The advent of induced pluripotent stem cells (iPSCs) together with recent advances in genome editing, microphysiological systems, tissue engineering and xenograft models present new opportunities for the investigation of hematological diseases and cancer in a patient-specific context. Here we review the progress in the field and discuss the advantages, limitations, and challenges of iPSC-based malignancy modeling. We will also discuss the use of iPSCs and its derivatives as cellular sources for drug target identification, drug development and evaluation of pharmacological responses. Hematological diseases and cancers are devastating diseases with a high economic and social burden. Generally basic and preclinical cancer research relies on model systems in order to understand the cellular and molecular mechanisms of the malignant state at the cellular, organ and organism level. The hope is that the information gained from such model systems will be helpful in devising precise, effective, and personalized therapeutic strategies. Prototypically, these model systems include immortalized cell lines and genetically engineered, mutant mice. More recently, advanced patient-derived models such as conditionally reprogrammed cells (CRs) –3, patieThe use of iPSCs in the study of hematological diseases, cancer, and tumorigenicity is gaining momentum. It started with the generation of iPSCs from a human melanoma and a human prostate cancer cell line in 2008 . Since tThe reprogramming of cancer cell lines was soon followed by the generation of iPSCs representing various hematological diseases, blood cell cancers, and non-hematopoietic cancers –77 Tabl. These iRUNX1 -iPSCs were impaired in both exocrine pancreatic and hematopoietic differentiation with reduced myeloid cell generation in vitro, increased apoptosis, and elevated protease activity recapitulating SDS patient phenotypes  that is caused by inherited monoallelic mutations in RUNX1 . FPD/AMLRUNX1 . Neitherpatients , 52, 64.enotypes . Forth wenotypes , 55, 73.enotypes , 82. Anoenotypes . They weKMT2A/MLL  features including increased mTORC1 activation, abnormal autophagy and LAM-associate biomarker expression . Additionally, if one agrees with Sánchez-García's tumor stem cell reprogramming viewpoint that cancer cell properties can reemerge upon differentiation and that this property is to a fixed, uni-differentiated cell fate then this may not reemerge in an iPSC model due to the fact that iPSCs by definition possess pluripotent differentiation ability. On the other hand, such a resetting might be looked at favorably in certain diseases/cancers of “pure” epigenetic origin for which one could envision of using cells differentiated from these epigenetically reset iPSCs as a regenerative therapy.Cell reprogramming is associated with resetting of the starting cell's epigenetic landscape to that of a pluripotent stem cell. This resetting might eliminate characteristic features of the disease/cancer cell phenotype that might not be recreated upon differentiation, thus producing a significant difference between the disease/cancer iPSC model and the original disease/cancer cell. Here, it is worth bringing forth the theory that the initial oncogenic insult to the cancer-initiating cell might (re)program the epigenome toward a specific cancer cell fate . This poin vitro is a challenge, as generally iPSC are maintained isolated as functionally autonomous entities in two-dimensional culture systems and not physiological integrated within the disease/tumor microenvironment. Recent progress and use of tissue engineering, three-dimensional organoids, MPS and in vivo xenografts offers a window to more sophisticated modeling that enables incorporation of malignant cells with cellular and extracellular components of the disease/tumor microenvironment, nutrient supply, and mimicking of blood/lymph flow thus attempting to recapitulate the in vivo architecture and physiological condition in which the malignant cells reside and grow.Last but not least, modeling systemic processes MLL rearrangements retained the leukemic mutations but also reset leukemic DNA methylation and gene expression patterns , exerts great influence on disease development and progression.Remarkably, a recent report showed that the cellular context could significantly impact on the genetic information and behavior of malignant cells . HashimoHBB in sickle cell disease (SCD) and β-thalassemia iPSC lines  EWSR1-FLI1 fusion and t NMP1-ALK fusion genomic translocations associated with Ewing sarcoma and anaplastic large cell lymphoma, respectively, or to revert the t NMP1-ALK translocation  EWSR1-FLI1 fusion translocation in iPSCs .Organoids derived from primary resected tumors or biopsies are hailed to create opportunities to build large biobanks with relevant patient material for cancer research, drug evaluation and therapy development –109. WitiPSCs in xenograft models, Zaret et al. modeled PDAC development using PDAC-iPSCs in combination with in vivo transplantation  have become a prominent model system as they are presumed to more faithfully capture the cellular, molecular and physiological characteristics of primary and metastatic malignancies , 121. Adin vivo environment and, hence, expand their experimental utility. PDAC has a very poor prognosis and until the elegant study by Zaret et al. lacked a human cell model of early disease progression (Transplantation of iPSCs or derived cells into appropriate animal models can provide a more physiological, three-dimensional gression . SubcutaKMT2A/MLL locus (vivo they found that the ability to give rise to leukemia in vivo is dependent on transplantation of AML-iPSC-derived hematopoietic cells as AML-iPSCs lacked leukemic potential. Additionally, despite retaining the leukemic-driver mutations, AML-iPSCs reset the leukemic DNA methylation and gene expression patterns. Surprisingly, hematopoietic differentiation of these AML-iPSCs and leukemia formation was sufficient to reestablish the leukemic DNA methylation and gene expression profile strongly suggesting that the genetic mutations/rearrangements of the KMT2A/MLL locus in AML-iPSCs reactivate a leukemic program in the context of hematopoietic cells (Majeti et al. established an AML model based on iPSCs generated form patients with rearrangements of the LL locus . Using iic cells .in vivo transplantation and propagation appears important in order to know whether the attempted PDX-model accurately retains the genetic lesions present in the original malignant cells or if they evolve, and if they evolve whether the evolution is specific to the patient or the host.It was recently reported that copy number alterations recurrently observed in primary human tumors gradually disappeared in PDXs, suggesting that events undergoing positive selection in humans can become dispensable during propagation in mice . In lighet al. (+ hematopoietic progenitors obtained from their patient's CML-iPSCs were partially sensitive to imatinib and Kumano et al. (− hematopoietic