Feasibility and operative outcomes of surgery in the liver area in advanced ovarian cancer.

OBJECTIVE: The study aimed to characterize intra-and postoperative complications according to a standardized anatomo-surgical classification for ovarian cancer metastases in the liver area. METHODS: Data from all patients with advanced ovarian cancer undergoing primary or secondary surgery with perihepatic liver involvement (May-2016 to May-2022), were retrospectively retrieved and classified according to a standardized anatomo-surgical classification, and clustered into four Classes: Class I "Peritoneal", Class II "Hepatoceliac-lymph-nodes", Class III "Parenchymal" and Class IV Mixed (≥ 2 classes). RESULTS: Data from 615 patients were collected. Intraoperative complications were observed in 15%, and severe postoperative complications in 17.6% of cases. While surgical complexity scores were similar, Class IV had longer operative times, higher blood loss, and a 30.4% intraoperative transfusion rate. Class II showed a higher prevalence of vascular injuries (8%). Classes II and IV were significantly associated with severe postoperative complications. Specific complications varied among classes, such as perihepatic collection and intrahepatic hematoma/abscess in Class III (p = 0.003, p < 0.001, respectively), and pleuric effusion, sepsis, anemia, and "other complications" in Class IV (p = 0.002, p = 0.004, p = 0.03, p = 0.03, respectively). Multivariable analysis identified Class II and IV (Class II: OR 4.991, p = 0.045; Class IV: OR 5.331, p = 0.030), Surgical Complexity Score group 3 (OR:3.922, p = 0.003), and the presence of residual tumor (OR:1.748, p = 0.048) as independent risk factors for severe postoperative complications. CONCLUSIONS: Liver procedures during advanced ovarian cancer surgery are feasible with acceptable complication rates According to the anatomo-surgical classification, metastatic patterns are related to both different surgical outcomes and postoperative complication profiles.

Proteomic Characterization of a 3D HER2+ Breast Cancer Model Reveals the Role of Mitochondrial Complex I in Acquired Resistance to Trastuzumab.

HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography-mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.

Mass spectrometry-based tear proteomics for noninvasive biomarker discovery.

The lacrimal film has attracted increasing interest in the last decades as a potential source of biomarkers of physiopathological states, due to its accessibility, moderate complexity, and responsiveness to ocular and systemic diseases. High-performance liquid chromatography-mass spectrometry (LC-MS) has led to effective approaches to tear proteomics, despite the intrinsic limitations in sample amounts. This review focuses on the recent progress in strategy and technology, with an emphasis on the potential for personalized medicine. After an introduction on lacrimal-film composition, examples of applications to biomarker discovery are discussed, comparing approaches based on pooled-sample and single-tear analysis. Then, the most critical steps of the experimental pipeline, that is, tear collection, sample fractionation, and LC-MS implementation, are discussed with reference to proteome-coverage optimization. Advantages and challenges of the alternative procedures are highlighted. Despite the still limited number of studies, tear quantitative proteomics, including single-tear investigation, could offer unique contributions to the identification of low-invasiveness, sustained-accessibility biomarkers, and to the development of personalized approaches to therapy and diagnosis.

Effective high-throughput isolation of enriched platelets and circulating pro-angiogenic cells to accelerate skin-wound healing.

Delayed wound healing and chronic skin lesions represent a major health problem. Over the past years, growth factors mediated by platelet-rich plasma (PRP) and cell-based therapies were developed as effective and affordable treatment able to improve wound healing capacity. We have advanced existing concepts to develop a highly efficient high-throughput protocol with proven application for the isolation of PRP and pro-angiogenic cells (AngioPRP). This protocol outlines the effectiveness of AngioPRP in promoting the critical healing process including wound closure, re-epithelialization, granulation tissue growth, and blood vessel regeneration. We coupled this effect with normalization of mechanical properties of rescued mouse wounds, which is sustained by a correct arrangement of elastin and collagen fibers. Proteomic analysis of treated wounds demonstrated a fingerprint of AngioPRP based on the up-regulation of detoxification pathway of glutathione metabolism, correlated to a decrease in inflammatory response. Overall, these results have enabled us to provide a framework for how AngioPRP supports wound healing, opening avenues for further clinical advances.

Interaction of SARS-CoV-2 Nucleocapsid Protein and Human RNA Helicases DDX1 and DDX3X Modulates Their Activities on Double-Stranded RNA.

