Sample-Treatment with the Virucidal ß-Propiolactone Does Not Preclude Analysis by Large Panel Affinity Proteomics, Including the Discovery of Biomarker Candidates.

Virus inactivation is a prerequisite for safe handling of high-risk infectious samples. ß-Propiolactone (BPL) is an established reagent with proven virucidal efficacy. BPL primarily reacts with DNA, RNA, and amino acids. The latter may modify antigenic protein epitopes interfering with binding properties of affinity reagents such as antibodies and aptamers used in affinity proteomic screens. We investigated (i) the impact of BPL treatment on the analysis of protein levels in plasma samples using the aptamer-based affinity proteomic platform SomaScan and (ii) effects on protein detection in conditioned medium samples using the proximity extension assay-based Olink Target platform. In the former setup, BPL-treated and native plasma samples from patients with ovarian cancer (n = 12) and benign diseases (n = 12) were analyzed using the SomaScan platform. In the latter, conditioned media samples collected from cultured T cells with (n = 3) or without (n = 3) anti-CD3 antibody stimulation were analyzed using the Olink Target platform. BPL-related changes in protein detection were evaluated comparing native and BPL-treated states, simulating virus inactivation, and impact on measurable group differences was assessed. While approximately one-third of SomaScan measurements were significantly changed by the BPL treatment, a majority of antigen/aptamer interactions remained unaffected. Interaction effects of BPL treatment and disease state, potentially altering detectability of group differences, were observable for less than one percent of targets (0.6%). BPL effects on protein detection with Olink Target were also limited, affecting 3.6% of detected proteins with no observable interaction effects. Thus, effects of BPL treatment only moderately interfere with affinity proteomic detectability of differential protein expression between different experimental groups. Overall, the results prove high-throughput affinity proteomics well suited for the analysis of high-risk samples inactivated using BPL.

Targeting the High-Density Lipoprotein Proteome for the Treatment of Post-Acute Sequelae of SARS-CoV-2.

Here, we target the high-density lipoprotein (HDL) proteome in a case series of 16 patients with post-COVID-19 symptoms treated with HMG-Co-A reductase inhibitors (statin) plus angiotensin II type 1 receptor blockers (ARBs) for 6 weeks. Patients suffering from persistent symptoms (post-acute sequelae) after serologically confirmed SARS-CoV-2 infection (post-COVID-19 syndrome, PCS, n = 8) or following SARS-CoV-2 vaccination (PVS, n = 8) were included. Asymptomatic subjects with corresponding serological findings served as healthy controls (n = 8/8). HDL was isolated using dextran sulfate precipitation and the HDL proteome of all study participants was analyzed quantitatively by mass spectrometry. Clinical symptoms were assessed using questionnaires before and after therapy. The inflammatory potential of the patients' HDL proteome was addressed in human endothelial cells. The HDL proteome of patients with PCS and PVS showed no significant differences; however, compared to controls, the HDL from PVS/PCS patients displayed significant alterations involving hemoglobin, cytoskeletal proteins (MYL6, TLN1, PARVB, TPM4, FLNA), and amyloid precursor protein. Gene Ontology Biological Process (GOBP) enrichment analysis identified hemostasis, peptidase, and lipoprotein regulation pathways to be involved. Treatment of PVS/PCS patients with statins plus ARBs improved the patients' clinical symptoms. After therapy, three proteins were significantly increased (FAM3C, AT6AP2, ADAM10; FDR < 0.05) in the HDL proteome from patients with PVS/PCS. Exposure of human endothelial cells with the HDL proteome from treated PVS/PCS patients revealed reduced inflammatory cytokine and adhesion molecule expression. Thus, HDL proteome analysis from PVS/PCS patients enables a deeper insight into the underlying disease mechanisms, pointing to significant involvement in metabolic and signaling disturbances. Treatment with statins plus ARBs improved clinical symptoms and reduced the inflammatory potential of the HDL proteome. These observations may guide future therapeutic strategies for PVS/PCS patients.

Fast mass spectrometry search and clustering of untargeted metabolomics data.