cells but not CD34+ hematopoietic progenitors, which recapitulated the pathophysiological feature of initial CML of that patient. In depth molecular characterization at the epigenome, transcriptome and proteome level will be necessary to discover the signaling networks responsible for the observed behavior. Induced PSCs and derived cells also present an opportunity for phenotypic drug testing and screening. This can be especially attractive for diseases with no previously characterized targets or drug treatment strategies. However, such phenotypic drug testing and screening requires the ability to identify cellular phenotypes or functional properties, such as proliferation, apoptosis, activation of a specific signaling pathway, a distinct metabolic profile that correlate with patient phenotypes and responses and thus can serve as surrogate readouts of therapeutic effectiveness (The cost of drug development from discovery, through clinical trials to approval and marketing is in excess of $2.6 billion . As costet al.  found tho et al.  found imtiveness , 138. Untiveness , 110.in vivo PDX models, automated high-throughput-screening tools and next-generation omics approaches will lead to a greater mechanistic understanding of disease/cancer, the relationship between malignant cells and their microenvironment, and drug responses. Undoubtedly, iPSC technology is revolutionizing the way we approach disease modeling, preclinical cancer research, drug development and precision medicine.iPSC technology started a new, exciting era in biomedicine. The ease by which patient-specific iPSCs from various primary or metastatic somatic tissues and blood of patients with hematological diseases and cancers can be derived provides a self-renewable, scalable and cryopreservable source of cells with the patient's genetic background. iPSCs are readily enable to genome-editing in order to either correct or introduce known or suspected disease-associated mutations. This novel tool enables attempts to successfully recapitulate various pathological disease states and features associated with malignancies in a patient-specific context. Integration of iPSC-based disease and cancer models with advanced, bioengineered physiological systems, All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Glial cell line-derived neurotrophic Family Ligands (GFL) are soluble neurotrophic factors that are required for development of multiple human tissues, but which are also important contributors to human cancers. GFL signaling occurs through the transmembrane RET receptor tyrosine kinase, a well-characterized oncogene. GFL-independent RET activation, through rearrangement or point mutations occurs in thyroid and lung cancers. However, GFL-mediated activation of wildtype RET is an increasingly recognized mechanism promoting tumor growth and dissemination of a much broader group of cancers. RET and GFL expression have been implicated in metastasis or invasion in diverse human cancers including breast, pancreatic, and prostate tumors, where they are linked to poorer patient prognosis. In addition to directly inducing tumor growth in these diseases, GFL-RET signaling promotes changes in the tumor microenvironment that alter the surrounding stroma and cellular composition to enhance tumor invasion and metastasis. As such, GFL RET signaling is an important target for novel therapeutic approaches to limit tumor growth and spread and improve disease outcomes. The neurotrophins are a family of soluble neurotrophic factors, originally recognized for their abilities to regulate growth, survival and differentiation of neural-derived cell types. Neurotrophins have well characterized roles as guidance, survival and differentiation factors in developing neurons in the central (CNS) and peripheral nervous systems (PNS) and may also promote survival or regrowth of mature neurons, by binding their cell-surface receptors and stimulating downstream signals in their target cells. However, with the increasing availability of tissue and cell-specific transcriptome and proteome data, it is becoming clear that neurotrophic factors and their receptors are also broadly expressed on other, non-neural cell types, where they can contribute to cell growth, differentiation and migration and tissue maturation. Importantly, the aberrant expression or activation of these signaling complexes can allow these normal growth signals to contribute to the growth or spread of cancer cells, making expression and functions of neurotrophins and their receptors important contributors to human cancer, and potentially valuable therapeutic targets.REarranged during Transfection) tyrosine kinase  are a family of neurotrophins with similarities to the transforming growth factor β growth regulatory proteins. There are four structurally similar family members: GDNF, neurturin (NRTN), artemin (ARTN), and persephin (PSPN) that are recruited to corresponding non-signaling co-receptors of the GDNF Family Receptors α GFRα1-4), which are tethered to the plasma membrane through glycosylphosphatidylinositol-anchors , an inherited cancer syndrome characterized by medullary thyroid cancers (MTC) and the adrenal tumor pheochromocytoma . Similarmonomers . As a remonomers . Howevermonomers . There aactivity . These autations . For exautations , leads tutations . In thesutations . In patiutations . The effutations .In addition to oncogenic mutations, increased expression or activity of wildtype RET is now being recognized in many additional tumor types where it may have a range of different implications. In response to GFLs, which are released at some level by many tumors as well as by many cell types in the tumor microenvironment, RET signaling can enhance growth, promote tumor spread or even affect response to therapies.in vitro cell and animal models of breast cancer, activation of RET signaling by GFLs enhances tumor cell proliferation, and survival and has been shown to promote estrogen-independent expression of a normally estrogen-ER-dependent transcriptional profile  and are an indicator of poorer prognosis and reduced overall survival . ExpressInterestingly, although activating RET mutations are very rare in PDAC, a common polymorphic glycine to serine sequence variant at codon 691 (G691S) in the RET intracellular juxtamembrane region is over represented in PDAC patients (∼20% cases) and has been linked to increased GDNF-dependent proliferation and invasion , suggestin vitro and preclinical animal models of prostate cancer and can be further enhanced in the presence of soluble forms of GFRα1 released by nerves (RET protein has been detected in 20–75% of high grade prostate adenocarcinomas (Gleason score >3) while GDNF is upregulated in both prostate tumors and surrounding stroma . GDNF-mey nerves . Upreguly nerves . Importay nerves .RET rearrangements in colorectal cancers similar to those in thyroid and lung , and a plethora of cellular components including cancer-associated fibroblasts (CAF), stromal, and immune or inflammatory cells