The nucleocapsid protein Np of SARS-CoV-2 is involved in the replication, transcription, and packaging of the viral genome, but it also plays a role in the modulation of the host cell innate immunity and inflammation response. Ectopic expression of Np alone was able to induce significant changes in the proteome of human cells. The cellular RNA helicase DDX1 was among the proteins whose levels were increased by Np expression. DDX1 and its related helicase DDX3X were found to physically interact with Np and to increase 2- to 4-fold its affinity for double-stranded RNA in a helicase-independent manner. Conversely, Np inhibited the RNA helicase activity of both proteins. These functional interactions among Np and DDX1 and DDX3X highlight novel possible roles played by these host RNA helicases in the viral life cycle.

A Shotgun Proteomic Platform for a Global Mapping of Lymphoblastoid Cells to Gain Insight into Nasu-Hakola Disease.

Nasu-Hakola Disease (NHD) is a recessively inherited systemic leukodystrophy disorder characterized by a combination of frontotemporal presenile dementia and lytic bone lesions. NHD is known to be genetically related to a structural defect of TREM2 and DAP12, two genes that encode for different subunits of the membrane receptor signaling complex expressed by microglia and osteoclast cells. Because of its rarity, molecular or proteomic studies on this disorder are absent or scarce, only case reports based on neuropsychological and genetic tests being reported. In light of this, the aim of this paper is to provide evidence on the potential of a label-free proteomic platform based on the Multidimensional Protein Identification Technology (MudPIT), combined with in-house software and on-line bioinformatics tools, to characterize the protein expression trends and the most involved pathways in NHD. The application of this approach on the Lymphoblastoid cells from a family composed of individuals affected by NHD, healthy carriers and control subjects allowed for the identification of about 3000 distinct proteins within the three analyzed groups, among which proteins anomalous to each category were identified. Of note, several differentially expressed proteins were associated with neurodegenerative processes. Moreover, the protein networks highlighted some molecular pathways that may be involved in the onset or progression of this rare frontotemporal disorder. Therefore, this fully automated MudPIT platform which allowed, for the first time, the generation of the whole protein profile of Lymphoblastoid cells from Nasu-Hakola subjects, could be a valid approach for the investigation of similar neurodegenerative diseases.

Single-Tear Proteomics: A Feasible Approach to Precision Medicine.

Lacrimal fluid is an attractive source of noninvasive biomarkers, the main limitation being the small sample amounts typically collected. Advanced analytical methods to allow for proteomics profiling from a few microliters are needed to develop innovative biomarkers, with attractive perspectives of applications to precision medicine. This work describes an effective, analytical pipeline for single-tear analysis by ultrahigh-resolution, shotgun proteomics from 23 healthy human volunteers, leading to high-confidence identification of a total of 890 proteins. Highly reproducible quantification was achieved by either peak intensity, peak area, or spectral counting. Hierarchical clustering revealed a stratification of females vs. males that did not emerge from previous studies on pooled samples. Two subjects were monitored weekly over 3 weeks. The samples clustered by withdrawal time of day (morning vs. afternoon) but not by follow-up week, with elevated levels of components of the immune system in the morning samples. This study demonstrates feasibility of single-tear quantitative proteomics, envisaging contributions of this unconventional body fluid to individualized approaches in biomedicine.

Comprehensive Profiling of Secretome Formulations from Fetal- and Perinatal Human Amniotic Fluid Stem Cells.

We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, anti-fibrotic and proliferative effects. hAFS may also be isolated from III trimester clinical waste samples during scheduled C-sections (perinatal hAFS), thus offering a more easily accessible alternative when compared to fetal hAFS. Nonetheless, little is known about the paracrine profile of perinatal hAFS. Here we provide a detailed characterization of the hAFS total secretome (i.e., the entirety of soluble paracrine factors released by cells in the conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it, from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. hAFS-CM and hAFS-EV formulations were analyzed for protein and chemokine/cytokine content, and the EV cargo was further investigated by RNA sequencing. The phenotype of fetal- and perinatal hAFS, along with their corresponding secretome formulations, overlapped; yet, fetal hAFS showed immature oxidative phosphorylation activity when compared to perinatal ones. The profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. Both cell sources provided formulations enriched with neurotrophic, immunomodulatory, anti-fibrotic and endothelial stimulating factors, and the immature fetal hAFS secretome was defined by a more pronounced pro-vasculogenic, regenerative, pro-resolving and anti-aging profile. Small RNA profiling showed microRNA enrichment in both fetal- and perinatal hAFS-EV cargo, with a stably- expressed pro-resolving core as a reference molecular signature. Here we confirm that hAFS represents an appealing source of regenerative paracrine factors; the selection of either fetal or perinatal hAFS secretome formulations for future paracrine therapy should be evaluated considering the specific clinical scenario.

