inhibited. The discussion is really worth a read!

EMBO Journal

AKT-phosphorylated FOXO1 suppresses ERK activation and chemoresistance by disrupting IQGAP1-MAPK interaction (Pan et al.)

Looking forward to reading this one at some point! The accompanying News and Views sums it all up: “And Akt-ion! IQGAP1 in control of signaling pathways” (Choi et al.

Mfn2 is critical for brown adipose tissue thermogenic function (Boutant et al.)

Worth a read! Adipose tissue-specific KO of Mfn2 results in increased fat accumulation in BAT even on a low-fat diet as well as an overall decrease in energy expenditure and an inability to sustain normal body temperature via cold-induced BAT thermogenesis. This is linked to an impaired O2 flux in BAT when specifically measuring the different complexes forming the electron transport chain. At the protein level, Complex I is almost absent upon Mfn2 ablation. Mfn2 is shown to interact with perilipin and the data suggest that this enhances mitochondria-lipid droplet interactions. It seems that glycolysis is increased due to mitochondrial dysfunction. There is also an increased accumulation of FAT in subcutaneous WAT and the authors suggest that this exlpains the improved metabolic profile of HFD-fed adipose-specific Mfn2 KO mice.

USP49 negatively regulates tumorigenesis and chemoresistance through FKBP51-AKT signaling (Luo et al.)

Had a quick skim – in cancer cell lines and cancer mouse models, but the mechanism might be valid in a more physiological context, too. Basically, USP49 deubiquinates FKBP51 which results in increased stabilisation. In turn, FKBP51 promotes the interaction between AKT and PHLPP, which dephosphorylates AKT at S473 thereby reducing its activation.

The phosphorylation status of T522 modulates tissue-specific functions of SIRT1 in energy metabolism in mice (Lu et al.)

Just skimmed the abstract, but might be interesting to adipogenesis enthusiasts.


Angiopoietin-2 in white adipose tissue improves metabolic homeostasis through enhanced angiogenesis (An et al.) – only read abstract, but might be relevant. Just in press, so still not formatted properly, i.e. annoying to read.




Other exciting bits

I have been reading a lot of developmental biology recently and came across the following Guardian coverage of the first experiments that make it possible to break the 14-day rule for embryo research. I just find the videos and images amazing + the useful infographic of lineage specification at the bottom. The two papers giving rise to the Guardian feature can be found at Nature and Nature Cell Biology. These are landmark studies and were recently followed up by a study from the same Cambridge group, now demonstrating that embryonic and extra-embryonic stem cells can be assembled in vitroto mimic embryogenesis (effectively generating embryos in vitro, see Harrison et al. 2017 Science).

In terms of interesting metabolism reviews, it is worth having a look at Nature Reviews Endocrinology – there are good ones on adipose tissue etc.

Modern peer review, science communication and this week’s science news

Modern peer review

At the time of writing this blog post, I am enjoying the lunch provided by the organisers of a workshop on “How to get the most out of modern peer review?” (it is being filmed so you can benefit from it, too!). I have learned a lot, and some things are worth sharing. The first presentation by Wei Mun Chan, Editorial Manager at eLife, offered valuable insight into consultative peer review where reviewers engage in a discussion with each other and feed back to the editors. The final decision letter and the author feedback is also openly accessible to promote greater transparency. This is just one example of a great initiative to promote faster peer reviews, less bias during the process and increased transparency. On to the second presentation, Dr Sabina Alam from F1000 introduced their open-science publishing platform where the paper/data comes first, followed by a transparent peer review process. The whole life-cycle of the paper can be followed openly by the readers who are also invited to comment and review the paper publicly. F1000 allows the publication of multiple types of data, including single findings, as long as it is reported correctly and meets scientific standards. So if you have old reagents gathering dust in the lab and projects that were terminated prematurely, why not share it with the rest of the world? Someone might be able to take it up and build on your efforts. You will get recognition for it, too! In the same vein, F1000 promotes the dissemination of scientific posters and PowerPoint slides; these do not undergo peer review, but are assigned a DOI and are therefore citable – GREAT!

Next we heard from Dr Laurent Gatto from the Proteomics Research Unit here at Cambridge. He gave us some tips for good reviewing, and you can view those online as Dr Gatto shares his material openly. In summary, make sure that the data is always there, that the names and numbers match up, that the metadata is available and the science reproducible!

The final talk before lunch was given by Tom Culley, Marketing Director at Publons. This is my first encounter with Publons, and I am impressed. What a great initiative to give scientists recognition for their peer review work! This can only improve the quality of the published science because reviewers have an incentive to do well and will be recognised for it. Publons creates a complete track record of all your reviews and you can even add past reviews to obtain the deserved credit for them. Another great initiative to foster trust and integrity in research!

I am looking forward to the last sessions and a workshop by Dr Varsha Khodiyar, Data Curation Editor at Scientific Data!

Writing for non-scientists

I have recently had to write a grant application which forced me to think carefully about wording when describing my research to the general public. I quickly realised that I will need much more trial-and-error experience to become as good as my supervisor (who has the added benefit of being a clinician who talks science to non-scientists all the time!). Luckily, there are many good guides available on the internet, and I have just come across a very useful collection provided by eLife on how to write plain language summaries! Looking forward to putting these into practice as I will be writing a guest blog for the Biochemical Society which is quite exciting.. Also, if I have time, I would really like to have a go at writing a story for the Biochemical Society’s Science Communication Competition!