all surrounded and supported by non-cellular components of the extracellular matrix (ECM) Figure . InteracImportantly, RET is implicated in promoting tumor-related inflammation, the infiltration of immune cells into the tumor environment, a key indicator of disease outcomes and therapeutic responses . RET actAs recognition of RET’s impact in diverse cancers expands, it has become an increasingly important therapeutic target. As yet, there are no agents that specifically target the RET kinase in clinical use, however, multikinase inhibitors originally developed against other kinases that also inhibit RET activity are proving valuable . SpecifiFurther, these approved multikinase inhibitors are associated with an array of significant off-target side effects, likely due to inhibition of other kinase family members . A numbe1. Early reports suggest these agents are better tolerated, with fewer off-target effects than multikinase inhibitors  have shown increased or prolonged benefit over single agents in thyroid and breast cancer models . Excitinin vitro and in vivo  and the GDNF Family Receptor α-like (GFRAL)  . GDF15-G (GFRAL) , suggestLM prepared the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CRISPR/Cas9 system requires short guide RNAs (sgRNAs) to direct genome modification. Most currently available tools for sgRNA design operate only with standard reference genomes, and are best suited for small-scale projects. To address these limitations, we developed Crisflash, a software tool for fast sgRNA design and potential off-target discovery, built for performance and flexibility. Crisflash can rapidly design CRISPR guides against any sequenced genome or genome sequences, and can optimize guide accuracy by incorporating user-supplied variant data. Crisflash is over an order of magnitude faster than comparable tools, even using a single CPU core, and efficiently and robustly scores the potential off-targeting of all possible candidate CRISPR guide oligonucleotides.https://github.com/crisflashBioinformatics online. CRISPR/Cas9 system has become a popular technology for mammalian genome editing, and depends on short, single-stranded guide RNAs (sgRNAs) directing the Cas9 nuclease to a desired locus (et al., 2014; Most available sgRNA design tools are web-based and created for sgRNA design in a window of up to ten thousand of base pairs (Historically, most CRISPR/Cas9-based research has focused on genetic manipulation of human and mouse cell lines and tissues. Accurately generating guide RNAs for gene editing targeted to specific human genomes, or to directly target spontaneous somatic mutations in the human cancer genome requires flexibility to tailor the reference genome and quickly evaluate the quality of candidate sequences. Here, we present Crisflash, a command-line sgRNA design tool built for rapid performance, and flexible and accurate handling of individual genomic variance.Crisflash first scans a FASTA file containing genomic sequences for PAM adjacent protospacers and adds identified gRNAs to an N-ary tree. Nodes correspond to sequence bases; hence, the depth of the tree is equal to the gRNA length. Genomic coordinates of protospacers are saved in terminal nodes. Candidate sgRNAs are then identified, and off-target sequences for these candidate guide RNAs are identified by walking the sequence into the depth of the tree. If a base in a sequence is different than that of a node, a mismatch is recorded, and children of the node are followed as long as the total number of mismatches has not been exceeded. The final score of each candidate sgRNA is computed based on the experimentally observed effects of the mismatch positions of sequences, when followed all the way to the terminal nodes (We benchmarked Crisflash against following tools: Cas-OFFinder . Off-tarThe speed advantage gained from tree-based, fast approximate string matching comes with a relatively high requirement for RAM. Existing tools typically rely on a few GB of operational memory. However, the 90 GB necessary for Crisflash to work with human reference genome is readily accommodated in modern server clusters.Crisflash is first of its kind to offer greater sgRNA accuracy using variant information. Most proposed applications of CRISPR in biomedical research will increasingly demand the flexibility that Crisflash provides in its highly tailored sgRNA design. Crisflash reduces undesirable off-target effects arising from individual human genetic variation and avoids designing non-operational guides.Crisflash is a new sgRNA design and potential off-target discovery software, which greatly outperforms similar existing software. Crisflash is built for speed and improved precision when incorporating individual-specific genetic variant data, and is suitable for large-scale guide design, including in non-standard model organisms.This work has been supported by Cancer Research UK core award [20412] and strategic award [22398]. Conflict of Interest: none declared.btz019_Supplementary_MethodsClick here for additional data file.


Imatinib, the first-in-class BCR-ABL tyrosine kinase inhibitor (TKI), had been a revolution for the treatment of chronic myeloid leukemia (CML) and had greatly enhanced patient survival. Second-  and third-generation (ponatinib) TKIs have been developed to be effective against BCR-ABL mutations making imatinib less effective. However, these treatments have been associated with arterial occlusive events. This review gathers clinical data and experiments about the pathophysiology of these arterial occlusive events with BCR-ABL TKIs. Imatinib is associated with very low rates of thrombosis, suggesting a potentially protecting cardiovascular effect of this treatment in patients with BCR-ABL CML. This protective effect might be mediated by decreased platelet secretion and activation, decreased leukocyte recruitment, and anti-inflammatory or antifibrotic effects. Clinical data have guided mechanistic studies toward alteration of platelet functions and atherosclerosis development, which might be secondary to metabolism impairment. Dasatinib, nilotinib, and ponatinib affect endothelial cells and might induce atherogenesis through increased vascular permeability. Nilotinib also impairs platelet functions and induces hyperglycemia and dyslipidemia that might contribute to atherosclerosis development. Description of the pathophysiology of arterial thrombotic events is necessary to implement risk minimization strategies. The last section discusses relevant off-targets that might be implicated in the cardiovascular toxicity. The discovery of the mechanism(s) by which arterial occlusive events arose in CML patients would help in the management of patients treated with BCR-ABL TKIs and implement risk minimization measures. Discovery of the pathophysiology of these events in CML patients might also led to the development of predictive biomarkers or to the development of new therapies with no or reduced cardiovascular toxicity profile while keeping an unaltered efficacy.BCR-ABL