The throughput of mass spectrometers and the amount of publicly available metabolomics data are growing rapidly, but analysis tools such as molecular networking and Mass Spectrometry Search Tool do not scale to searching and clustering billions of mass spectral data in metabolomics repositories. To address this limitation, we designed MASST+ and Networking+, which can process datasets that are up to three orders of magnitude larger than those processed by state-of-the-art tools.

Serum proteomics hint at an early T-cell response and modulation of SARS-CoV-2-related pathogenic pathways in COVID-19-ARDS treated with Ruxolitinib.

Background: Acute respiratory distress syndrome (ARDS) in corona virus disease 19 (COVID-19) is triggered by hyperinflammation, thus providing a rationale for immunosuppressive treatments. The Janus kinase inhibitor Ruxolitinib (Ruxo) has shown efficacy in severe and critical COVID-19. In this study, we hypothesized that Ruxo's mode of action in this condition is reflected by changes in the peripheral blood proteome. Methods: This study included 11 COVID-19 patients, who were treated at our center's Intensive Care Unit (ICU). All patients received standard-of-care treatment and n = 8 patients with ARDS received Ruxo in addition. Blood samples were collected before (day 0) and on days 1, 6, and 10 of Ruxo treatment or, respectively, ICU admission. Serum proteomes were analyzed by mass spectrometry (MS) and cytometric bead array. Results: Linear modeling of MS data yielded 27 significantly differentially regulated proteins on day 1, 69 on day 6 and 72 on day 10. Only five factors (IGLV10-54, PSMB1, PGLYRP1, APOA5, WARS1) were regulated both concordantly and significantly over time. Overrepresentation analysis revealed biological processes involving T-cells only on day 1, while a humoral immune response and complement activation were detected at day 6 and day 10. Pathway enrichment analysis identified the NRF2-pathway early under Ruxo treatment and Network map of SARS-CoV-2 signaling and Statin inhibition of cholesterol production at later time points. Conclusion: Our results indicate that the mechanism of action of Ruxo in COVID-19-ARDS can be related to both known effects of this drug as a modulator of T-cells and the SARS-CoV-2-infection.

Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK-STAT signaling in macrophages by perturbing lipid raft structures.

Survival of ovarian carcinoma is associated with the abundance of immunosuppressed CD163high CD206high tumor-associated macrophages (TAMs) and high levels of arachidonic acid (AA) in the tumor microenvironment. Here, we show that both associations are functionally linked. Transcriptional profiling revealed that high CD163 and CD206/MRC1 expression in TAMs is strongly associated with an inhibition of cytokine-triggered signaling, mirrored by an impaired transcriptional response to interferons and IL-6 in monocyte-derived macrophages by AA. This inhibition of pro-inflammatory signaling is caused by dysfunctions of the cognate receptors, indicated by the inhibition of JAK1, JAK2, STAT1, and STAT3 phosphorylation, and by the displacement of the interferon receptor IFNAR1, STAT1 and other immune-regulatory proteins from lipid rafts. AA exposure led to a dramatic accumulation of free AA in lipid rafts, which appears to be mechanistically crucial, as the inhibition of its incorporation into phospholipids did not affect the AA-mediated interference with STAT1 phosphorylation. Inhibition of interferon-triggered STAT1 phosphorylation by AA was reversed by water-soluble cholesterol, known to prevent the perturbation of lipid raft structure by AA. These findings suggest that the pharmacologic restoration of lipid raft functions in TAMs may contribute to the development new therapeutic approaches.

Conducting a Pragmatic Trial in Integrated Primary Care: Key Decision Points and Considerations.