Finally, a bit from the research world…

I haven’t had a chance to look at these in detail yet, but here are some papers that caught my attention this week. Fingers crossed for solid data reporting!

Phosphorylation of the exocyst protein Exo84 by TBK1 promotes insulin-stimulated GLUT4 trafficking (

Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states (

Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions (

Lysosomal cholesterol activates mTORC1 via an SLC38A9–Niemann-Pick C1 signaling complex (

Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells (

The cold-induced lipokine 12,13-diHOME promotes fatty acid transport into brown adipose tissue (

Some recent reads.. So much new science!

Metabolism and physiology

An adipo-biliary-uridine axis that regulates energy homeostasis (Deng et al. 2017 Science Signaling)

Copied lay description: “The nucleoside uridine is well known for its role in critical cellular functions such as nucleic acid synthesis. Its role in whole-animal physiology has received comparatively little attention. In mammals, plasma uridine levels are tightly regulated, but the underlying mechanisms are unclear. Studying mouse models, Deng et al. show that plasma uridine levels are controlled by feeding behavior (see the Perspective by Jastroch and Tschöp). Fasting causes an adipocyte-mediated rise in plasma uridine, which triggers a lowering of body temperature. Feeding causes a bile-mediated drop in plasma uridine, which enhances insulin sensitivity in a leptin-dependent manner. Thus, uridine is part of a complex regulatory loop that affects energy balance and potentially contributes to metabolic disease.” – read through it briefly, and it is quite interesting!

Pik3r1 is required for glucocorticoid-induced perilipin 1 phosphorylation in lipid dropled for adipocyte lipolysis (in press in Diabetes; by Kuo et al.)

  • Not read in detail, but claims to demonstrate a role of p85a independent of its regulatory function as class IA PI3K components.. Would be worth reading in detail as I can imagine multiple confounding factors given the deinhibition of the catalytic p110 subunit in the absence of p85a.

Stem cell and developmental biology

Autophagy maintains the metabolism and function of young and old stem cells (Ho et al. 2017 Nature)

  • Aim of study: to identify how autophagy controls hematopoietic stem cell function, and how changes in autophagy levels control HSC ageing.
  • Results: autophagy plays a critical role in preserving HSC functionality by clearing aged mitochondria. This maintains HSC quiescience and stemness. Decreased autophagy with age, resulting in lower regeneration potential.

Human haematopoietic stem cell lineage commitment is a continuous process (Velten et al. Nature 2017)

  • This is an interesting topic, particularly because I recently attended a talk by Dr Ana Cvejic who talked about the current debate about the accuracy of the current haematopoietic lineage tree. Are there distinct intermediate steps from a multipotent stem cell to a unipotent cell? Or does the transition occur directly? This confusion largely stems from the reliance on cell surface markers to identify distinct cell populations. Instead, a call was made to incorporate single-cell transcriptional analyses as part of future studies.
  • This is exactly what this study has done and it describes that the tree-like structure of haematopoiesis is incorrect as haematopoietic stem cells appear to acquire differentiation biases in a gradual manner without passing through discrete intermediate states.

 PI3K/AKT/mTORC signalling pathway

mTORC1 and muscle regeneration are regulated by the LINC00961-encoded SPAR polypeptide (Matsumoto et al. Nature 2017) – from January but only got around to looking at it now. The second long non-coding RNA that seems to play a role in regulating this signalling pathway (see Lin et al. Nature Cell Biology 2017 for another example). What’s special about this one is that its regulatory role depends not on its non-coding properties, but on a hidden protein-coding sequence. The peptide that is produced by LINC00961 is expressed in a tissue-specific manner and restricts mTORC1 activation in response to amino acids.  It is demonstrated that this mechanism has a functional importance in muscle regeneration in mouse model organisms.

 PARK2 depletion connects energy and oxidative stress to PI3K/Akt activation via PTEN S-Nitrosylation (Gupta et al. Molecular Cell 2017; Lewis Cantley one of the last authors)

  • PARK2 is commonly deleted in cancer, and this paper demonstrates a novel regulatory mechanism that involves indirect PARK2-induced activation (via decreased removal of damaged mitochondria) of eNOS. Activation of eNOS in this setting promotes S-nitrosylation of PTEN and its subsequent Ubiquitin-dependent degradation. This mechanism was demonstrated in specific cancer cell lines and other immortalised cell models. Supporting its pathological importance, PARK2 and PTEN loss occur together in many cancers – if one PTEN allele is lost, the protein product of the remaining allele will suffer increased degradation upon concomitant PARK2 The only thing that is missing from this paper is a clear statement of the number of independent experimental replicates that they produced!

Amino acid – insensitive mTORC1 regulation enables nutritional stress resilience in hematopoietic stem cells (Kalaitzidis et al. 2017 JCI; Sabatini as one of the last authors)

  • Interesting paper because of increasing evidence in the stem cell worls that mTORC signalling is carefully regulated, partly to maintain low protein synthesis rate.
  • The authors suggest that a nutrient-insensitive mTORC1 in HSCs is part of a protective mechanism against variable nutrient availability and oncogenes due to excess nutrient stimulation.