TKIs are associated with both bleeding and thrombotic complications.Experimental assessments of platelet functions with imatinib demonstrate less pronounced effects on platelets. Imatinib inhibits platelet aggregation only at high doses,Even if ponatinib induces very few bleeding disorders, assessment of primary hemostasis in CML patients demonstrated that ponatinib induces defect in platelet aggregation. This impairment was found at all ponatinib dosage, in patients with or without low platelet counts.To summarize, dasatinib and imatinib induce hemorrhagic events through alteration of platelet functions, but the molecular mechanism needs to be better determined. Ponatinib also impairs platelet functions. Therefore, no current data involve platelets in the pathogenesis of arterial thrombosis occurring with dasatinib and ponatinib. Oppositely, nilotinib might induce arterial thrombosis through alteration of platelet secretion, adhesion, and activation.BCR-ABL TKIs have contradictory effect on glucose metabolism. Imatinib and dasatinib improve glucose metabolism and type 2 diabetes management in CML patients .Oppositely to imatinib and dasatinib, case reports and clinical trials indicate that nilotinib increases blood glucose level and promotes diabetes mellitus.Similarly with glucose metabolism, effects on lipid metabolism are conflicting between TKIs. Oppositely to in vivo study which demonstrated no impact of imatinib on total cholesterol and triglycerides levels in diabetic mice,Oppositely, dasatinib and mostly nilotinib are associated with an increase of cholesterol level.Nilotinib and ponatinib reduce EC proliferation and might impaired endothelial regeneration.In addition to their effect on EC viability, nilotinib and ponatinib also influence EC functions, inhibit their migration, and decrease angiogenesis.Effects of new-generation TKIs on inflammatory cells were less studied, but first experiments indicate similarities with imatinib about its impact on monocytes and macrophages. Both dasatinib and nilotinib have similar inhibitory profile on macrophage-colony formation that has been linked to CSFR inhibition.Globally, BCR-ABL TKIs possess reassuring profile on inflammatory cells. However, impact of new-generation TKIs on several functions of macrophages have not been assessed , whereas effect of ponatinib on inflammatory cells is unknown. The assessment of lipid uptake and foam cell formation is particularly relevant with new-generation TKIs because there are numerous interactions between TKIs and ABC transporters.Impact of new-generation TKIs on fibrosis was less studied but demonstrate similar inhibitory effect on VSMCs and fibroblasts. Indeed, dasatinib inhibits PDGFR more potently than imatinib,BCR-ABL TKIs bind the highly conserved ATP binding site and are therefore not very specific to BCR-ABL and possess multiple cellular targets (kinases and nonkinase proteins).However, inhibitory profiles are difficult to determine and several researches published discrepancies. For conflicting results, a conservative approach has been applied inAs described over this review, PDGF signaling has countless effects on several cells and tissues and is involved in several proatherogenic mechanisms  and vascular homeostasis, which led to the suggestion of its implication in the potential beneficial cardiovascular effect of imatinib.Other tyrosine kinases have been incriminated in the occurrence of arterial thrombosis with new-generation TKIs. DDR-1This review summarizes the data underlying the potential preventive effect of imatinib on the occurrence of arterial thrombosis. Globally, in vitro and in vivo experiments demonstrate that imatinib possesses antiplatelet activity, hypolipidemic and hypoglycemic effects, and inhibits inflammation and atherosclerosis development in several cell types . These benefits were largely attributed to PDGFR inhibition. It is currently unknown why new-generation TKIs that also inhibit PDGFR present opposite cardiovascular safety profile and this point needs to be elucidated.New-generation BCR-ABL TKIs increase the risk of arterial thromboembolism with different clinical features  and are associated with different safety profiles, suggesting different pathways to explain the pathophysiology. The safety profile of nilotinib is mostly characterized by impaired glucose and lipid metabolism. However, both the molecular mechanism of these alterations and their impact on the occurrence of arterial thrombosis are unknown. Both dasatinib and ponatinib exhibit antiplatelet effect, whereas it was recently suggested that nilotinib potentially induces prothrombotic phenotype of platelets. Based on the clinical characteristics and case reports, atherosclerosis appears the most plausible mechanisms by which new-generation TKIs induce arterial thrombosis. However, in vitro and in vivo studies of viability and functions of SMCs and inflammatory cells demonstrate reassuring impact of dasatinib and nilotinib, even if additional studies are required to complete this evaluation. However, first experiments indicate that dasatinib, nilotinib, and ponatinib influence EC survival and/or endothelium integrity, suggesting a reasonable hypothesis by which new-generation TKIs induce atherosclerosis development and, subsequently, arterial thrombosis. Additional studies on the shedding of functional extracellular vesicles by endothelial cells might be interesting regarding their important role in coronary artery diseases.To conclude, new-generation TKIs increase the risk of arterial thrombosis in patients with CML, whereas imatinib, the first-generation TKI, might prevent the development of cardiovascular events. To date, the cellular events and signaling pathways by which these events occurred are unknown and researches are extremely limited focusing mainly on imatinib and nilotinib. Researches need to be extended to all new-generation BCR-ABL TKIs . The understanding of the mechanisms by which new-generation BCR-ABL TKIs induce or promote arterial occlusive events will improve the clinical uses of these therapies. To date, only general risk minimization measures have been proposed .Relevant articles published from the database inception to July 11, 2017, were identified from an electronic database (PubMed) using the keywords “vascular,” “thrombosis,” “atherosclerosis,” “arteriosclerosis,” “venous,” “arterial,” “hemostasis,” “metabolic,” “metabolism,” “glycemia,” “glycaemia,” “cholesterol,” “triglycerides,” and “platelet” combined with the five approved BCR-ABL TKIs. The search strategy is presented in supplementary files. Articles published in languages other than English were excluded from the analysis. Primary criteria were pathophysiological explanation of arterial thrombotic events. Abstracts and full-text articles were reviewed with a focus on atherogenesis, plaque rupture, platelet functions, and their link with the development of arterial thrombosis with BCR-ABL TKIs. The reference section of identified articles was also examined.