Pragmatic trials testing the effectiveness of interventions under "real world" conditions help bridge the research-to-practice gap. Such trial designs are optimal for studying the impact of implementation efforts, such as the effectiveness of integrated behavioral health clinicians in primary care settings. Formal pragmatic trials conducted in integrated primary care settings are uncommon, making it difficult for researchers to anticipate the potential pitfalls associated with balancing scientific rigor with the demands of routine clinical practice. This paper is based on our experience conducting the first phase of a large, multisite, pragmatic clinical trial evaluating the implementation and effectiveness of behavioral health consultants treating patients with chronic pain using a manualized intervention, brief cognitive behavioral therapy for chronic pain (BCBT-CP). The paper highlights key choice points using the PRagmatic-Explanatory Continuum Indicator Summary (PRECIS-2) tool. We discuss the dilemmas of pragmatic research that we faced and offer recommendations for aspiring integrated primary care pragmatic trialists.

Transcriptional drug repositioning and cheminformatics approach for differentiation therapy of leukaemia cells.

Differentiation therapy is attracting increasing interest in cancer as it can be more specific than conventional chemotherapy approaches, and it has offered new treatment options for some cancer types, such as treating acute promyelocytic leukaemia (APL) by retinoic acid. However, there is a pressing need to identify additional molecules which act in this way, both in leukaemia and other cancer types. In this work, we hence developed a novel transcriptional drug repositioning approach, based on both bioinformatics and cheminformatics components, that enables selecting such compounds in a more informed manner. We have validated the approach for leukaemia cells, and retrospectively retinoic acid was successfully identified using our method. Prospectively, the anti-parasitic compound fenbendazole was tested in leukaemia cells, and we were able to show that it can induce the differentiation of leukaemia cells to granulocytes in low concentrations of 0.1 µM and within as short a time period as 3 days. This work hence provides a systematic and validated approach for identifying small molecules for differentiation therapy in cancer.

SIRT1 promotes lipid metabolism and mitochondrial biogenesis in adipocytes and coordinates adipogenesis by targeting key enzymatic pathways.

The NAD+-dependent deacetylase SIRT1 controls key metabolic functions by deacetylating target proteins and strategies that promote SIRT1 function such as SIRT1 overexpression or NAD+ boosters alleviate metabolic complications. We previously reported that SIRT1-depletion in 3T3-L1 preadipocytes led to C-Myc activation, adipocyte hyperplasia, and dysregulated adipocyte metabolism. Here, we characterized SIRT1-depleted adipocytes by quantitative mass spectrometry-based proteomics, gene-expression and biochemical analyses, and mitochondrial studies. We found that SIRT1 promoted mitochondrial biogenesis and respiration in adipocytes and expression of molecules like leptin, adiponectin, matrix metalloproteinases, lipocalin 2, and thyroid responsive protein was SIRT1-dependent. Independent validation of the proteomics dataset uncovered SIRT1-dependence of SREBF1c and PPARα signaling in adipocytes. SIRT1 promoted nicotinamide mononucleotide acetyltransferase 2 (NMNAT2) expression during 3T3-L1 differentiation and constitutively repressed NMNAT1 and 3 levels. Supplementing preadipocytes with the NAD+ booster nicotinamide mononucleotide (NMN) during differentiation increased expression levels of leptin, SIRT1, and PGC-1α and its transcriptional targets, and reduced levels of pro-fibrotic collagens (Col6A1 and Col6A3) in a SIRT1-dependent manner. Investigating the metabolic impact of the functional interaction of SIRT1 with SREBF1c and PPARα and insights into how NAD+ metabolism modulates adipocyte function could potentially lead to new avenues in developing therapeutics for obesity complications.

Targeting Chronic Pain in Primary Care Settings by Using Behavioral Health Consultants: Methods of a Randomized Pragmatic Trial.

