Santosh Phuyal

• Radulovic, Maja; Pust, Sascha; Kournoutis, Athanasios; Chen, Di; Giner, M. Isabel & Liang, Qijian [Show all 19 contributors for this article] (2026). DFCP1-containing ER microdomains mediate lysosomal membrane repair. Nature Cell Biology. ISSN 1465-7392. doi: 10.1038/s41556-026-02062-z. Full text in Research Archive Show summary

  Abstract Lysosomal membrane integrity is essential for preserving cellular homeostasis in response to different stressors. Upon lysosomal membrane permeabilization, cells activate several mechanisms for lysosomal membrane repair, including ESCRT proteins, phosphatidylinositol 4-phosphate (PI4P)-dependent lipid transfer from the endoplasmic reticulum (ER) and conjugation of ATG8 family proteins to single membranes (CASM). The interplay between these pathways and the regulation of the lipid transfer machinery remain incompletely understood. Here we show that phosphatidylinositol 3-phosphate (PI3P)-containing ER domains play a major role in lysosomal membrane repair. PI3P is formed on lysosome-proximal ER domains by the phosphatidylinositol 3-kinase PIK3C3/VPS34 in response to membrane damage, and inhibition or depletion of PIK3C3 inhibits lysosome repair. Mechanistically, the ATPase DFCP1/ZFYVE1 accumulates on lysosome-proximal ER domains by its PI3P binding, triggered by Ca 2+ efflux from lysosomes and requiring the ULK1 kinase complex and ER proteins of the VAP family. Downstream of CASM, PI4P, ESCRTs and PI3P, DFCP1 promotes focal accumulation of the lipid channel VPS13C on ER domains proximal to damaged lysosomes to promote their repair. The function and dynamics of DFCP1 depend on its ability to bind and hydrolyse ATP, and absence of DFCP1 compromises cellular resistance to vacuolar damage induced by Listeria   monocytogenes . We conclude that DFCP1 mediates concentration of the ER-associated lipid transport machinery at damaged lysosomes to promote their sealing in response to Ca 2+ flux and PIK3C3 activation.

• Pfefferle, Aline; Phuyal, Santosh; Netskar, Herman Krogstad & Malmberg, Karl-Johan (2024). Egr2 to the rescue: nanoparticles revitalize natural killer cells in the fight against cancer. EMBO Journal. ISSN 0261-4189. 43, p. 2527–2529. doi: 10.1038/s44318-024-00144-y. Full text in Research Archive Show summary

  Reversal of tumor-induced natural killer (NK) cell dysfunction remains a major hurdle to overcome for the clinical success of immunotherapies based on NK cells targeting solid tumors. New research in The EMBO Journal identifies the transcription factor early growth response gene 2 (Egr2) and diacylglycerol kinase DGKα as two negative regulators of NK cell dysfunction (Sabag et al, 2024).

• Phuyal, Santosh; Romani, Patrizia; Dupont, Sirio & Farhan, Hesso (2023). Mechanobiology of organelles: illuminating their roles in mechanosensing and mechanotransduction. Trends in Cell Biology. ISSN 0962-8924. 33(12), p. 1049–1061. doi: 10.1016/j.tcb.2023.05.001. Full text in Research Archive
• Ghannoum, Salim; Fantini, Damiano; Zahoor, Muhammad; Reiterer, Veronika; Phuyal, Santosh & Netto, Waldir Leoncio [Show all 13 contributors for this article] (2023). A combined experimental-computational approach uncovers a role for the Golgi matrix protein Giantin in breast cancer progression. PLoS Computational Biology. ISSN 1553-734X. 19(4). doi: 10.1371/journal.pcbi.1010995. Full text in Research Archive Show summary

  Our understanding of how speed and persistence of cell migration affects the growth rate and size of tumors remains incomplete. To address this, we developed a mathematical model wherein cells migrate in two-dimensional space, divide, die or intravasate into the vasculature. Exploring a wide range of speed and persistence combinations, we find that tumor growth positively correlates with increasing speed and higher persistence. As a biologically relevant example, we focused on Golgi fragmentation, a phenomenon often linked to alterations of cell migration. Golgi fragmentation was induced by depletion of Giantin, a Golgi matrix protein, the downregulation of which correlates with poor patient survival. Applying the experimentally obtained migration and invasion traits of Giantin depleted breast cancer cells to our mathematical model, we predict that loss of Giantin increases the number of intravasating cells. This prediction was validated, by showing that circulating tumor cells express significantly less Giantin than primary tumor cells. Altogether, our computational model identifies cell migration traits that regulate tumor progression and uncovers a role of Giantin in breast cancer progression.