Shotgun Proteomics of Isolated Urinary Extracellular Vesicles for Investigating Respiratory Impedance in Healthy Preschoolers.

Urine proteomic applications in children suggested their potential in discriminating between healthy subjects from those with respiratory diseases. The aim of the current study was to combine protein fractionation, by urinary extracellular vesicle isolation, and proteomics analysis in order to establish whether different patterns of respiratory impedance in healthy preschoolers can be characterized from a protein fingerprint. Twenty-one 3-5-yr-old healthy children, representative of 66 recruited subjects, were selected: 12 late preterm (LP) and 9 full-term (T) born. Children underwent measurement of respiratory impedance through Forced Oscillation Technique (FOT) and no significant differences between LP and T were found. Unbiased clustering, based on proteomic signatures, stratified three groups of children (A, B, C) with significantly different patterns of respiratory impedance, which was slightly worse in group A than in groups B and C. Six proteins (Tripeptidyl peptidase I (TPP1), Cubilin (CUBN), SerpinA4, SerpinF1, Thy-1 membrane glycoprotein (THY1) and Angiopoietin-related protein 2 (ANGPTL2)) were identified in order to type the membership of subjects to the three groups. The differential levels of the six proteins in groups A, B and C suggest that proteomic-based profiles of urinary fractionated exosomes could represent a link between respiratory impedance and underlying biological profiles in healthy preschool children.

Methionine oxidation in α-synuclein inhibits its propensity for ordered secondary structure.

α-Synuclein (AS) is an intrinsically disordered protein highly expressed in dopaminergic neurons. Its amyloid aggregates are the major component of Lewy bodies, a hallmark of Parkinson's disease (PD). AS is particularly exposed to oxidation of its methionine residues, both in vivo and in vitro Oxidative stress has been implicated in PD and oxidized α-synuclein has been shown to assemble into soluble, toxic oligomers, rather than amyloid fibrils. However, the structural effects of methionine oxidation are still poorly understood. In this work, oxidized AS was obtained by prolonged incubations with dopamine (DA) or epigallocatechin-3-gallate (EGCG), two inhibitors of AS aggregation, indicating that EGCG promotes the same final oxidation product as DA. The conformational transitions of the oxidized and non-oxidized protein were monitored by complementary biophysical techniques, including MS, ion mobility (IM), CD, and FTIR spectroscopy assays. Although the two variants displayed very similar structures under conditions that stabilize highly disordered or highly ordered states, differences emerged in the intermediate points of transitions induced by organic solvents, such as trifluoroethanol (TFE) and methanol (MeOH), indicating a lower propensity of the oxidized protein for forming either α- or ß-type secondary structures. Furthermore, oxidized AS displayed restricted secondary-structure transitions in response to dehydration and slightly amplified tertiary-structure transitions induced by ligand binding. This difference in susceptibility to induced folding could explain the loss of fibrillation potential observed for oxidized AS. Finally, site-specific oxidation kinetics point out a minor delay in Met-127 modification, likely due to the effects of AS intrinsic structure.

Urinary Proteomics Profiles Are Useful for Detection of Cancer Biomarkers and Changes Induced by Therapeutic Procedures.

Boron neutron capture therapy (BNCT) is a binary cancer treatment modality where two different agents (10B and thermal neutrons) have to be present to produce an effect. A dedicated trial design is necessary for early clinical trials. The concentration of 10B in tissues is an accepted surrogate to predict BNCT effects on tissues. Tissue, blood, and urines were sampled after infusion of two different boron carriers, namely BSH and BPA in the frame of the European Organisation for Research and Treatment of Cancer (EORTC) trial 11001. In this study, urine samples were used to identify protein profiles prior and after drug infusion during surgery. Here, an approach that is based on the mass spectrometry (MS)-based proteomic analysis of urine samples from head and neck squamous cell carcinoma (HNSCC) and thyroid cancer patients is presented. This method allowed the identification of several inflammation- and cancer-related proteins, which could serve as tumor biomarkers. In addition, changes in the urinary proteome during and after therapeutic interventions were detected. In particular, a reduction of three proteins that were involved in inflammation has been observed: Galectin-3 Binding Protein, CD44, and osteopontin. The present work represents a proof of principle to follow proteasome changes during complex treatments based on urine samples.