BACKGROUND: Manualized cognitive and behavioral therapies are increasingly used in primary care environments to improve nonpharmacological pain management. The Brief Cognitive Behavioral Therapy for Chronic Pain (BCBT-CP) intervention, recently implemented by the Defense Health Agency for use across the military health system, is a modular, primary care-based treatment program delivered by behavioral health consultants integrated into primary care for patients experiencing chronic pain. Although early data suggest that this intervention improves functioning, it is unclear whether the benefits of BCBT-CP are sustained. The purpose of this paper is to describe the methods of a pragmatic clinical trial designed to test the effect of monthly telehealth booster contacts on treatment retention and long-term clinical outcomes for BCBT-CP treatment, as compared with BCBT-CP without a booster, in 716 Defense Health Agency beneficiaries with chronic pain. DESIGN: A randomized pragmatic clinical trial will be used to examine whether telehealth booster contacts improve outcomes associated with BCBT-CP treatments. Monthly booster contacts will reinforce BCBT-CP concepts and the home practice plan. Outcomes will be assessed 3, 6, 12, and 18 months after the first appointment for BCBT-CP. Focus groups will be conducted to assess the usability, perceived effectiveness, and helpfulness of the booster contacts. SUMMARY: Most individuals with chronic pain are managed in primary care, but few are offered biopsychosocial approaches to care. This pragmatic brief trial will test whether a pragmatic enhancement to routine clinical care, monthly booster contacts, results in sustained functional changes among patients with chronic pain receiving BCBT-CP in primary care.

multicrispr: gRNA design for prime editing and parallel targeting of thousands of targets.

Targeting the coding genome to introduce nucleotide deletions/insertions via the CRISPR/Cas9 technology has become a standard procedure. It has quickly spawned a multitude of methods such as prime editing, APEX proximity labeling, or homology directed repair, for which supporting bioinformatics tools are, however, lagging behind. New CRISPR/Cas9 applications often require specific gRNA design functionality, and a generic tool is critically missing. Here, we introduce multicrispr, an R/bioconductor tool, intended to design individual gRNAs and complex gRNA libraries. The package is easy to use; detects, scores, and filters gRNAs on both efficiency and specificity; visualizes and aggregates results per target or CRISPR/Cas9 sequence; and finally returns both genomic ranges and sequences of gRNAs. To be generic, multicrispr defines and implements a genomic arithmetic framework as a basis for facile adaptation to techniques recently introduced such as prime editing or yet to arise. Its performance and design concepts such as target set-specific filtering render multicrispr a tool of choice when dealing with screening-like approaches.

Metabolic Signatures of Tumor Responses to Doxorubicin Elucidated by Metabolic Profiling in Ovo.

BACKGROUND: Dysregulated cancer metabolism is associated with acquired resistance to chemotherapeutic treatment and contributes to the activation of cancer survival mechanisms. However, which metabolic pathways are activated following treatment often remains elusive. The combination of chicken embryo tumor models (in ovo) with metabolomics phenotyping could offer a robust platform for drug testing. Here, we assess the potential of this approach in the treatment of an in ovo triple negative breast cancer with doxorubicin. METHODS: MB-MDA-231 cells were grafted in ovo. The resulting tumors were then treated with doxorubicin or dimethyl sulfoxide (DMSO) for six days. Tumors were collected and analyzed using a global untargeted metabolomics and comprehensive lipidomics. RESULTS: We observed a significant suppression of tumor growth in the doxorubicin treated group. The metabolic profiles of doxorubicin and DMSO-treated tumors were clearly separated in a principle component analysis. Inhibition of glycolysis, nucleotide synthesis, and glycerophospholipid metabolism appear to be triggered by doxorubicin treatment, which could explain the observed suppressed tumor growth. In addition, metabolic cancer survival mechanisms could be supported by an acceleration of antioxidative pathways. CONCLUSIONS: Metabolomics in combination with in ovo tumor models provide a robust platform for drug testing to reveal tumor specific treatment targets such as the antioxidative tumor capacity.

Stimulation of glycolysis promotes cardiomyocyte proliferation after injury in adult zebrafish.

Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration model, we first find an increase in the levels of mRNAs encoding enzymes regulating glucose and pyruvate metabolism, including pyruvate kinase M1/2 (Pkm) and pyruvate dehydrogenase kinases (Pdks), especially in tissues bordering the damaged area. We further find that impaired glycolysis decreases the number of proliferating cardiomyocytes following injury. These observations are supported by analyses using loss-of-function models for the metabolic regulators Pkma2 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha. Cardiomyocyte-specific loss- and gain-of-function manipulations of pyruvate metabolism using Pdk3 as well as a catalytic subunit of the pyruvate dehydrogenase complex (PDC) reveal its importance in cardiomyocyte dedifferentiation and proliferation after injury. Furthermore, we find that PDK activity can modulate cell cycle progression and protrusive activity in mammalian cardiomyocytes in culture. Our findings reveal new roles for cardiac metabolism and the PDK-PDC axis in cardiomyocyte behavior following cardiac injury.