• Radulovic, Maja; Wenzel, Eva; Gilani, Sania; Holland, Lya KK; Lystad, Alf Håkon Lohsen & Phuyal, Santosh [Show all 12 contributors for this article] (2022). Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair. EMBO Journal. ISSN 0261-4189. 41:e112677, p. 1–18. doi: 10.15252/embj.2022112677. Full text in Research Archive Show summary

  Lysosome integrity is essential for cell viability, and lesions in lysosome membranes are repaired by the ESCRT machinery. Here, we describe an additional mechanism for lysosome repair that is activated independently of ESCRT recruitment. Lipidomic analyses showed increases in lysosomal phosphatidylserine and cholesterol after damage. Electron microscopy demonstrated that lysosomal membrane damage is rapidly followed by the formation of contacts with the endoplasmic reticulum (ER), which depends on the ER proteins VAPA/B. The cholesterol-binding protein ORP1L was recruited to damaged lysosomes, accompanied by cholesterol accumulation by a mechanism that required VAP–ORP1L interactions. The PtdIns 4-kinase PI4K2A rapidly produced PtdIns4P on lysosomes upon damage, and knockout of PI4K2A inhibited damage-induced accumulation of ORP1L and cholesterol and led to the failure of lysosomal membrane repair. The cholesterol–PtdIns4P transporter OSBP was also recruited upon damage, and its depletion caused lysosomal accumulation of PtdIns4P and resulted in cell death. We conclude that ER contacts are activated on damaged lysosomes in parallel to ESCRTs to provide lipids for membrane repair, and that PtdIns4P generation and removal are central in this response.

• Phuyal, Santosh; Djaerff, Elena; Roux, Anabel-Lise Le; Baker, Martin J; Fankhauser, Daniela & Mahdizadeh, Sayyed Jalil [Show all 16 contributors for this article] (2022). Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1. EMBO Journal. ISSN 0261-4189. 41(18). doi: 10.15252/embj.2022110596. Full text in Research Archive Show summary

  Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER-to-Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER-to-Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.

• Besse, Lenka; Besse, Andrej; Stolze, Sara C.; Sobh, Amin; Zaal, Esther A & Ham, Alwin J.Van Der [Show all 25 contributors for this article] (2021). Treatment with HIV-protease inhibitor nelfinavir identifies membrane lipid composition and fluidity as a therapeutic target in advanced multiple myeloma. Cancer Research. ISSN 0008-5472. 81(17), p. 4581–4593. doi: 10.1158/0008-5472.CAN-20-3323. Full text in Research Archive Show summary

  The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)–refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9–based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI–refractory multiple myeloma. Significance: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.

• Alswady-Hoff, Mayes; Erdem, Johanna Maria Samulin; Phuyal, Santosh; Knittelfelder, Oskar; Sharma, Animesh & Fonseca, Davi de Miranda [Show all 9 contributors for this article] (2021). Long-Term Exposure to Nanosized TiO2 Triggers Stress Responses and Cell Death Pathways in Pulmonary Epithelial Cells. International Journal of Molecular Sciences. ISSN 1661-6596. 22(10). doi: 10.3390/ijms22105349. Full text in Research Archive Show summary

  There is little in vitro data available on long-term effects of TiO2 exposure. Such data are important for improving the understanding of underlying mechanisms of adverse health effects of TiO2 . Here, we exposed pulmonary epithelial cells to two doses (0.96 and 1.92 µg/cm2 ) of TiO2 for 13 weeks and effects on cell cycle and cell death mechanisms, i.e., apoptosis and autophagy were determined after 4, 8 and 13 weeks of exposure. Changes in telomere length, cellular protein levels and lipid classes were also analyzed at 13 weeks of exposure. We observed that the TiO2 exposure increased the fraction of cells in G1-phase and reduced the fraction of cells in G2-phase, which was accompanied by an increase in the fraction of late apoptotic/necrotic cells. This corresponded with an induced expression of key apoptotic proteins i.e., BAD and BAX, and an accumulation of several lipid classes involved in cellular stress and apoptosis. These findings were further supported by quantitative proteome profiling data showing an increase in proteins involved in cell stress and genomic maintenance pathways following TiO2 exposure. Altogether, we suggest that cell stress response and cell death pathways may be important molecular events in long-term health effects of TiO2

