Walko Lab

Mapping cryptic phosphorylation sites in the human proteome | The EMBO Journal embopress.org/doi/full/10.10…

Our RAEFISH spatial transcriptomics technology is now published in Cell @CellCellPress! RAEFISH enables sequencing-free whole genome spatial transcriptomics at single molecule resolution. This work represents the first time that transcripts from more than 23,000 genes were directly probed and imaged in situ with any technology, and the first time numerous different gRNAs were directly probed and distinguished by imaging in a high-content CRISPR screen. The challenge: Recent breakthroughs in spatial transcriptomic technologies, from us and others, have greatly improved our ability to profile cell types, states, cellular interactions, and the underlying gene programs within the native tissue contexts. However, these technologies have limitations. Methods based on 2D-array-capture/tagging and ex situ sequencing offer genome-scale coverage, but lack the resolution needed to accurately study fine spatial organization. In contrast, image-based methods that rely on highly multiplexed fluorescence in situ hybridization or in situ sequencing provide single-molecule resolution and resolve fine spatial organization, but require pre-selecting a limited set of target genes (typically hundreds to a few thousand genes), which limits discovery and sometimes leads to only validations of prior knowledge due to the pre-selected targets being well studied in the context. The solution: RAEFISH, our lab's new flagship image-based spatial transcriptomics technology, simultaneously enables single-molecule spatial resolution and whole-genome level coverage of long and short, endogenous and engineered RNA species in cell cultures and intact tissues. The results: 🔥 We performed RAEFISH targeting 23,312 human genes in cell cultures, and demonstrated hypothesis-free discovery of cell cycle associated genes and subcellular localization patterns of transcripts, including nearly the entire protein coding transcriptome and additional long noncoding RNAs. 🔥 We performed RAEFISH targeting 21,955 mouse genes in mouse liver, placenta, and lymph node tissues. Our analyses on immediately neighboring cells uncovered intriguing cell-cell interactions and previously unknown gene expression programs underlying the interactions, such as those between cholangiocytes and immune cells. 🔥 Finally, we further developed RAEFISH to directly read out guide RNAs (gRNAs), demonstrating Perturb-RAEFISH in an image-based high-content CRISPR screen. The capacity of Perturb-RAEFISH to directly read out gRNAs addresses a crucial limitation of previous techniques that read out a barcode/identifier sequence paired with each gRNA species, as the pairing can be shuffled due to RNA recombination intrinsic to lentivirus used in such screens, which limits screen sensitivity and accuracy. In summary, RAEFISH provides the biomedical research community with a generalizable research tool, which will bring more spatial and mechanistic insights across health and disease. This work was co-led by my postdocs Drs. @ChengYubao, Shengyuan Dang, and Yuan Zhang, and was supported by the @NIH, @genome_gov, @sennetresearch, and @psscra. I would like to thank our co-authors, funding agencies, editor, reviewers, and my whole lab @YaleGenetics @YaleCellBio @YaleCancer @YaleMed @Yale. Link to paper: cell.com/cell/fulltext/…

I am delighted to share this new review in @NatureCancer that I co-author with @AnqiDong18 In which we discuss the different EMT tumor states and their implications for metastasis and cancer therapy.

Now out in its final form, with a new title "Mechanical control of cell fate decisions in the skin epidermis" and simulations/quantifications! See below for thread of how unbalanced tensions can bias fate choices in minimal 3D models of tissues/tumors! nature.com/articles/s4146…

Syndromic epidermal differentiation disorders: a new classification toward pathogenesis-based therapy The authors present a new gene- and protein product function-based classification for the group of disorders formerly called ichthyosis. Read more: doi.org/10.1093/bjd/lj…

Matrix-induced nuclear remodeling and mechano-therapeutics dlvr.it/TMgmjj

Mechanotransduction in the nucleus Multiple TFs transmit mechanical forces into transcriptional regulation. Nuclear shape, size, and chromatin abnormalities are common features of many diseases.

A lack of career progression within UK universities is “demoralising” staff and threatening to push people out of the sector, as institutions respond to the financial crisis by freezing promotions and concentrating hiring on lower-level positions timeshighereducation.com/news/staff-dem…

I wrote an end-to-end tutorial to replicate this genomics figure during the last winter holidays 👇 Take a look at the website and follow the step-by-step guide here crazyhottommy.github.io/reproduce_geno…

Predicting the translation efficiency of messenger RNA in mammalian cells. #TranslationEfficiency #TranslationalControl @NatureBiotech nature.com/articles/s4158…

Researchers have claimed that the number of redundancies being made in UK higher education could be much higher than previously thought, potentially impacting more than 20,000 jobs timeshighereducation.com/news/universit…

📢 For all HaloTag lovers💙💚💛❤️💜🩶 we are excited to share that our work, in which we developed a new biotin-HaloTag ligand, is now published in @JCellBiol! Full link here: rupress.org/jcb/article-ab…

.@yadav_anoop101, Jadhav, @SvendaJakub, @HanaSedlackova1 et al. present a novel cell-permeable biotin-HaloTag ligand. The reported ligand enables in vivo labeling & provides capabilities for efficient affinity capture of protein variants from live cells. hubs.la/Q03xv9Sd0

The pan-cancer proteome atlas, a mass spectrometry-based landscape for discovering tumor biology, biomarkers, and therapeutic targets dlvr.it/TLyB5j

1/11 Our study on subclonal immune evasion in non-small cell lung cancer has just been published in @Cancer_Cell cell.com/cancer-cell/fu…

Quality of scientific papers questioned as academics ‘overwhelmed’ by the millions published theguardian.com/science/2025/j…

Why do wounds in the mouth heal without scars? New work in @ScienceTM shows that oral fibroblasts suppress fibrosis through the proteins AXL and GAS6, and finds stimulating AXL recapitulates these benefits in facial skin wounds in mice. scim.ag/46xnJ3t

Find the housekeeping genes for your qPCR ACTB, B2M, GAPD, HMBS, HPRT1, RPL13A, SDHA, TBP, UBC, YWHAZ "internal control gene-stability measure M as the average pairwise variation of a particular gene with all other control genes" "we recommend the minimal use of the three most stable internal control genes for calculation of an RT-PCR normalization factor" genorm.cmgg.be @jvdesomp @fspelema @GenomeBiology 2002 genomebiology.biomedcentral.com/articles/10.11…

New study reveals that vimentin plays a crucial role in DNA #repair and lipolysis. Depleting vimentin leads to DNA #damage and impairs repair mechanisms. nature.com/articles/s4138…

Online Now: The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks dlvr.it/TLZkmH