Giant fluctuations and structural effects in a flocking epithelium

We thank S Henkes for useful discussions. FGia and RC acknowledge funding from the Italian Ministry of University and Scientific Research (MIUR) under the program Futuro in Ricerca—Project ANISOFT (RBFR125H0M) and from Regione Lombardia and CARIPLO foundation under the joint action Avviso congiunto per l'incremento dell'attrattivitá del sistema ricerca lombardo e della competitivitá dei ricercatori candidati su strumenti ERC—Project 2016-0998. CM, SC and GS acknowledge funding from Associazione Italiana per la Ricerca sul Cancro (AIRC 10168 and 18621), MIUR, the Italian Ministry of Health, Ricerca Finalizzata (RF0235844), Worldwide Cancer Research (AICR-14-0335), and the European Research Council (Advanced-ERC-268836). CM was also supported by Fondazione Umberto Veronesi and SC by an AIRC fellowship. FGin acknowledges support from the Marie Curie Career Integration Grant (CIG) PCIG13-GA-2013-618399, and wish to thank the University of Milan and LibrOsteria for their hospitality while this work was underway.Peer reviewedPostprin

Endocytic reawakening of motility in jammed epithelia.

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination

How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p < 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p < 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p < 0.0001) or urgent (20.4% vs. 38.5%; p < 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p < 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Identification of New Substrates of the Protein-tyrosine Phosphatase PTP1B by Bayesian Integration of Proteome Evidence*

There is growing evidence that tyrosine phosphatases display an intrinsic enzymatic preference for the sequence context flanking the target phosphotyrosines. On the other hand, substrate selection in vivo is decisively guided by the enzyme-substrate connectivity in the protein interaction network. We describe here a system wide strategy to infer physiological substrates of protein-tyrosine phosphatases. Here we integrate, by a Bayesian model, proteome wide evidence about in vitro substrate preference, as determined by a novel high-density peptide chip technology, and “closeness” in the protein interaction network. This allows to rank candidate substrates of the human PTP1B phosphatase. Ultimately a variety of in vitro and in vivo approaches were used to verify the prediction that the tyrosine phosphorylation levels of five high-ranking substrates, PLC-γ1, Gab1, SHP2, EGFR, and SHP1, are indeed specifically modulated by PTP1B. In addition, we demonstrate that the PTP1B-mediated dephosphorylation of Gab1 negatively affects its EGF-induced association with the phosphatase SHP2. The dissociation of this signaling complex is accompanied by a decrease of ERK MAP kinase phosphorylation and activation

The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins-0

Cubated with TRITC labeled EGF for 20 minutes at 37°C. Cells were fixed, without acid wash, and observed on an Olympus IX 70 with Nanomoverand softWoRx Delta Vision. Transfected cells are visible in the 528 nm channel as green colored. TRITC-EGF was visualized at 617 nm and is colored in red. Endocytosed vescicles with TRITC-EGF, visible as fluorescent spots, are not detectable in cells over expressing the mutated Dynamin K44A or the central proline rich region of POB1 (POB1 PRD1). A black arrow points the transfected cells in the zoomed sections. HeLa cells transfected with GFP-POB1 PRD1 (308–365) were serum starved and incubated with TRITC-Transferrin (100 mg/ml). POB1-PRD1 transfected cells are visualized as above, internalized transferrin is visible as fluorescent red spots.<p><b>Copyright information:</b></p><p>Taken from "The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins"</p><p>http://www.biomedcentral.com/1471-2091/9/21</p><p>BMC Biochemistry 2008;9():21-21.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2494995.</p><p></p

The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins-1

L of Grb2 (Grb2SH3-N), GST-SH3 carboxy-terminal of Grb2 (Grb2SH3-C) and GST alone, adsorbed to glutathione-Sepharose 4B beads. The retained proteins were separated by SDS-PAGE and probed with an anti-Myc antibody (for POB1). The input is 10% of the affinity-selected lysate. The AmphiphysinII SH3 binds 25% of the input while the carboxy-terminal SH3 of Grb2 binds 4%. The ammino-terminal SH3 of Grb2 does not bind POB1. The Ponceau staining shows the GST fusions and the input lysates. . HEK293 cells were transiently transfected with the indicated POB1 constructs expressed as fusions to GFP protein: GFP (lanes 1, 2, 3), GFP-POB1 full length (lanes 4, 5, 6) and GFP-PRD1, the poly-proline rich PRD1 region of POB1 from residue 308 to 365 (7, 8, 9). Cellular extracts were incubated with GST-SH3 of AmphiphysinII (left) or GST-SH3 carboxy-terminal of Grb2 (right) and GST alone. The retained proteins were subjected to SDS-PAGE and probed with an anti-GFP antibody (for POB1 or PRD1). The sample loaded in the input lane is 5% of the material used for affinity selection. A faint unspecific band is visible between the POB1 full length and the PRD1. POB1 PRD1 (308–365) is bound by both the SH3 tested: Amphiphysin II (last lane in leftmost gel) and Grb2 (last lane in rightmost gel). The Ponceau staining shows the GST fusions and the input lysates. . Left: POB1-GFP was co-expressed in HEK293 cells together with Grb2-HA or Amphiphysin II-FLAG (Amph-FLAG). Cellular extracts were immunoprecipitated (IP) with anti-HA antibody (for Grb2) or anti-FLAG resin (for Amphiphysin II), respectively and the co-immunoprecipitated POB1 was revealed with an anti-GFP antibody. Two lanes for each immuno-precipitation correspond to the same experiment repeated twice but at different washing stringency (without or with 1% Triton). Mock HA and FLAG lanes represent controls transfected with empty vectors. In the input lane we have loaded 1.5% of the cell lysate used for the immunoprecipitation. Right: An empty vector (lane 1) and two independent transfections of POB1-Myc were carried out in HEK293 cells (lanes 2, 3). Upper panel: POB1-Myc in cell lysate was revealed with an anti-Myc antibody (5% of the total cell lysate). Second panel: Cells extracts were immunoprecipitated with anti-Myc (for POB1) and the immunoprecipitated POB1 (an amount corresponding to 20% of the total lysate) were revealed with an anti-Myc antibody; Third panel: the cell lysate was probed with anti-Grb2 antibody, to detect endogenous Grb2 (1% of the total lysate). Panel below: Cells extracts were immunoprecipitated (IP) with anti-Myc (for POB1) and the co-precipitated material was blotted (WB) with an anti-Grb2 antibody to detect endogenous Grb2 co-immunoprecipitated by POB1.<p><b>Copyright information:</b></p><p>Taken from "The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins"</p><p>http://www.biomedcentral.com/1471-2091/9/21</p><p>BMC Biochemistry 2008;9():21-21.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2494995.</p><p></p