Proteome Investigation of Rat Lungs subjected to Ex Vivo Perfusion (EVLP).

Ex vivo lung perfusion (EVLP) is an emerging procedure that allows organ preservation, assessment and reconditioning, increasing the number of marginal donor lungs for transplantation. However, physiological and airflow measurements are unable to unveil the molecular mechanisms responsible of EVLP beneficial effects on lung graft and monitor the proper course of the treatment. Thus, it is urgent to find specific biomarkers that possess these requirements but also accurate and reliable techniques that identify them. The purpose of this study is to give an overview on the potentiality of shotgun proteomic platforms in characterizing the status and the evolution of metabolic pathways during EVLP in order to find new potential EVLP-related biomarkers. A nanoLC-MS/MS system was applied to the proteome analysis of lung tissues from an optimized rat model in three experimental groups: native, pre- and post-EVLP. Technical and biological repeatability were evaluated and, together with clustering analysis, underlined the good quality of data produced. In-house software and bioinformatics tools allowed the label-free extraction of differentially expressed proteins among the three examined conditions and the network visualization of the pathways mainly involved. These promising findings encourage further proteomic investigations of the molecular mechanisms behind EVLP procedure.

Emerging MS-based platforms for the characterization of tumor-derived exosomes isolated from human biofluids: challenges and promises of MudPIT.

INTRODUCTION: Exosomes are small extracellular vesicles of endosomal origin that are produced and released by several type of cells. These vesicles contain different macromolecules: proteins, mRNA, miRNA, mitochondrial DNA, and lipids. Exosomes play an important role in cell-to-cell communication, also promoting cancer progression. Areas covered: Various proteomic approaches have been applied to study exosomes isolated from different human biofluids in search of possible cancer biomarkers. The results of these studies are reported, and pros and cons of each employed technique are described. Gel-free and gel-based mass spectrometry systems are discussed, giving particular emphasis on the innovative multidimensional protein identification technology (MudPIT). Expert commentary: Proteomic studies on exosomes as candidate cancer biomarkers from urine and other body fluids in cancer have shown the potential of MS-based techniques. In particular, MudPIT is a promising tool to be applied in clinical proteomics of cancer.

Proteomic analysis of Mesembryanthemum crystallinum leaf microsomal fractions finds an imbalance in V-ATPase stoichiometry during the salt-induced transition from C3 to CAM.

The halophyte Mesembryanthemum crystallinum adapts to salt stress by salt uptake and switching from C3 photosynthesis to CAM (crassulacean acid metabolism). An important role in this process is played by transport proteins in the tonoplast of the central vacuole. In the present study we examine dynamic changes in the protein composition during salt-stress adaptation in microsomes from M. crystallinum leaves. Plants challenged with 400 mM NaCl accumulate salt by day 4 of treatment and malic acid only at day 12; a switching to CAM hence follows any initial steps of salt adaptation with a delay. Using a label-free and semiquantitative approach, we identified the most dramatic changes between the proteome of control plants and plants harvested after 12 days of the treatment; the abundance of 14 proteins was significantly affected. The proteomic data revealed that the majority of the subunits of V-ATPase (vacuolar H(+)-ATPase) holoenzyme. The salt treatment somewhat decreased the abundance of all subunits in the short term (4 days). Long-term adaptation, including the switching to CAM, goes together with a strong increase in the representation of all detectable subunits. Because this increase is subunit-specific, with the highest rise occurring for subunits E and c, the data suggest that long-term adaptation to salt stress correlates with a change in V-ATPase subunit stoichiometry and highlight the structural plasticity of this holoenzyme.

Magnetic-field-driven targeting of exosomes modulates immune and metabolic changes in dystrophic muscle.