A key leading edge review from Sabatini: mTOR signalling in growth and disease (Cell 2017)



GuideScan software for improved single and paired CRISPR guide RNA design (Perez et al. 2017 Nat Biotechnology)

  • For the CRISPR users out there, the Ventura lab offer a new and superior tool for CRISPR gRNA design with improved specificity compared to commonly used competitor tools. Utility demonstrated for non-coding DNA regions.

Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin (Chamma et al. 2017 Nature Protocols)

  • This seems quite cool and relevant if you want to perform cell imaging experiments with the aim to define target-specific trafficking events. The monomeric streptavidin labelling method has the advantage that the tag is very small, hence the fluorescent probe will be right in the vicinity what you want to study which is important for reliable superresolution results. The tag is only 15 aa big.

I came across an “older” paper from 2015 on CRISPR sgRNA designs by Farboud, B. and Meyer, B. (Genetics 2015); they describe improved targeting efficiency if the sgRNAs have a 3’ GG motif in addition to the 3’ terminal NGG PAM site. Worth a read..

Diet/exercise and other interesting bits and pieces

Went back to read Raubenheimer and Simpson’s review in Annual Rev Nutr 2016 which describes their nutritional geometry framework that advocates a holistic approach to nutrition science. Instead of focusing on the effects of a single nutrient in isolation, we should explore how different food components interact and how an animal’s behaviour, including foraging, is dependent on multiple environmental variables that are often left out in reductionist-type nutrient science. It is also worth having a look at the same authors’ Cell Metabolism paper from 2014: The Ratio of Macronutrients, Not Caloric Intake, Dictates Cardiometabolic Health, Aging, and Longevity in Ad Libitum-Fed Mice.

 Dieting, independent of genetic factors, has been shown to result in long-term weight gain – by yet another study (International Journal of Obesity, Pietiläinen et al. Does dieting make you fat? A Twin Study).

More papers.. :-)

As always, the world of science never sleeps, and hundreds of papers are published each week. Here is a list of what I found interesting this week, including a couple of reviews. Again, just my notes as I have been reading, but perhaps some of it is useful to others, too..

Stem cells (incl. gene editing):

Favourite because  it is simply cool! – Harrison et al. (Science 2017): Assembly of embryonic and extra-embryonic stem cells to mimic embryogenesis in vitro. It is almost like science fiction except that it is real. Cambridge researchers succeed in mimicking early embryogenesis in vitro by fostering close interaction between mouse embryonic and extramebryonic cells in a 3D Matrigel scaffold and specialised medium allowing co-development of such cells. The ESCs and TSCs self-assemble into a structure that faithfully mimics the natural embryo. Several developmental processes demonstrated (cavitation, early specification of endoderm and mesoderm, formation of primordial germ cells), including the underlying signalling mechanisms. This is crucial as it will allow future modelling of developmental process in vitro, reducing the requirement for animal studies.

Guénantin et al. (Diabetes, 2017): Functional human beige adipocytes from induced pluripotent stem cells. Very unfortunate not to have access to this; I have not actually come across a protocol for beige adipocytes from iPSCs before, so this would seem to be particularly novel and relevant. According to abstract, no overexpression of exogenous factors required and cells are functional upon engraftment in mice.

Mitzelfelt et al. (Stem Cell Reports, 2017): Efficient precision genome editing in iPSCs via genetic co-targeting with selection. Adding to the pile of papers dealing with improving the efficiency of CRISPR/Cas9-mediated gene editing in stem cells. Particularly relevant for disease modelling in the research lab. Note that this method doesn’t allow subsequent removal of the co-targeted antibiotic resistance gene which is incorporated into the safe-harbour AAVS1 locus. Interestingly, another group simultaneously published a similar approach in JBC, but their method relies on a transposable HDR reporter that can be used to enrich successfully edited cells (demonstrated in immortalised and immortalised cell lines; NB – not in stem cells, though). It is an elegant approach and worth keeping in mind. Importantly, the HDR reporter can be removed following successful knockins by adding Piggybac transposase to the cells. Paper details: Wen et al. JBC 2017 – A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair).

Araki et al. (Stem Cells, 2017): The number of point mutations in iPS cells and ntES cells depends ont he method and somatic cell type employed for their generation. Need to read properly, but would appear to be useful for people considering the use of iPSCs for disease modelling. It seems to suggest that point mutations are intrinsic to the process of reprogramming and are not a Yamanaka-specific phenomenon. The extent of point mutations during reprogramming might be reduced by careful optimisation of various reprogramming conditions, including consideration of the age of the parental line used for reprogramming.


Pappalardo et al. (Diabetes, 2017): A Whole Genome RNA Interference Screen Reveals a Role for Spry2 in Insulin Transcription and the Unfolded Protein Response. Unfortunately, no access but appears to be interesting in that Spry2 is a known GWAS hit for T2D, yet no previous connections to metabolic phenotypes. Mechanistic studies in cells and in mice according to the abstract.