• Phuyal, Santosh & Baschieri, Francesco (2020). Endomembranes: Unsung Heroes of Mechanobiology? Frontiers in Bioengineering and Biotechnology. 8. doi: 10.3389/fbioe.2020.597721. Full text in Research Archive Show summary

  Mechanical stimuli have profound effects on the cellular architecture and functions. Over the past two decades, considerable progress has been made in unraveling the molecular machineries that confer cells the ability to sense and transduce mechanical input into biochemical signals. This has resulted in the identification of several force-sensing proteins or mechanically activated ion channels distributed throughout most cell types, whereby the plasma membrane, cytoskeleton, and the nucleus have garnered much attention. Although organelles from the endomembrane system make up significant portion of cell volume and play pivotal roles in the spatiotemporal distribution of signaling molecules, they have received surprisingly little attention in mechanobiology. In this mini-review, we summarize results that document participation of the endomembrane system in sensing and responding to mechanical cues.

• Eck, Franziska; Phuyal, Santosh; Smith, Matthew D.; Kaulich, Manuel; Wilkinson, Simon & Farhan, Hesso [Show all 7 contributors for this article] (2020). ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis. Journal of Cell Science. ISSN 0021-9533. 133(18), p. 1–16. doi: 10.1242/jcs.243477. Full text in Research Archive Show summary

  ABSTRACT While studies of the autophagy-related (ATG) genes in knockout models have led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3 and GABARAP family proteins (human ATG8 equivalents) are still poorly understood. A major drawback in understanding their roles is that the available interactome data has largely been acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was employed to map endogenous GABARAPL2 protein complexes using interaction proteomics. This approach identified the ER-associated protein and lipid droplet (LD) biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. GABARAPL2 bound ACSL3 in a manner dependent on its LC3-interacting regions, whose binding site in GABARAPL2 was required to recruit the latter to the ER. Through this interaction, the UFM1-activating enzyme UBA5 became anchored at the ER. Furthermore, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together these data allow us to define ACSL3 as a novel regulator of the enigmatic UFM1 conjugation pathway.

• Phuyal, Santosh & Farhan, Hesso (2019). Multifaceted Rho GTPase Signaling at the Endomembranes. Frontiers in Cell and Developmental Biology. 7(127). doi: 10.3389/fcell.2019.00127. Full text in Research Archive Show summary

  The Rho family of small GTPases orchestrates fundamental biological processes such as cell cycle progression, cell migration, and actin cytoskeleton dynamics, and their aberrant signaling is linked to numerous human diseases and disorders. Traditionally, active Rho GTPase proteins were proposed to reside and function predominantly at the plasma membrane. While this view still holds true, it is emerging that active pool of multiple Rho GTPases are in part localized to endomembranes such as endosomes and the Golgi. In this review, we will focus on the intracellular pools and discuss how their local activation contributes to the shaping of various cellular processes. Our main focus will be on Rho signaling from the endosomes, Golgi, mitochondria and nucleus and how they regulate multiple cellular events such as receptor trafficking, cell proliferation and differentiation, cell migration and polarity.

• Phuyal, Santosh; Kasem, Mayes; Knittelfelder, Oskar; Sharma, Animesh; Fonseca, Davi de Miranda & Vebraite, Vaineta [Show all 10 contributors for this article] (2018). Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes. Nanotoxicology. ISSN 1743-5390. 12(2), p. 138–152. doi: 10.1080/17435390.2018.1425500. Full text in Research Archive Show summary

  The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an in vitro normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.

• Moen, Aurora Lie; Jacobsen, Daniel; Phuyal, Santosh; Legfeldt, Anna; Haugen, Fred & Røe, Cecilie [Show all 7 contributors for this article] (2017). MicroRNA-223 demonstrated experimentally in exosome-like vesicles is associated with decreased risk of persistent pain after lumbar disc herniation. Journal of Translational Medicine. 15(89), p. 1–11. doi: 10.1186/s12967-017-1194-8. Full text in Research Archive Show summary