Myeloid cells represent a diverse range of innate leukocytes that are crucial for mounting successful immune responses against viruses. These cells are responsible for detecting pathogen-associated molecular patterns, thereby initiating a signaling cascade that results in the production of cytokines such as interferons to mitigate infections. The aim of this review is to outline recent advances in our knowledge of the roles that neutrophils and inflammatory monocytes play in initiating and coordinating host responses against viral infections. A focus is placed on myeloid cell development, trafficking and antiviral mechanisms. Although known for promoting inflammation, there is a growing body of literature which demonstrates that myeloid cells can also play critical regulatory or immunosuppressive roles, especially following the elimination of viruses. Additionally, the ability of myeloid cells to control other innate and adaptive leukocytes during viral infections situates these cells as key, yet under-appreciated mediators of pathogenic inflammation that can sometimes trigger cytokine storms. The information presented here should assist researchers in integrating myeloid cell biology into the design of novel and more effective virus-targeted therapies. Upon viral infection, there are a number of myeloid cell subsets that play various roles in the subsequent inflammatory, cellular, and humoral responses. Myeloid cells are granulocytic and phagocytic leukocytes that traverse blood and solid tissues. When they recognize virus-infected cells or tissues damaged by viruses, these sentinels rapidly initiate an innate immune response . . 152]. UAlthough memory is traditionally considered a hallmark of the adaptive immune response, recent advances have shed light on the contributions of innate memory. Innate memory, also referred to as trained immunity, is a multifaceted response. A recent component of trained immunity involves its modulation of hematopoiesis . AlthougClearly, the cross-talk that is occurring between monocytes, neutrophils, and T-cells constitutes a crucial bridge between innate and adaptive immunity. Future investigations are encouraged to examine the full extent of communication between these cells, further elucidate the mechanisms, and the anatomical locations of these interactions. Depletion assays will be beneficial to determine which cell subsets can mount effective anti-viral responses, not just by T-cell and APC interactions, but also by direct interactions with neutrophils and monocytes.Extensive studies have highlighted the role type I IFNs play in initiating an anti-viral state in cells through the inhibition of viral replication . In someThe fatal outcome of severe influenza infections is shown to be correlated with the early persistent production of inflammatory cytokines and chemokines that recruit neutrophils and monocytes ,160. LetMX1) and ISG-15, are correlated with the heightened production and activation of type I IFNs after viral infections [Investigation into the mechanisms involved in host responses to viral infections demonstrates a complex and carefully balanced interaction between type I IFNs and inflammatory neutrophils and monocytes. Recent analysis of mRNAs in the blood of humans responding to infections with influenza viruses revealed that early gene expression patterns of anti-viral molecules, such as the genes encoding for myxovirus resistance protein-1 (fections . Late gefections , suggestIt is important to study the receptors mediating the neutrophil antiviral response to reduce aberrant host responses and damage. NLRP12 is a nucleotide-binding domain leucine-rich repeat protein that is expressed on blood-derived leukocytes, including monocytes, and modulates neutrophil recruitment by increasing the chemokine CXCL1 through the IL-17-NLRP12 axis and increasing vascular permeability . Another-/- x IRF-7-/- double-knockout mice and WNV [The early production of virus-induced type I IFNs has been observed to upregulate genes in neutrophils that encode pro-apoptotic molecules, such as IFN-induced dsRNA-activated protein kinase, and the oligoadenylate synthase-like proteins and the RNase L system . Experim and WNV  conclude and WNV . The del and WNV . In cont and WNV ,90. The  and WNV ,172, hig and WNV  demonstrhi monocytes to sites of viral infections, but also with the prevention of excessive monocyte and neutrophil activation, thereby controlling inflammation caused by type II IFNs, such as IFN-γ [-/-, Ifnar1-/-, Ifngr1-/-and Stat1-/- C57Bl/6 mice [lo monocytes transitioned to being more inflammatory than Ly6Chi monocytes. In the absence of type I IFN signaling, Ly6Clo monocytes traditionally associated with tissue re-modeling became phenotypically and functionally more pro-inflammatory during infection with influenza A viruses [The protective functions of type I IFNs have been associated not only with the recruitment of neutrophils and inflammatory Ly6Cas IFN-γ . The intl/6 mice . Both IF viruses . Notably viruses . Measure viruses , further viruses . DetermiNeutrophils, inflammatory monocytes, and their roles in mitigating bacterial infections have been extensively studied and well characterized. Exciting new research in immunology and virology has demonstrated that these first responders of the innate immune system are also crucial in limiting viral infections, replication, and associated off-target pathological damage. A multifaceted range of tactics is utilized to combat an equally diverse range of viruses, including phagocytosis, the formation of extracellular traps, the production of cytokines such as IFNs, and modulation of ILCs and lymphocytes.Despite rapid advances in the field, many exciting unknown aspects of the involvement of neutrophils and inflammatory monocytes in combating viral infections remain to be clarified. Current research has documented the impact of neutrophil/monocyte retention in the bone marrow as it pertains to viral infections, but we still do not completely understand all mechanisms by which myeloid cells are recruited from the blood stream to the primary sites of infection. Future studies should aim to elucidate the specific signaling cascades that recruit myeloid cells into infected tissues and the mechanistic consequences of disruptions in these cascades via the chemokine gradient as well as depletions of specific ligands. If the scientific community can determine how different cell subsets can influence the production of chemokine populations and hone in on the essential ligands required for migration into the primary sites of infection, drugs could potentially be developed to exploit this localized production of chemokines. The discovery of pharmaceuticals that could fine-tune myeloid cell trafficking could prove beneficial to inducing rapid antiviral responses. Differential ligation versus the blockade of PRRs associated with protective versus pathological inflammation constitutes another strategy to balance rapid viral clearance and minimize host damage. Current knowledge from myeloid cell studies in bacterial diseases demonstrated that neutrophils are essential for monocyte recruitment and function. Additionally, it has been shown that the ratio of neutrophils to lymphocytes is higher in bacterial than viral infections among patients hospitalized for fevers . It is cImportantly, viruses and virus-mediated tissue damage stimulate both neutrophils and monocytes, triggering a cascade of cytokine/chemokine-mediated innate immune responses. This antiviral activity is not always beneficial for a host and, when improperly regulated, may contribute to immunopathologies such as cytokine storms that have been observed in many severe viral infections and could be related to type I IFN signaling. Mechanisms, including the potential relationship between type I IFN signaling and the regulation of excessive cytokine responses, should be further examined to develop strategies to minimize detrimental tissue damage by neutrophils and monocytes, while maximizing their beneficial anti-viral features.