Robinson et al. (Cell Metabolism, 2017): Enhanced protein translation underlies improved metabolic and physical adaptations to different exercise training models in yound and old humans. Although not scrutinised in detail, it is very interesting. This group sets out to assess the effect of different exercise modalities on skeletal muscle adaptations in young vs  old adults. Although n-numbers are modest, several significant effects emerge, and there are important insights into the molecular transducers of exercise adaptations. Mitochondrial proteins are, perhaps not surprisingly, topping the list. Ultimately, the study concludes that supervised HIIT appears to be an effective recommendation to improve cardiometabolic health in ageing adults.

Suzuki et al. (Cell Reports, 2017): ER Stress Protein CHOP Mediates Insulin Resistance by Modulating Adipose Tissue Macrophage Polarity. Haven’t read, but potentially relevant.


Barilari et al. (The EMBO Journal, 2017): ZRF1 is a novel S6 kinase substrate that drives the senescence programme. Decent paper and relevant for understanding the signalling mechanisms underlying oncogene-induced senescence (OIS); the protective mechanisms employed by cells against malignant transformation in response to hyperactivation of growth pathways such as PI3K/AKT. Hyperactivation of mTOR in vivo and in vitro leads to senescence in the absence of concomitant p53 mutations. This group demonstrates that the increase in p16 (cell cycle inhibitor involved in triggering OIS) is dependent on ZRF1  phosphorylation by S6K.

In the context of oncogene-induced senescence, it is interesting to note a Previews article in Cell Stem Cell covering a publication from last week that demonstrates an intricate link between senescence and cellular plasticity, whereby senescence-induced secretory factors trigger dedifferentiation in neighbouring cells – in a physiological context, this would enhance tissue regeneration, but it is easy to envisage how such a mechanism can be hijacked in cancer. Preview details: Taguchi & Yamada (Cell Stem Cell, 2017): Unveiling the role of senescence-induced cellular plasticity. Another paper that deals with this topic, published earlier this year: Ritschka et al. The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration (Genes & Development 2017).

Mitochondrial homeostasis in adipose tissue remodelling (Svetlana Altshuler-Keylin and Shingo Kajimura): pertinent review given the need for research into the relationship between mitophagy and energy metabolism. The authors outline the balance between mitochondrial generation and degradation (via global autophagy or selective autophagy, i.e. mitophagy). Mitochondrial damage = major physiological trigger for mitophagy. Such mechanisms are important in mitochondria-enriched cells, incl. brown and beige adipocytes. Mitophagy occurs through two different mechanisms: adapter-mediated (ubiquitin-dependent) and adapter-independent (ubiquitin-independent). The review highlights the need for controlled Cre line usage to elucidate the role of autophagy/mitophagy in defined cell types, such as preadipocytes and differentiated adipocytes. Previous genetic autophagy-deficient animal models have yielded inconsistent results due to use of multiple Cre lines with temporal differences in induction and affected cell type. Physiologically, autophagy regulation is tightly coupled to nutrient sensing via mTOR signalling. Another physiologically relevant pathway: PKA downstream of beta3-AR signalling, which is a known mediator of beige adipocyte biogenesis in response to cold exposure. PKA directly phosphorylates mTOR and its binding partner RAPTOR, activating the complex and thereby promoting autophagy inhibition. Mitophagy has to be activated when during beige-to-white adipocyte conversion. Mechanism under investigation. Dysregulation in obesity and metabolic diseases; autophagy blocks beige adipocyte development. Mitochondria are also critically important in the pancreas – for glucose-stimulated insulin secretion; on the other hand, autophagy maintains β-cell homeostasis by removing damaged mitochondria and/or ER. Autophagy is also important in liver metabolic control – here it prevents diet-induced liver steatosis. The review ends by listing methodologies that can be used for detecting mitophagy in adipocytes.

Other bits and pieces: 

GuideScan – new software to design single and paired CRISPR guide RNAs (Perez et al. Nature Biotechnology 2017)

Highly efficient RNA-guided base editing in mouse embryos (Kim et al. Nature Biotechnology 2017)

Reading nuggets from this week (for scientists)

My favourite occupation is to read scientific papers and here is a biassed list of what I have come across recently.. Unfortunately, it would require technical knowledge, so probably most appropriate for scientists themselves.

My favourites because they are relevant for my stuff: 

A very important contribution in Nature Cell Biology by Liu et al.: G1 cyclins link proliferation, pluripotency and differentiation of embryonic stem cells. Demonstration that multiple cells are actually capable of proliferating in the absence of any of the G1 cyclins (D + E) – contrary to the prevailing model; however, pluripotent stem cells lose their pluripotent proficiency and acquire a trophectodermal cell fate (as well as a propensity to generate neural tissue in chimerism studies). The underlying mechanism comprises G1 Cyclin/CDK-mediated phosphorylation of the core transcription factors NANOG, OCT4 and SOX2. This is suggested as a potential contributing mechanism to the acquisition of pluripotency traits in malignant cells. Very interesting given the link between PI3K/AKT activation and Cyclin D + CDK2 upregulation. Note that individual loss of either Cyclin D or E doesn’t result in pluripotency loss. Also, the studies are performed in MEFs isolated from genetically engineered mice of the right genotype.