  Background: Previous findings have demonstrated that lumbar radicular pain after disc herniation may be associated with up-regulation of inflammatory mediators. In the present study we examined the possible role of extracellular microRNAs (miRs) in this process. Methods: Single unit recordings, isolation of exosome-like vesicles, electron microscopy, nanoparticle tracking analysis, western blot analysis and qPCR were used in rats to demonstrate the effect of nucleus pulposus (NP) applied onto the dorsal nerve roots. ELISA and qPCR were used to measure the level of circulating IL-6 and miRs in a 1-year observational study in patients after disc herniation. Results: In the rats, enhanced spinal cord nociceptive responses were displayed after NP applied onto the dorsal nerve roots. An increased release of small non-coding RNAs, including miR-223, miR-760 and miR-145, from NP in exosome-like vesicles was demonstrated. In particular, the NP expression of miR-223, which inhibited the nociceptive spinal signalling, was increased. In the patients, increased extracellular miR-223 was also verified in the acute phase after disc herniation. The increased miR-223 expression was, however, only observed in those who recovered (sex, age and smoking were included as covariates). Conclusions: Our findings suggest that miR-223, which can be released from the NP after disc herniation, attenuates the neuronal activity in the pain pathways. Dysregulation of miR-223 may predict chronic lumbar radicular pain.

• Phuyal, Santosh; Kasem, Mayes; Rubio, Laura; Karlsson, Hanna; Marcos, Ricard & Skaug, Vidar [Show all 7 contributors for this article] (2017). Effects on human bronchial epithelial cells following low-dose chronic exposure to nanomaterials: A 6-month transformation study. Toxicology in Vitro. ISSN 0887-2333. 44, p. 230–240. doi: 10.1016/j.tiv.2017.07.016. Full text in Research Archive Show summary

  The most plausible exposure route to manufactured nanomaterials (MNM) remains pulmonary inhalation. Yet, few studies have attempted to assess carcinogenic properties in vitro following long-term exposure of human pulmonary cells to low and occupationally relevant doses. The most advanced in vitro tests for carcinogenicity, the cell transformation assay (CTA), rely mostly on rodent cells and short-term exposure. We hypothesized that long-term exposure of human bronchial epithelial cells with a normal phenotype could be a valuable assay for testing carcinogenicity of nanomaterials. Therefore, this study (performed within the framework of the FP7-NANoREG project) assessed carcinogenic potential of chronic exposure (up to 6 months) to low doses of multi-walled carbon nanotubes (MWCNT, NM-400 and NM-401) and TiO2 materials (NM62002 and KC7000). In order to harmonize and standardize the experiments, standard operating protocols of MNM dispersion (NANOGENOTOX) were used by three different NANoREG project partners. All nanomaterials showed low cytotoxicity in short-term tests for the tested doses (0.96 and 1.92 μg/cm2). During long-term exposure, however, NM-401 clearly affected cell proliferation. In contrast, no cell transformation was observed for NM-401 by any of the partners. NM-400 and NM62002 formed some colonies after 3 months. We conclude that agglomerated NM-401 in low doses affect cell proliferation but do not cause cell transformation in the CTA assay used.

• Vestad, Beate; Llorente, Alicia; Neurauter, Axl; Phuyal, Santosh; Kierulf, Bente & Kierulf, Peter [Show all 10 contributors for this article] (2017). Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study. Journal of Extracellular Vesicles. 6(1), p. 1–11. doi: 10.1080/20013078.2017.1344087. Full text in Research Archive Show summary

  Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, n = 6) and inter-assay (day-to-day, n = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1–12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted.

• Phuyal, Santosh; Skotland, Tore; Hessvik, Nina Pettersen; Simolin, Helena; Øverbye, Anders & Brech, Andreas [Show all 10 contributors for this article] (2015). The ether lipid precursor hexadecylglycerol stimulates the release and changes the composition of exosomes derived from PC-3 cells. Journal of Biological Chemistry. ISSN 0021-9258. 290, p. 4225–4237. doi: 10.1074/jbc.M114.593962. Full text in Research Archive
• Phuyal, Santosh; Hessvik, Nina Pettersen; Skotland, Tore; Sandvig, Kirsten & Llorente, Alicia (2014). Regulation of exosome release by glycosphingolipids and flotillins. The FEBS Journal. ISSN 1742-464X. 281(9), p. 2214–2227. doi: 10.1111/febs.12775. Full text in Research Archive
• Hessvik, Nina Pettersen; Phuyal, Santosh; Brech, Andreas; Sandvig, Kirsten & Llorente, Alicia (2012). Profiling of microRNAs in exosomes released from PC-3 prostate cancer cells. Biochimica et Biophysica Acta. Gene Regulatory Mechanisms. ISSN 1874-9399. 1819(11-12), p. 1154–1163. doi: 10.1016/j.bbagrm.2012.08.016. Full text in Research Archive