Author Correction: Effect of induced hypoglycemia on inflammation and oxidative stress in type 2 diabetes and control subjects.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effect of induced hypoglycemia on inflammation and oxidative stress in type 2 diabetes and control subjects.

Intensive diabetes control has been associated with increased mortality in type 2 diabetes (T2DM); this has been suggested to be due to increased hypoglycemia. We measured hypoglycemia-induced changes in endothelial parameters, oxidative stress markers and inflammation at baseline and after a 24-hour period in type 2 diabetic (T2DM) subjects versus age-matched controls. Case-control study: 10 T2DM and 8 control subjects. Blood glucose was reduced from 5 (90 mg/dl) to hypoglycemic levels of 2.8 mmol/L (50 mg/dl) for 1 hour by incremental hyperinsulinemic clamps using baseline and 24 hour samples. Measures of endothelial parameters, oxidative stress and inflammation at baseline and at 24-hours post hypoglycemia were performed: proteomic (Somalogic) analysis for inflammatory markers complemented by C-reactive protein (hsCRP) measurement, and proteomic markers and urinary isoprostanes for oxidative measures, together with endothelial function. Between baseline and 24 -hours after hypoglycemia, 15 of 140 inflammatory proteins differed in T2DM whilst only 1 of 140 differed in controls; all returned to baseline at 24-hours. However, elevated hsCRP levels were seen at 24-hours in T2DM (2.4 mg/L (1.2-5.4) vs. 3.9 mg/L (1.8-6.1), Baseline vs 24-hours, P < 0.05). In patients with T2DM, between baseline and 24-hour after hypoglycemia, only one of 15 oxidative stress proteins differed and this was not seen in controls. An increase (P = 0.016) from baseline (73.4 ng/mL) to 24 hours after hypoglycemia (91.7 ng/mL) was seen for urinary isoprostanes. Hypoglycemia resulted in inflammatory and oxidative stress markers being elevated in T2DM subjects but not controls 24-hours after the event.

A Systems-level Characterization of the Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities that are beneficial for the treatment of various diseases. To avoid the issues that come with using tissue-derived MSCs in therapy, MSCs may be generated by the differentiation of human embryonic stems cells (hESCs) in culture. However, the changes that occur during the differentiation process have not been comprehensively characterized. Here, we combined transcriptome, proteome and phosphoproteome profiling to perform an in-depth, multi-omics study of the hESCs-to-MSCs differentiation process. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation. Among the earliest and strongest induced proteins with extensive differential phosphorylation was AHNAK, which we hypothesized to be a defining factor in MSC biology. We observed two distinct expression waves of developmental HOX genes and an AGO2-to-AGO3 switch in gene silencing. Exploring the kinetic of noncoding ORFs during differentiation, we mapped new functions to well annotated long noncoding RNAs (CARMN, MALAT, NEAT1, LINC00152) as well as new candidates which we identified to be important to the differentiation process. Phosphoproteome analysis revealed ESC and MSC-specific phosphorylation motifs with PAK2 and RAF1 as top predicted upstream kinases in MSCs. Our data represent a rich systems-level resource on ESC-to-MSC differentiation that will be useful for the study of stem cell biology.

Metabolic and proteomic signatures of hypoglycaemia in type 2 diabetes.