Review in Nature Communication by Dejana et al.: The molecular basis of endothelial cell plasticity. Covers early endothelial development, and the remarkable cell fate plasticity exhibited by endothelial cells. Essentially, all aspects of endothelial cell commitment and their ability to transition into haematopoietic stem cells or cardiac mesenchyme are dependent on the same set of pathways, which include VEGFR1/VEGFR2 and c-KIT signalling, i.e. PI3K activation.

A Stem Cell paper from Harding et al.: Highly Efficient Differentiation of Endothelial Cells from Pluripotent Stem Cells Requires the MAPK and the PI3K Pathways. The most useful bit of this paper is the development of an efficient endothelial cell differentiation protocol from human pluripotent stem cells that doesn’t require cell sorting. As of the claim that PI3K and MAPK pathways are essential, I would like to see more detailed evidence beyond the use of broad-spectrum inhibitors.

Signalling studies and molecular biology

The Yudushkin lab (same guy who recently published a nice Molecular Cell paper PIP3-dependent restriction of AKT activity to cell membranes) have contributed a paper to JCB, examining the localisation of mTORC2 activity (Ebner et al. 2017: Localization of mTORC2 activity inside cells). The starting question was how growth factors couple to mTORC2 in order to induce downstream phosphorylation of AKT. Previous studies had provided some evidence that mTORC2 associates with mitochondria, ribosomes, endosomal compartments and the plasma membrane. However, not much known regarding which of these membranes link to mTORC2 activity in a cellular context and whether there are pool-specific contributions to AKT activation. One outcome of the current study is, therefore, the development of a tool that allows tracking of mTORC2 enzymatic activity towards AKT within the cell. Important findings from this paper include: PM-associated mTORC2 is constitutively active towards AKT; hence neither growth factors nor PI3K inhibition has an effect on mTORC2 activity at this cellular site – however, PI3K activity is required for mTORC2 activity at early and late endosomes. Studies performed in HEK293s, so will be interesting to see if this is replicated in additional cell types in the future.

Metabolism, T2D, obesity incl. adipocyte studies 

Nature paper by Wong et al. The role of fatty acid β-oxidation in lymphangiogenesis. Haven’t read beyond abstract and discussion, but interesting concept! Turns out that β-oxidation, as expected, contributed to energy generation and nucleotide synthesis, as well as epigenetic regulation through Acetyl-CoA-dependent p300-mediated histone acetylation of the PROX1 gene, which is important for VEC to LEC differentiation. Multiple reports out recently that elegantly demonstrate the integration of metabolism and cell fate commitment.

Cell Metabolism paper by Mauro et al. 2017 (K. Okkenhaug is a co-author): Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4+ T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals. Haven’t read it in detail, but this is the summary provided by the journal (seems relevant!): “Lymphocyte infiltration of non-lymphoid tissues, including adipose and vascular tissues, is a prominent feature of chronic inflammation in diet obesity. Mauro et al. find that the saturated fatty-acid palmitate activates a PI3K p110δ-Akt pathway leading to CD4+ T cell differentiation into effector memory-like T cells upon priming in obese mice and humans.”

A nice Cell Metabolism review on ketogenesis and alternative functions of ketone bodies; Puchalska, P. & Crawford, P. 2017: Multi-dimensional Roles of Ketone Bodies in Fuel Metabolism, Signaling, and Therapeutics

A paper in Diabetes (Ehrlund et al. 2017: Transcriptional Dynamics During Human Adipogenesis and Its Link to Adipose Morphology and Distribution), published a few a weeks ago, explores the transcriptional dynamics of adipocytes subjected to differentiation in vitro. Unfortunately, I can’t get access to the paper even when I login. From the abstract, however, I gather that it might be relevant as the study profiles the expressional changes of genes, enhancers, and long noncoding RNAs and demonstrates that enhancers expressed during adipogenesis overlap with SNPs associated with white adipose tissue distribution. The paper should contain useful time courses with important distinctions between downregulated, transient and late-induced transcripts, such as relationships to hypertrophy and insulin sensitivity.

Another report in Diabetes by Ralph de Fronzo (big name in the field I believe; Gastaldelli et al. 2017: Role of Adipose Tissue Insulin Resistance in the Natural History of T2DM: Results from the San Antonio Metabolism Study) investigates the role of adipose tissue insulin resistance in the natural progression to Type 2 Diabetes in humans. Specifically, they recruited 302 subjects varying glucose tolerance (normal, impaired, Type 2 Diabetes), subjected them to an OGTT (oral glucose tolerance test) and an euglycaemic insulin clamp and profiled their insulin sensitivity alongside plasma free-fatty acids (FFAs). A progressive decline in insulin sensitivity was accompanied by impaired FFA suppression, but overt hyperglycaemia was only established with the progression to T2D. Results are kind of expected in light of the burgeoning mouse literature on the topic as well as epidemiological studies, but I suppose it had not been formally demonstrated in human beings.

PIK3CD papers that might be relevant

Compagno et al. Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells (Nature 2017); mechanism relies on the ability of PIK3CD to suppress activation-induced cytidine deaminase (AID) in B cells, where this enzyme promotes class switching of immunoglobulin genes.