The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins-2

AmphIISH3) or the carboxy-terminal SH3 of Grb2 (Grb2SH3-C) expressed in bacteria as GST fusions. GST alone is used as a negative control. Anti-GST antibodies were utilized to identify bound proteins. The peptide sequences are indicated. The best binding peptides are shown in bold. The three positive control peptides are: GST binding peptides (positive control for GST and secondary antibody) and two peptides containing SH3 interacting motifs. Each binding experiment was carried out in duplicate. The sequence in bold correspond to the best binders. The best peptide 338PPTPPPR344 shows an intensity above 11.000 arbitrary units. Another peptide containing the sequence 214LKARPS219 binds to the SH3 of Amphiphysin, albeit producing a spot intensity around 9000 arbitrary units. Quantitative data are available in additional file . ) The POB1 fifteenmer peptide (PPTPPPRPQKTHSRA), containing the 338PPTPPPR344 binding motif, was systematically mutagenized by introducing an alanine at each of the indicated 15 positions. The upper membrane was probed with GST as a control, the middle one with the SH3 domain of Amphiphysin II, the bottom one with the carboxy-terminal SH3 of Grb2 fused to GST. The substitution of arginine 344 with alanine reduces the binding of Amphiphysin-SH3 down to 2.5%; a substitution of proline 339 with alanine reduces the binding to 31%. A reduction in Grb2 binding is observed when arginine 344 is substituted with alanine (13%) and when Pro343 or Pro 435 are substituted with Ala (30%). Supplemental quantitative data are provided in additional file . ) HEK293 cells were transiently transfected with plasmids expressing GFP fused to the proline rich domain PRD1 of POB1 (lane 1, 2, 3) or with an equivalent construct directing the synthesis of the POB1 PRD1 region mutated in Arg344 (PRD1 R344A), in lanes 4, 5, 6. Cell extracts were adsorbed to glutathione resins containing either the SH3 of Amphiphysin II (top gel) or the C-terminal SH3 of Grb2 (bottom gel). Adsorbed proteins were identified with anti GFP antibodies (for POB1 PRD1). Input is 3% of the lysate utilized in the GST pull down.<p><b>Copyright information:</b></p><p>Taken from "The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins"</p><p>http://www.biomedcentral.com/1471-2091/9/21</p><p>BMC Biochemistry 2008;9():21-21.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2494995.</p><p></p

The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins-5

And observed on an Olympus IX 70 with Nanomoverand softWoRx DeltaVision. GFP transfected cells are visible in the 528 nm channel (green). TRITC EGF was visualized at 617 nm (fluorescent spots). Nuclei were visualized by DAPI staining at 457 nm (blue). Transfected cells that do not show endocytic vescicles are indicated by a white arrow. HeLa cells were transiently transfected with GFP-POB1 PRD1 S354A or GFP-POB1 PRD1 R344A and treated as above. Transfected cells are visible in the 528 nm channel as green cells. These cells show a wild type distribution of fluorescent endocytic vesicles (TRITC-EGF visualized at 617 nm). Nuclei were visualized by DAPI staining at 457 nm (blue). Overexpression of 14-3-3 abolish the dominant negative effect of POB1-PRD1 on EGF endocytosis. HeLa cells were transiently transfected with GFP-POB1 PRD1 together with 3xFLAG-14-3-3 ζ. Cells were treated as above. Cells were then permeabilized and treated with Cy5-conjugated antibodies targeting the FLAG epitope of the 14-3-3 proteins, visible at 685 nm. Cells were observed as described above. These cells show a wild type distribution of fluorescent endocytic vesicles (TRITC-EGF visualized at 617 nm).<p><b>Copyright information:</b></p><p>Taken from "The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins"</p><p>http://www.biomedcentral.com/1471-2091/9/21</p><p>BMC Biochemistry 2008;9():21-21.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2494995.</p><p></p