Exosomes are promising therapeutics for tissue repair and regeneration to induce and guide appropriate immune responses in dystrophic pathologies. However, manipulating exosomes to control their biodistribution and targeting them in vivo to achieve adequate therapeutic benefits still poses a major challenge. Here we overcome this limitation by developing an externally controlled delivery system for primed annexin A1 myo-exosomes (Exomyo). Effective nanocarriers are realized by immobilizing the Exomyo onto ferromagnetic nanotubes to achieve controlled delivery and localization of Exomyo to skeletal muscles by systemic injection using an external magnetic field. Quantitative muscle-level analyses revealed that macrophages dominate the uptake of Exomyo from these ferromagnetic nanotubes in vivo to synergistically promote beneficial muscle responses in a murine animal model of Duchenne muscular dystrophy. Our findings provide insights into the development of exosome-based therapies for muscle diseases and, in general, highlight the formulation of effective functional nanocarriers aimed at optimizing exosome biodistribution.

Lung-to-Heart Nano-in-Micro Peptide Promotes Cardiac Recovery in a Pig Model of Chronic Heart Failure.

BACKGROUND: The lack of disease-modifying drugs is one of the major unmet needs in patients with heart failure (HF). Peptides are highly selective molecules with the potential to act directly on cardiomyocytes. However, a strategy for effective delivery of therapeutics to the heart is lacking. OBJECTIVES: In this study, the authors sought to assess tolerability and efficacy of an inhalable lung-to-heart nano-in-micro technology (LungToHeartNIM) for cardiac-specific targeting of a mimetic peptide (MP), a first-in-class for modulating impaired L-type calcium channel (LTCC) trafficking, in a clinically relevant porcine model of HF. METHODS: Heart failure with reduced ejection fraction (HFrEF) was induced in Göttingen minipigs by means of tachypacing over 6 weeks. In a setting of overt HFrEF (left ventricular ejection fraction [LVEF] 30% ± 8%), animals were randomized and treatment was started after 4 weeks of tachypacing. HFrEF animals inhaled either a dry powder composed of mannitol-based microparticles embedding biocompatible MP-loaded calcium phosphate nanoparticles (dpCaP-MP) or the LungToHeartNIM only (dpCaP without MP). Efficacy was evaluated with the use of echocardiography, invasive hemodynamics, and biomarker assessment. RESULTS: DpCaP-MP inhalation restored systolic function, as shown by an absolute LVEF increase over the treatment period of 17% ± 6%, while reversing cardiac remodeling and reducing pulmonary congestion. The effect was recapitulated ex vivo in cardiac myofibrils from treated HF animals. The treatment was well tolerated, and no adverse events occurred. CONCLUSIONS: The overall tolerability of LungToHeartNIM along with the beneficial effects of the LTCC modulator point toward a game-changing treatment for HFrEF patients, also demonstrating the effective delivery of a therapeutic peptide to the diseased heart.

Cytoreductive surgery for advanced epithelial ovarian cancer in the poly(ADP-ribose) polymerase inhibitors era-Is it time for a new paradigm shift? A systematic review and meta-analysis.

INTRODUCTION: In patients with newly diagnosed advanced high-grade serous and endometrioid epithelial ovarian cancer (EOC) first-line maintenance therapy with poly(ADP-ribose) polymerase inhibitors (PARPi) tremendously improved progression-free survival (PFS). Yet, data on the effect of PARPi in proportion to postoperative residual disease status were lacking. MATERIAL AND METHODS: A systematic review and meta-analysis was conducted in accordance with the Preferred Reporting Items of Systematic reviews and Meta-Analysis (PRISMA) guidelines. We searched Medline/Pubmed, Embase and Cochrane databases as well as meeting abstracts until 18th March 2023. Hazard ratios (HRs) alongside their 95% confidence intervals (CIs) for PFS were extracted from the studies. A subgroup analysis was conducted to examine the effect of PARPi according to postoperative residual disease. RESULTS: A total of six phase III randomised controlled trials were included and comprised SOLO 1, PAOLA 1, PRIMA, PRIME, ATHENA-MONO and VELIA. Patients who received PARPi following complete gross resection showed greatest PFS benefit. Compared with placebo, maintenance with PARPi significantly improved PFS in patients with macroscopic residual disease (pooled HR 0.55; 95% CI 0.44-0.68). This magnitude was comparable to that found in patients with complete gross resection (pooled HR 0.53; 95% CI 0.41-0.67). CONCLUSIONS: Patients with macroscopic residual disease benefit from PARPi at the same extent as cases with complete gross resection. However, patients with complete gross resection who were treated with PARPi show the most favourable PFS rates. Hence, the pursuit of achieving complete cytoreduction remains valid in the PARPi era.