Just out of interest…

The Biochemical Society’s recent Biochemist issue focussed on Gender Medicine. All too often, the variable “sex” is ignored in biomedical research, and that needs to change. Personally, I can see why it can be difficult to always include XX and XY in your research – after all, this doubles the resources required for your study. I suppose funders need to be more supportive in this respect! The issue also contains additional interesting reads on science policy and outreach, so have a look if you can gain access to it via the following link (not sure if you are not a member):

One step closer to understanding consciousness? A group in the U.S. discovered the existence of mouse neurons that seem to wrap around the whole brain; the group believes that this might underlie the mechanism of consciousness as the three neurons seemed to connect to most or all of the outer parts of the brain that take in sensory  information and control behaviour. Furthermore, the Read the Nature News & Views here:



Interesting papers, week 8

I realise that week 8 was last week, but I thought I would share the regular paper digest that I send out to people in my lab each week. I love papers and reading about exciting, new science. Sometimes, I also come across publications, even high-impact ones, that are actually flawed in some way or other, and I think it is important to evaluate all new findings critically and highlight potential misinformation.

So join the discussion and add to this list of papers if you have come across something that excited you recently, or perhaps a paper that should be highlighted as problematic!

In future posts, I will try to be more detailed as this is taken straight from an email sent out to the lab!

Transcriptional activation of lipogenesis by insulin requires phosphorylation of MED17 by CK2 (Science Signaling)
Report on how MED17 is phosphorylated by CK2 in response to insulin, but only if it hasn’t been phosphorylated by p38 – something that happens during fasting. MED17 interacts with USF1 in order to be recruited to the FASN promoter. Mechanism demonstrated in liver in vivo and in hepatocytes in vitro. MED17 is a core component of the Mediator complex, by the way. The Mediator complex recruits RNA pol II to active promoters and initiates gene transcription. Lots of biochemical stuff in this paper, too – seems decent.
Pentraxin-3 is a PI3K signaling target that promotes stem cell – like traits in basal-like breast cancers (Science Signaling)
The role of PTX3 appears to be dependent on the particular tumours, but in basal-like breast cancers it seems to enhance PI3K-dependent hyperactivation of key cancer phenotypes. Stem cell-like properties not investigated directly – inferred by observation that PTX3 is highly enriched in mesenchymal / mesenchymal stem cell-like breast cancers.
Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras (Cell)
It is not directly related to what we do, but good inspiration for what is possible in the future if you want to get a step closer to personalised medicine; i.e. analyse gene essentiality relationship in mutant iPSCs subjected to a CRISPR screen and identify specific liabilities that can guide potential future treatment. Also good discussion on synthetic lethality in cancer and how it may differ for oncogenes compared to mutations in “caretaker” genes.
Two back-to-back Nature papers on GATORs and mTORC1 regulation in relation to nutrient sensing (haven’t read in detail, but seem interesting, and apparently the identified components have been identified in neurological disorders that lead to mTORC1 hyperactivation!): 
KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1 (Sabatini lab)
SZT2 dictates GATOR control of mTORC1 signalling (Quite impressive with only 3 authors: Min Peng, Na Yin, Ming Li)
The LINK-A lncRNA interacts with PtdIns ( 3 , 4 , 5 ) P 3 to hyperactivate AKT and confer resistance to AKT inhibitors (Lin et al. Nature)
Seems cool, but not read in detail yet as it has A LOT of complex biochemistry so don’t read if your brain is mushy!
Lipid transport by TMEM24 at ER–plasma membrane contacts regulates pulsatile insulin secretion (Lees et al., Science)
Same as above.

A high-throughput, image-based screen to identify kinases involved in brown adipocyte development (Perdikari et al., Science Signaling)

Also not read properly. Using two orthogonal approaches – lentivirus-mediated shRNAs to knockdown kinases (the whole mouse kinome actually!) as well as kinase-specific inhibitors. Apparently, they see that knockdown of the insulin receptor results in improved BAT differentiation!

DNA damage is a major cause of sequencing errors, directly confounding variant identification. (Science)
Seems quite important for the sequencing crew.. Not read beyond abstract, but it suggests that DNA damage during the preparatory stage of DNA for sequencing leads to spurious findings of low-frequency variants, i.e. false-positives. This has implications for the identification of true somatic variants.
Adipose-derived circulating miRNAs regulate gene expression in other tissues (Nature)
I do believe the demonstration that adipose tissue is an important source of miRNAs and that these are likely to regulate other tissues, in an adipose tissue-specific and receptor tissue-specific manner; I will believe the miR-99b story outlined in this paper upon further validation as the stats are not great and n-numbers are low.
Labeling proteins inside living cells using external fluorophores for microscopy. (eLife)
Teng et al. optimise the use of the pore-forming bacterial toxin streptolysin O (SLO) to create small pores in cell membranes in order to facilitate the labelling of intracellular proteins with membrane-impermeant probes, allowing the study of the targeted proteins in live cells. Probes range in size from 2 kDa to 150 kDa; optimised for labelling of cytoplasmic or nuclear proteins, transfected or non-transfected (hence possibly to avoid overexpression artefacts). Cells retain intact membrane structures following recovery. Relevant following our discussion of click chemistry and the issues relating to intracellular labelling of proteins. Also relevant for super resolution imaging applications. Notably, labelling with probes <2 kDa results in background-free labelling because the unbound probe is able to diffuse out through the formed pores during the recovery phase.


New Year, new blogpost, new language: my project in Danish!