AIMS: To determine the biochemical changes that underlie hypoglycaemia in a healthy control group and in people with type 2 diabetes (T2D). MATERIALS AND METHODS: We report a hypoglycaemic clamp study in seven healthy controls and 10 people with T2D. Blood was withdrawn at four time points: at baseline after an overnight fast; after clamping to euglycaemia at 5 mmol/L; after clamping to hypoglycaemia at 2.8 mmol/L; and 24 hours later, after overnight fast. Deep molecular phenotyping using non-targeted metabolomics and the SomaLogic aptamer-based proteomics platform was performed on collected samples. RESULTS: A total of 955 metabolites and 1125 proteins were identified, with significant alterations in >90 molecules. A number of metabolites significantly increased during hypoglycaemia, but only cortisol, adenosine-3',5'-cyclic monophosphate (cyclic AMP), and pregnenolone sulphate, were independent of insulin. By contrast, identified protein changes were triggered by hypoglycaemia rather than insulin. The T2D group had significantly higher levels of fatty acids including 10-nonadecenoate, linolenate and dihomo-linoleate during hypoglycaemia compared with the control group. Molecules contributing to cardiovascular complications such as fatty-acid-binding protein-3 and pregnenolone sulphate were altered in the participants with T2D during hypoglycaemia. Almost all molecules returned to baseline at 24 hours. CONCLUSIONS: The present study provides a comprehensive description of molecular events that are triggered by insulin-induced hypoglycaemia. We identified deregulated pathways in T2D that may play a role in the pathophysiology of hypoglycaemia-induced cardiovascular complications.

Myh10 deficiency leads to defective extracellular matrix remodeling and pulmonary disease.

Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.

A comprehensive metabolomic data set of date palm fruit.

This article provides detailed information on the phenotypes and the metabolic profiles of 196 date fruits from 123 unique date fruit varieties. These date fruits are extensively diverse in their country of origin, variety and post harvesting conditions. We used a non-targeted mass-spectrometry based metabolomics approach to metabolically characterize date fruits, and measured 427 metabolites from a wide range of metabolic pathways. The metabolomics data for all the date fruit samples are available at the NIH Common Fund's Data Repository and Coordinating Center (supported by NIH grant, U01-DK097430) website, http://www.metabolomicsworkbench.org), under Metabolomics Workbench StudyID: ST000867. The data are directly accessible at http://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Study&StudyID=ST000867&StudyType=MS&ResultType=1.

Accelerated lipid catabolism and autophagy are cancer survival mechanisms under inhibited glutaminolysis.

Suppressing glutaminolysis does not always induce cancer cell death in glutamine dependent tumors because cells may switch to alternative energy sources. To reveal compensatory metabolic pathways, we investigated the metabolome-wide cellular response to inhibited glutaminolysis in cancer cells. Glutaminolysis inhibition with C.968 suppressed cell proliferation but was insufficient to induce cancer cell death. We found that lipid catabolism was activated as a compensation for glutaminolysis inhibition. Accelerated lipid catabolism, together with oxidative stress induced by glutaminolysis inhibition, triggered autophagy. Simultaneously inhibiting glutaminolysis and either beta oxidation with trimetazidine or autophagy with chloroquine both induced cancer cell death. Here we identified metabolic escape mechanisms contributing to cancer cell survival under treatment and we suggest potentially translational strategy for combined cancer therapy, given that chloroquine is an FDA approved drug. Our findings are first to show efficiency of combined inhibition of glutaminolysis and beta oxidation as potential anti-cancer strategy as well as add to the evidence that combined inhibition of glutaminolysis and autophagy may be effective in glutamine-addicted cancers.

Proteomic profiling of human cancer pseudopodia for the identification of anti-metastatic drug candidates.

Cancer metastasis causes approximately 90% of all cancer-related death and independent of the advancement of cancer therapy, a majority of late stage patients suffers from metastatic cancer. Metastasis implies cancer cell migration and invasion throughout the body. Migration requires the formation of pseudopodia in the direction of movement, but a detailed understanding of this process and accordingly strategies of prevention remain elusive. Here, we use quantitative proteomic profiling of human cancer pseudopodia to examine this mechanisms essential to metastasis formation, and identify potential candidates for pharmacological interference with the process. We demonstrate that Prohibitins (PHBs) are significantly enriched in the pseudopodia fraction derived from cancer cells, and knockdown of PHBs, as well as their chemical inhibition through Rocaglamide (Roc-A), efficiently reduces cancer cell migration.