Risk of retinal vein occlusion following COVID-19 vaccination: a self-controlled case series.

BACKGROUND: To evaluate the association between COVID-19 vaccination and retinal vein occlusion (RVO). METHODS: This multicentre self-controlled case series included patients with RVO seen in five tertiary referral centres in Italy. All adults who received at least one dose of the BNT162b2, ChAdOx1 nCoV-19, mRNA-1273 or Ad26.COV2.S vaccine and had a first diagnosis of RVO between January 01, 2021, and December 31, 2021 were included. Incidence rate ratios (IRRs) of RVO were estimated using Poisson regression, comparing rates of events in a 28-day period following each dose of vaccination and in the unexposed control periods. RESULTS: 210 patients were included in the study. No increased risk of RVO was observed after the first dose (1-14 days IRR: 0.87, 95% CI: 0.41-1.85; 15-28 days IRR: 1.01, 95% CI: 0.50-2.04; 1-28 days IRR: 0.94, 95% CI: 0.55-1.58) and second dose of vaccination (1-14 days IRR: 1.21, 95% CI: 0.62-2.37; 15-28 days IRR: 1.08, 95% CI: 0.53-2.20; 1-28 days IRR: 1.16, 95% CI: 0.70-1.90). No association between RVO and vaccination was found in subgroup analyses by type of vaccine, gender and age. CONCLUSIONS: This self-controlled case series found no evidence of an association between RVO and COVID-19 vaccination.

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking.

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking.

Investigating the Paracrine Role of Perinatal Derivatives: Human Amniotic Fluid Stem Cell-Extracellular Vesicles Show Promising Transient Potential for Cardiomyocyte Renewal.

Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell paracrine therapy has attracted increasing attention to resurge rescue mechanisms within the heart. We previously characterized the paracrine effects that human amniotic fluid-derived stem cells (hAFSC) can exert to provide cardioprotection and enhance cardiac repair in preclinical models of myocardial ischemia and cardiotoxicity. Here, we analyze whether hAFSC secretome formulations, namely, hAFSC conditioned medium (hAFSC-CM) over extracellular vesicles (hAFSC-EVs) separated from it, can induce cardiomyocyte renewal. c-KIT+ hAFSC were obtained by leftover samples of II trimester prenatal amniocentesis (fetal hAFSC) and from clinical waste III trimester amniotic fluid during scheduled C-section procedures (perinatal hAFSC). hAFSC were primed under 1% O2 to enrich hAFSC-CM and EVs with cardioactive factors. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from cardiac tissue of R26pFUCCI2 mice with cell cycle fluorescent tagging by mutually exclusive nuclear signal. mNVCM were stimulated by fetal versus perinatal hAFSC-CM and hAFSC-EVs to identify the most promising formulation for in vivo assessment in a R26pFUCCI2 neonatal mouse model of myocardial infarction (MI) via intraperitoneal delivery. While the perinatal hAFSC secretome did not provide any significant cardiogenic effect, fetal hAFSC-EVs significantly sustained mNVCM transition from S to M phase by 2-fold, while triggering cytokinesis by 4.5-fold over vehicle-treated cells. Treated mNVCM showed disorganized expression of cardiac alpha-actinin, suggesting cytoskeletal re-arrangements prior to cell renewal, with a 40% significant downregulation of Cofilin-2 and a positive trend of polymerized F-Actin. Fetal hAFSC-EVs increased cardiomyocyte cell cycle progression by 1.8-fold in the 4-day-old neonatal left ventricle myocardium short term after MI; however, such effect was lost at the later stage. Fetal hAFSC-EVs were enriched with a short isoform of Agrin, a mediator of neonatal heart regeneration acting by YAP-related signaling; yet in vitro application of YAP inhibitor verteporfin partially affected EV paracrine stimulation on mNVCM. EVs secreted by developmentally juvenile fetal hAFSC can support cardiomyocyte renewal to some extension, via intercellular conveyance of candidates possibly involving Agrin in combination with other factors. These perinatal derivative promising cardiogenic effects need further investigation to define their specific mechanism of action and enhance their potential translation into therapeutic opportunity.