For friends and family in Denmark (+ any other interested Danish readers), finally a description of what I spend most of my wake hours doing. I hope it makes sense!

Hver gang jeg kommer hjem til Danmark, bliver jeg mødt af velmente spørgsmål og bemærkninger vedrørende min forskning:

”Hvad er det præcist, du laver?”

”Er det ikke diabetes, du forsker i?”

”Du finder vel sikkert kuren mod kræft!”

”Ej, det lyder godt nok specifikt og komplekst.”

Det er skønt, at folk er oprigtigt interesserede i mit arbejde! Og det er på tide, at jeg takker for interessen ved at trylle mystikken væk og give jer alle en (forhåbentligt) forståelig beskrivelse af, hvad jeg bruger de fleste af mine vågne timer på.

Tænk dig først tilbage til den tid, hvor du lå gemt i din mors mave. Er det ikke utroligt, at sammensmeltningen af en ægcelle og en sædcelle på 9 måneder kan føre til fødslen af en oftest velskabt menneskebaby? Utallige komplekse processer finder sted i løbet af denne udvikling, hvor en enkelt celle bliver til en hel organisme bestående af op mod 10000000000000 celler organiseret på en helt bestemt måde. Hvad er op og ned, venstre og højre? Hvor skal de forskellige organer placeres, og hvor store skal de være?

Vores tidlige udvikling er derfor et godt eksempel på, hvorfor det er vigtigt, at vækst er nøje reguleret helt nede på cellulært niveau. Modsat er kræft et eksempel på, hvor galt det kan gå, når cellers vækst pludselig ikke kan holdes i skak. Typisk sker det, fordi vores celler med alderen akkumulerer flere og flere fejl i sit DNA (”computerkoden”), hvorved diverse vækstregulerende mekanismer går tabt, mens andre bliver forstærket. Det kan kun ende i en katastrofe, når bremserne ikke virker, og speederen er i bund.

Det viser sig, at der på verdensplan findes sjældne individer med fejl i dette vækstprogram – fejl, som har fundet sted meget tidligt i fosterudviklingen. Disse mennesker har ikke kræft, men er ved fødslen kendetegnet ved abnorm vækst, som fortsætter livet igennem. Det var et gennembrud, da min vejleder og hans team in 2011 opdagede, at en af de mest hyppige programmeringsfejl i kræft også er årsagen til disse sjældne vækstsygdomme. Det drejer sig om en genetisk mutation – en stavefejl i DNA’et – i lige netop den del, der koder for proteinet PIK3CA (de fleste gener koder for proteiner, og proteiner udfører de fleste funktioner i vores celler).

PIK3CA er ikke et tilfældigt protein. Det fungerer som tænd-knappen for cellens vækst, stofskifte, deling og bevægelse. De omtalte genetiske ”stavefejl” fører til, at PIK3CA er aktivt hele tiden, eller med andre ord: tænd-knappen sidder fast. Konsekvenserne er til at få øje på. Den første patient, som blev diagnosticeret med denne mutation, har to ben, der hver især vejer over 50 kg. Til sammenligning er hendes overkrop overraskende tynd. Som følge af, at den genetiske fejl opstår under udviklingen, er der altså ingen garanti for, at det er hele kroppen, der kommer til at lide af abnorm vækst. Vi har derfor at gøre med et helt spektrum af sjældne patienter, hvor nogle kun har en enkelt finger, der er for stor, mens andre kæmper med alvorlige misdannelser omfattende hjerne og blodkar.

Formålet med mit projekt er at forstå, hvorledes de forskellige genetiske ”stavefejl” i PIK3CA omprogrammerer en celles udvikling og vækst. For at komme så tæt på den tidlige udvikling som muligt bruger jeg pluripotente stamceller, dvs. celler med potentiale til at blive til enhver anden celle i menneskekroppen. For at få stamceller med de rette genetiske fejl, kan man benytte sig af to forskellige teknikker. Den ene er at få en vævsprøve fra patienten og omprogrammere hudceller tilbage til stamceller – en metode, der førte til udgivelsen af en Nobelpris! Den anden teknik benytter sig af nutidens biologis mest revolutionerende redskab (en kommende Nobelpris): CRISPR. CRISPR kan sammenlignes med en saks, der er i stand til at klippe meget præcist i DNA’et i lige netop dét gen, man ønsker at ændre. Man lapper derefter DNA-bruddet og indsætter samtidigt den ønskede ”stavefejl”. Voilá – jeg har de celler, jeg skal bruge! (Helt så let er det heller ikke, og det tog mig et helt år at nå dertil!)

Min forskning er betydningsfuld, fordi den nye viden potentielt kan føre til udviklingen af nye behandlingsmetoder for sjældne patienter med abnorm vækst. I bredere forstand vil vi lære noget fundamentalt om udviklings- og vækstkontrol på cellulært niveau. Sidst men ikke mindst vil denne viden bidrage til en bedre forståelse af individuelle kræftgeners virkningsmekanismer.

Mange, der kender mig, tænker sikkert: ”Det har godt nok ingenting med diabetes at gøre.”