Mesoporous silica nanoparticles (MSN) have attracted a lot of attention during the past decade which is attributable to their versatile and high loading capacity, easy surface functionalization, excellent biocompatibility, and great physicochemical and thermal stability. In this review, we discuss the factors affecting the loading of protein into MSN and general strategies for targeted delivery and controlled release of proteins with MSN. Additionally, we also give an outlook for the remaining challenges in the clinical translation of protein-loaded MSNs. Recombinant human insulin (humulin), the first recombinant protein therapeutic was developed and launched by Herbert Boyer, and Eli Lilly opened the Pandora’s box in pharmaceutical proteins 30 years ago . Thus faGenerally speaking, all of these kinds of nanoparticles could achieve relatively improved protein therapeutic benefits in some degree, including: (a) protect protein from premature degradation, (b) prolong protein circulation time, (c) mask their epitope and subsequent immunogenicity, (d) realize their targeted delivery and controlled release, (e) enhance therapeutic efficacy, whereas reducing side effects. However, premature protein leakage, degradation, and limited release deriving from the soft property of liposomes, degradation of polymer matrixes, and chemical modification, respectively, are major challenges that these nanoparticles have to address when they serve as protein drug-delivery vehicles.Due to its ease of synthesis, facile surface multi-functionalization, preeminent biocompatibility as well as excellent physicochemical and thermal stability, mesoporous silica nanoparticles (MSN) have gained great attention as excellent delivery vehicles ,18,19,203/g) of MSNs. ii) The chemically and mechanically stable inorganic oxide framework of MSNs shelters the cargo protein from exposure to harmful species. iii) It has been previously proved that MSNs are capable of escaping endo-lysosomal entrapment due to the pH buffering properties . Th. Th95]. Enzymes play a very important role in physiological and pathological processes. The unusual expression or upregulated expression of an enzyme in the target site makes it elegant endogenous stimuli due to its high sensitivity and selectivity . Matrix N-isopropylacrylamide) (PEI/NIPAM) was decorated onto the surface of MSN and acted as a temperature-responsive gatekeeper for trapping small molecules in the pore matrix as well as a retainer for proteins buried in the polymer shell by electrostatic and hydrogen bonds interactions. When the nanodevices were placed in an oscillating or alternating magnetic field, the iron oxide nanocrystals would transduce magnetic energy to heat due to the hysteresis loss and/or Néel relaxation to promote polarity inversion and tridimensional polymeric network changes if the heat reaches LCST, subsequently provoking the pore opening and a significant molecules and protein release. Moreover, the magnetic nanocrystals would endow the nanodevices’ magnetically targeted ability to the desired place using a permanent magnet. In addition, it can also be employed as contrast agents for magnetic resonance imagining. Similarly, Khashab and co-workers also developed a silica-iron oxide hybrid nanovectors with large mesopores (20–60 nm) for protein (mTFP-Ferritin) loading and delivery to cancer cell [Heat is a very effective stimulus which has frequently been used as a trigger for protein release from MSN-based delivery system. To achieve that, such a complex of the MSN system usually includes MSNs, a thermal conversion agent, a drug, and a temperature-sensitive polymer which can swell above certain temperatures  and shrink below certain temperatures to facilitate drug release. Stroeve et al. used thermosensitive poly(n-isopropylacrylamide) (PNIPAM) as a gatekeeper to realize controlled bovine hemoglobin (BHb) release . Other tcer cell . The iroco-TENBMA) that could form a protein-polyelectrolyte complex (PPC) with bovine serum albumin (BSA) was anchored on the surface of MSN as a capping agent for the nanopores of the MSNs. The PPC is stable at physiology environment, but P(OEGMA-co-TENBMA) trend to hydrolyze to ionic P(OEGMA-co-MAA) upon ultraviolet (UV)-irradiation on photo-labile 2-nitrobenzyl ester moieties. This charge conversion would lead to the disruption of PPC and subsequent co-release of BSA and small molecule rhodamine B.Light is an attractive stimulus as a trigger for controlled drug release due to its non-invasive nature, desirable controllability, high spatial resolution and temporal precision with a pulsatile switch on/off model ,104. CarFor in vivo biomedical applications, it is crucial that nanoparticles can not only achieve their designed mission but also do not produce non-specific and deleterious changes to the body, indicating good biocompatibility. Although silica has been used as an excipient in the pharmaceutical industry for decades, and generally considered as of low toxicity, its in vivo toxic effects have been reported. Thus its toxicity to the body (including acute toxicity and chronic toxicity) should be carefully assessed when it was applied in the body. The exact mechanism for MSN toxicity is still uncertain, and some theories were proposed and evaluated, including the induction of membrane damage , generatParticle size is one of the most important parameters of MSN, which can have a great influence on MSN biocompatibility. Mou et al. investigated the influence of MSN size on its intracellular internalization by Hela cells . They foStructural properties of MSN could also affect its biocompatibility, and this effect is especially prominent when it compares to amorphous silica in its hemolytic property. RBCs would be lysed when amorphous silica is at a high concentration. The mechanism accounts for this hemolysis cytotoxicity is mainly based on the interaction between the numerous surface silanol groups of silica materials and the tetra-alkyl ammonium groups on the membrane of RBCs. Although compared with amorphous silica material, highly ordered MSN had more silanols due to a large surface area, but most silanol groups were on the internal surface, which could be attributed to the less negative ζ-potential of the highly ordered MSN (−35 mV) than the one of amorphous silica (−49 mV). The relatively low concentration of external silanols made MSN no-hemolytic on RBCs under determined concentration. Furthermore, the discontinuous external surface silanols due to mesoporosity would lower the hemolytic activity of MSN . SimilarThe surface property or surface functionalization is another crucial parameter apart from size, which not only has significant influence on cargoes’ delivery but is also involved in MSN vehicle biocompatibility. As mentioned above, abundant silanol groups exposed on a bared MSN surface provide facile rivet for surface modification. Different modifications could interact with cellular membrane lipids and proteins and disturb their structure and conformation. Positive charge reagents or polymers are commonly used in protein delivery MSN systems. It is well known that particles with cationic surface charge would induce intense immune response, cytotoxicity, and short circulation times compared with anionic and neutral ones. However, positive surface charge is of obvious superiority for transvascular transport and intracellular transport due to the generally negative potential of the cell membrane . Thus, hIn this review, we mainly focus on research advances on MSN-based protein delivery. It is clear that MSN materials are promising candidate carriers for protein delivery in vitro and in vivo due to their specific structure and physicochemical property. First, their versatile and adjustable pore size and porosity make MSNs suitable for loading proteins cargos. Second, the simple surface functionality broadens their protein delivery ability and possibility, such as different kinds of proteins, co-delivery of proteins and other drugs, targeted protein transport, and various stimuli-responsive protein release methods. Third, their great biocompatibility bestows promising potential for future biomedical applications.Besides the aforementioned advantages, there remain some significant challenges that should be investigated to enable the development of practical applications for MSN-based protein delivery systems. Apart from in vitro protein release profiles’ investigation, much more attention should be paid to track in vivo navigation of proteins, especially their release in target tissues by using integrated multifunctional or multimode imaging methods. Although there has been some research into the development of such multifunctional MSNs, novel-innovative methods for functionalization remain limited. Protein structure is the base of its function. In view of the frangibility of proteins and complexity in a physiological and pathological conditions, there should be caution characterizing their structures, as well as the biofunction after their release from MSN in the targeted site. Toxicity is the major obstacle for the translation of nanomaterials from preclinical research to clinical application. Although most recent reports showed that MSN materials have little cytotoxicity, and are of great biocompatibility in vivo, much more work still needs to be performed to demonstrate comprehensively the biological safety of MSNs, such as the ultimate fates of MSNs after in vivo application.