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• Chan, Hanna; Borowicz, Pawel; Gilmour, Brian Christopher; Stensland, Maria; Garcia-Loza, Ivan & Phuyal, Santosh [Show all 10 contributors for this article] (2026). The TSAd-DOK2 axis regulates CD8+ T cell effector function. Full text in Research Archive
• Chan, Hanna; Borowicz, Pawel; Gilmour, Brian Christopher; Stensland, Maria; Loza, Ivan Garcia & Phuyal, Santosh [Show all 10 contributors for this article] (2024). Identification of novel interactors of T cell Specific Adaptor protein TSAd - An adaptor molecule with elusive function. Full text in Research Archive
• Chan, Hanna; Borowicz, Pawel; Gilmour, Brian Christopher; Stensland, Maria; Loza, Ivan Garcia & Phuyal, Santosh [Show all 10 contributors for this article] (2024). Identification of novel interactors of T cell specific adaptor protein TSAd - an adaptor molecule with elusive function. Full text in Research Archive
• Gilmour, Brian Christopher; Chan, Hanna; Borowicz, Pawel; Phuyal, Santosh; Lossius, Andreas & Spurkland, Anne (2023). β2, or not β2? Connecting the Lck-adaptor TSAd to integrin β2 via a combined wet lab & bioinformatic approach. Full text in Research Archive
• Auer, Susann; Haelterman, Nele A.; Weissgerber, Tracey L.; Erlich, Jeffrey C.; Susilaradeya, Damar & Julkowska, Magdalena [Show all 38 contributors for this article] (2021). Science forum: A community-led initiative for training in reproducible research. eLIFE. 10. doi: 10.7554/eLife.64719. Full text in Research Archive Show summary

  Open and reproducible research practices increase the reusability and impact of scientific research. The reproducibility of research results is influenced by many factors, most of which can be addressed by improved education and training. Here we describe how workshops developed by the Reproducibility for Everyone (R4E) initiative can be customized to provide researchers at all career stages and across most disciplines with education and training in reproducible research practices. The R4E initiative, which is led by volunteers, has reached more than 3000 researchers worldwide to date, and all workshop materials, including accompanying resources, are available under a CC-BY 4.0 license at https://www.repro4everyone.org/.

• Phuyal, Santosh & Farhan, Hesso (2021). Want to leave the ER? We offer vesicles, tubules, and tunnels. 220(6). doi: 10.1083/jcb.202104062. Full text in Research Archive
• Zienolddiny, Shanbeh; Phuyal, Santosh; Kasem, Mayes; Knittelfelder, Oskar; Sharma, Animesh & Fonseca, Davi de Miranda [Show all 8 contributors for this article] (2017). Toxicoproteomic effects of carbon nanotubes on chronically exposed human lung cells. Toxicology Letters. ISSN 0378-4274. 280, p. S141–S141. doi: 10.1016/j.toxlet.2017.07.395. Full text in Research Archive
• Gilmour, Brian Christopher; Spurkland, Anne; Dissen, Erik & Phuyal, Santosh (2024). β2, or not β2: that is the question. Expanding the SH2D2A-interactome to adhesion & β-integrins using a combined bioinformatic & wet-lab approach. Universitetet i Oslo. ISSN 9788234804847. Full text in Research Archive Show summary

  While the functions of many genes are known, others remain unexplored. Adaptor proteins, an under-studied group, lack enzymatic activity but are crucial in forming and modulating signalling complexes. These proteins are difficult to study because their knockout often does not produce a clear phenotype—an “anchor” which can then be used for further study—making further exploration challenging. Adaptor proteins are especially important in immune cells, which rely on complex signalling pathways. SH2D2A (TSAd) is one such adaptor protein that is highly expressed in immune cells. Its exact function is unclear, though it interacts with key kinases like LCK, ITK, and SRC. This study aimed to expand the known interactome of SH2D2A to better understand its role. Initial experiments suggested SH2D2A might interact with the SRC family kinase FYN, but this could not be confirmed under physiological conditions. Further analysis of bulk RNA-seq data from the Cancer Genome Atlas (TCGA) revealed that higher SH2D2A expression in tumours correlates with better prognosis in bladder urothelial cancer (BLCA), an effect associated with FOXP3+ IL2RAhi T regulatory (Treg) cells, which are the main expressors of SH2D2A in the BLCA bladder. This bioinformatic approach could help uncover functions of other immune-related adaptor proteins.

• Phuyal, Santosh; Llorente, Alicia & Sandvig, Kirsten (2014). Composition and cellular release of exosomes from cancer cells. Akademika publishing. Full text in Research Archive

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