Og så alligevel – det har i den grad noget med det at gøre. Den hyppigste form for diabetes, Type 2, er en stofskiftesygdom karakteriseret ved manglende evne til at fjerne sukker fra blodet, fordi bugspytkirtlen ikke er i stand til at producere nok af hormonet insulin og den smule, der stadig bliver lavet, er ikke længere i stand til at virke på de muskel- og fedtceller, der skal optage sukkeret. PIK3CA er et af nøgleproteinerne, der gør en celle i stand til at respondere på insulin: ved at optage sukker og vokse. PIK3CA orkestrerer derfor cellens stofskifte, og mit projekt giver mig den unikke mulighed for at få et indblik i de underliggende mekanismer.

Det er vildt spændende. Og krævende! Dog er jeg taknemmelig for at kunne sige, at jeg bliver betalt for at lave noget, som jeg elsker, som er sjovt, og som har en mening – for mig personligt, og også for de patienter, der bliver berørt af det.

Wow, n=3 independent experiments finally makes sense…

3 December – really??! Where did this year go? I don’t know the answer to this question, but I certainly look forward to Christmas. It has been an extremely busy but exciting year. My PhD project seems to have gained momentum, the models I investigate are more or less established, and I enjoy what I am doing. Can’t help the feeling of insecurity even when I am writing this; the recurrent question: “What if it goes wrong just because you state that it currently goes well?” Rubbish thought, dismiss it. Need to believe in myself, but it has proven more difficult than anticipated..

One of my main discoveries this past year is that n=3 independent experiments really matters. Let me explain. In biomedical science, particular in experiments involving cells, you usually repeat something at least 3 times on different days – ideally also on different cells.. This way, if you observe the same result with every repeat, you can be more confident that your findings are robust. We tend to laugh at this because from a statistical point of view n=3 is by no means a magical number.. Although scientists love to perform statistical tests on n=3, from a statistical point of view it doesn’t make much sense. So when I first entered the field, I thought that n=3 was quite a peculiar idea because it remains statistically irrelevant.

Statistically irrelevant doesn’t mean biologically irrelevant, though. Science is expensive, so performing the same experiment n=10 is unlikely to happen because your finances will suffer. Nonetheless, performing it minimum 3 times really is a good idea as I have discovered following endless worries that I might have messed something up. Perhaps a consequence of my pathological perfectionism, I often myself doubting every single thing I do and whether I might have messed something up without noticing? It doesn’t help that I spend most of my wake hours culturing cells, which at some point becomes quite automated so you just do stuff; yet, what if my autopilot has failed me without me noticing? Is everything I do wrong? Disaster. Rubbish thought, dismiss again. You see how easy it is to enter a vicious cycle of worries and self-doubt? Thankfully, I need to repeat it anyway, right? So hopefully, even if my autopilot might have failed me at some point, it won’t do so in a subsequent repeat. So if n=2 is different from n=1, n=3 surely will either conform to n=2 or n=1, if not – go for n=4 and n=5!

I wonder if others come across similar doubts and worries? Do share your comments if that’s the case. It usually helps to discover that you are not alone! Just remember, n=3 is there for a reason..


Fighting your sweet tooth


If you are anything like me, you love chocolate, cake, biscuits, anything sugary and fatty! You know that it is bad for you, yet it is difficult to resist the temptations lurking around the corners. At my work place, cakes and sweets find their way at bigger seminars, at  group meetings, at birthdays, leaving dos, the last Friday of the month, the monthly Student Cake Club… Whatever the reason, the sugar bombs will be accessible on a daily basis, inviting you to try them as were each time the last to ever have a cake experience.

I have often wondered if my brain’s appetite circuit is failing completely as I am remarkably limited in my ability to resist the aforementioned temptations. It is scary as I have all the necessary knowledge to resist, yet it is so hard to turn my back to the moist and tasty chocolate brownie appearing in the Institute’s social room on a regular afternoon.

Right until a good friend and colleague of mine and I joined forces and declared a war on those sugary treats. Here is what we are doing: we stick to eating sugary food once a week only; for every 6/7 sugar-free days, we collect £20 from each other and save those in a “reward” jar towards a trip to Copenhagen. There is also an additional £10 bonus for every 7 bouts of exercise, and our goal is to reach a total of £500 each to cover flights, accommodation and pocket money for the trip. The whole process is rather addictive and incredibly motivating.

The key aspect here is reward and positive feedback. Rather than establishing a punishment system against failure, we reward ourselves for being strong. We sustain a positive feedback through a shared calendar to log our exercises as well as a spreadsheet detailing our savings so far. We have also decided that breaking the rules even once requires all money to be donated to a charity organisation and the Copenhagen trip to be cancelled. Thus, a failure by one will take away a good experience from the other, so rather than only doing it from a “selfish” perspective, we are actually also doing it for each other.

This is just an example of one scheme, and similar approaches can be adopted by others willing to limit their daily consumption of unhealthy food. Find a reward cycle that suits you  and your fellow cake fighters – plan trips to the cinema, spoil each other  with useful little presents or something completely different that does not require you to spend money at all. You will be surprised at how good your brain becomes at resisting the temptations when you are all in it together.

Bottom line: you can make the right dietary choices with the right support network and incentives in place. So find your partner in anti-cake crime, establish your favourite reward scheme, share a calendar or spreadsheet to track your progress and start fighting that sweet tooth now!