Transcranial direct current stimulation (tDCS) is a modality of non-invasive brain stimulation involving the application of low amplitude direct current via surface electrodes on the scalp. tDCS has been studied in healthy populations and in multiple brain disorders and has the potential to be a treatment for several neuropsychiatric conditions by virtue of its capability of influencing cognitive, motor and behavioral processes. tDCS is a generally safe technique when performed within standardized protocols in research or clinical settings. Furthermore, tDCS portability, high acceptability and user-friendly interface makes it highly appealing for telemedicine practices. The term “telemedicine” refers to the procedures, educational strategies, and care services that are remotely administered by means of different communication technologies, with the final goal of increasing access to care for individuals and for improving public health. The use of telemedicine combined with tDCS protocols is increasing, although the safety of this approach in different clinical settings awaits further assessment. While “do-it-yourself” tDCS should be discouraged due to the unknown risk of adverse events, the implementation of tele-monitored tDCS (tele-tDCS) within standardized frameworks ensuring safety, tolerability, and reproducibility may allow this technology to reach larger clinical populations and bypass some of the common barriers preventing access to health services and clinical trials. This review will discuss the current evidence supporting the feasibility of tele-tDCS paradigms and their therapeutic potential, with particular emphasis on the implications for patients with Parkinson’s disease. The past decade of this century has been marked by the steady development of new technologies that are now able to connect broad sectors of populations to medical providers and health care institutions. The term “telemedicine”  encompasses a broad range of telecommunication technologies that allow for health services to be delivered remotely . These sThere are further potential advantages related to the implementation of telemedicine protocols. These include the possibility of providing real-time monitoring of adverse events, facilitating therapeutic compliance, reducing social disparities in the access to care, promoting patients’ active role in diagnostic and therapeutic processes, and reducing waiting time and economic burden, particularly for people affected by chronic illnesses , 5. EvenOn the other hand, the rapid development of telemedicine poses radical new challenges. The intensive exchange of medical information through different platforms carries potential risks related to loss of confidentiality on large volumes of sensitive data. These risks must be minimized by implementing appropriate data protection strategies . FurtherNevertheless, in the past years, the availability of technology-enabled communications has grown dramatically and their cost/effectiveness has improved accordingly. As a result, the remote administration of diagnostic and therapeutic procedures has become a widely consolidated clinical practice in different medical fields, including urgent care, robotic surgery, imaging and physical rehabilitation –12. ReceTranscranial direct current stimulation (tDCS) is an investigational device broadly used in experimental and clinical neuroscience with a wide range of potential therapeutic applications. To date, tDCS has been applied for the study and treatment of several conditions by virtue of its capability of influencing cognitive, motor and behavioral processes related to the pathophysiology of different brain disorders . One of The aim of this narrative review is to present the reader with the latest evidence supporting the feasibility of tele-tDCS paradigms and the potential near future applications of this technique for both experimental and clinical purposes. Finally, the specific challenges and theoretical implications for patients affected by PD will be critically discussed.tDCS is a modality of NIBS involving the application of a low amplitude direct current (DC) via surface electrodes on the scalp for a predetermined time in a relatively safe manner. In tDCS, the cerebral cortex is stimulated through a continuous, weak current  which alters brain function by changing the neuronal resting membrane potential to either cause depolarization (under the anode) or hyperpolarization (under the cathode) . The priGrowing evidence accumulated through randomized controlled trials (RCT) supports the potential of tDCS for the treatment of various disorders, such as chronic pain, fatigue, cognitive abnormalities, substance-related disorders, and depression . Many ofA major limitation to the extensive clinical application of NIBS protocols has been that most research studies have involved small sample sizes and short duration, resulting in trials without proper power analysis and predefined intention to treat designs. The large majority of available trials has indeed involved a limited number of sessions and participants as both clinicians and investigators face challenges in the recruitment and retention of participants in large-scale NIBS trials –30. FromOne prerequisite to tele-tDCS is the availability of equipment specifically designed to allow for remote use and a customized headset for easy and reliable placement of the electrodes. Simply utilizing the same devices designed to be operated by health professionals may compromise subject safety and affect experimental reproducibility since inter-individual differences in technological skills and environments may not be adequately addressed . CustomiIn addition to dedicated devices, tele-tDCS should employ an adequate level of control by specifically trained research staff or expert clinical professionals. On this topic, “do it yourself” (DIY) tDCS practices should be highly discouraged in light of the inherent risks involving subject’s safety and experimental reproducibility . Visual The biological effects of tDCS, as any other NIBS technique, are essentially determined by two factors: extrinsic (related to the intervention) and intrinsic (related to the stimulated subject) . ExtrinsIn general, the best way to ensure consistent and reliable electrode placement is to implement video-supervised sessions during which the user, with or without caregivers, are followed in real time by study personnel until electrodes are properly placed and adequate contact is confirmed. Indeed, tele-tDCS protocols have consistently showed optimal adherence, high tolerability and uniformity across sessions, while self-administered tDCS without any degree of supervision has been associated with a high dropout rate , 38.As far as the device-controlled voltage waveform is involved, the so-called “hardware approach” involves the administration of a pre-determined voltage by programmed devices operating for a limited number of sessions. After completion of the last session, the device no longer provides any output. Alternatively, in the so-called “software approach”, each tDCS session is enabled with a pre-set duration and intensity by a specific code provided to the user and/or caregiver by the research staff. After a single session, the device remains inactive until a new code is provided to unlock the next session .remotely-supervised tDCS” (RS-tDCS) in order to ensure the same level of uniformity, compliance and reproducibility that would be expected by tDCS sessions administered in the clinic [In 2015, Charvet et al. proposed a standardized framework for trials utilizing a tele-tDCS protocol defined as “e clinic . This pre clinic .Fig. 2AlShaw et al. analyzed the feasibility and tolerability of a remotely supervised protocol involving anodal tDCS over left dorsolateral prefrontal cortex (DLPFC) paired with computer-based cognitive training . The autWhile protocols involving the remote use of tDCS should be structured enough to ensure reproducibility and safety, certain population-specific considerations should also be made when applying tele-tDCS to patients with different clinical profiles. Charvet et al. provided some examples of these population-specific adaptations, where peculiar issues encountered while delivering RS-tDCS in people affected by attention-deficit/hyperactivity disorder, depression, multiple sclerosis, and severe chronic illnesses requiring palliative care were critically discussed . It is lThe available evidence supporting the telemedicine-based use of tDCS remains limited to small exploratory trials showing significant heterogeneity in terms of patient populations, primary outcome, and experimental design. In 2018, Palm et al. conducted a systematic review including available protocols and original research involving the home use of tele-tDCS for the treatment of various neuropsychiatric disorders . From fidomiciliary use” of tDCS for compassionate or interventional purposes, in which stimulation parameters are advised by the medical staff but remain entirely dependent on patient’s compliance, vs a “remotely supervised use”, in which the patient or the caregiver activates a preprogrammed device secured against manipulation. In remotely supervised protocols, supervision is offered by means of different technological platforms  in order to overcome potential technical difficulties and ensure optimal adherence. A third identified setting is the “remotely controlled tDCS”, in which the device, while still operated by patients or caregivers, remains under constant remote control of the medical staff. Regardless of its practical implications, this nomenclature reflects the need to refine the terminology used in the various trials to foster a greater experimental uniformity and facilitate the systematic analysis of the available data. Additionally, while the evidence supporting the safety and feasibility of tele-tDCS is gradually emerging, the efficacy of this new modality of neuromodulation remains to be validated though well-powered, well designed studies.Furthermore, Palm et al. proposed a new nomenclature to highlight the methodological differences between different uses of tele-tDCS. More specifically, the authors proposed a distinction between a “Neurodegenerative diseases, particularly movement disorders, are known to be characterized by chronic, progressive disability and reduced mobility. These conditions are frequently compounded by a number of psychosocial factors that can further reduce both accessibility and adherence to medical care. Furthermore, in certain geographical and socioeconomic backgrounds, the effective care of movement disorders is hindered by the scarcity of specifically trained health care professionals as well as by limited access to dedicated facilities .PD is the second most common neurodegenerative disorder after Alzheimer’s Dementia. Given the current demographic trend, the global burden of PD will increase rapidly, particularly in those developing economies where access to care is more limited . It was We reviewed the state of research on tele-tDCS applied to patients affected by PD. The research included the following databases: Pubmed, Google Scholar, Scopus, Semantic Scholar, Clinical Trials.gov and Research Gate. The terms “remotely-supervised”, “at-home”, “domiciliary”, “tele-monitored”, “telehealth” and “telemedicine” were searched in cross-combination with “Parkinson’s Disease” and “transcranial direct current stimulation”. We found three studies regarding the use of telemedicine-based tDCS in this specific population.Shaw and colleagues published the first feasibility and safety trial with a paradigm of remotely supervised tDCS (RS-tDCS) that included six PD patients in 2017. The study reported feasibility and safety of RS-tDCS over 10 daily sessions paired with computer-based cognitive training in 52 patients. Of the 60 RS-tDCS sessions in PD participants, side effects did not differ from commonly reported side effects of conventional tDCS .The authors of the present review have recently completed an exploratory, open label study of remotely supervised anodal tDCS over the left DLPFC paired with cognitive training in PD participants . Based ohttps://clinicaltrials.gov/show/NCT03189472). The protocol involves 10 daily sessions of bi-frontal 20-min RS-tDCS  followed by an optional open label phase consisting of 10 additional sessions. Seventeen participants completed 330 tele-tDCS sessions (170 double blind and 160 open label). Preliminary feasibility and safety results showed no serious adverse events, only mild to moderate side effects and 100% compliance. All participants but one opted to undergo the open label phase [After the successful completion of the first open label exploratory study, our group is conducting an ongoing double-blind, pilot, RCT testing the effects of RS-tDCS using a DLPFC montage to ameliorate fatigue and cognitive slowing in PD , the support of a caregiver, spouse or significant other might be important for tele-tDCS paradigms. Increased caregiver’s burden is a well-documented occurrence in PD . This faFluctuating motor performance, severe and/or unpredictable OFF states, severe freezing and bothersome dyskinesia or tremor could also be present temporarily in a given patient. Special precautions (e.g. providing video-conferencing platforms to detect these states) and schedule flexibility to conduct sessions might improve compliance, safety and tolerability in these particular cases.Finally, from an experimental viewpoint, patient’s pharmacological state while receiving tDCS neuromodulation can affect the brain activation state and connectivity by modulating neuronal propensity to fire and undergo plastic phenomena. In patients with PD, this is particularly noteworthy, as changes in cortical excitability and neuroplasticity are critically influenced by dopamine bioavailability, and the institution of a dopaminergic therapy can influence the subsequent neurophysiologic and behavioral effects of stimulation . The potThe broad application of non-invasive brain stimulation techniques, including tDCS, is currently limited by different factors. First, the body of the available evidence still rests on small-sized studies carried out with exploratory designs. As such, these studies are known to be particularly prone to the risk of type I and type II statistical errors. A second order of limitation is posed by the high heterogeneity of stimulation parameters and methods between the published trials. These differences result in a limited comparability between the various experimental protocols. Finally, a third important constrain is the paucity of studies with multiple tDCS sessions (i.e. beyond 10 sessions) as the current evidence suggests higher chances of harnessing cumulative biological effects following multiple sessions of stimulation over time. The development of tele-tDCS paradigms may specifically address some of these challenges improving protocol standardization and adherence while minimizing attrition. This may be particularly noteworthy in patients burdened by chronic motor disability or living in remote geographic areas. In the near future, the broad availability of different communication technologies may favor the implementation of new personalized models of care in neurology and rehabilitation. In this setting, the therapeutic value and overall safety of tele-tDCS remain to be determined through appropriately designed trials. The current limited evidence suggests high acceptance rate and overall optimal feasibility but further studies are needed to corroborate these preliminary findings.tDCS is a relatively safe and tolerable non-invasive neuro-modulation technique that could be incorporated into telemedicine protocols in light of its portability and easy operability. The use of tele-tDCS within standardized frameworks ensuring safety, tolerability, and reproducibility in adequately selected patients may expand access to care and allow for the inclusion of larger populations into clinical trials while minimizing attrition and improving cost/effectiveness. Most patients affected by PD worldwide face multiple barriers preventing a consistent access to specialized, effective, and interdisciplinary care. Current evidence supports feasibility and safety of tele-tDCS protocols in the setting of remotely supervised videoconference sessions delivered to patients affected by PD. In these patients, specific challenges to the extensive implementation of tele-tDCS include the possibility of motor and non-motor fluctuations, cognitive deficits, polypharmacy, psychiatric comorbidities and risk of caregiver’s burden. Although the safety and feasibility of these protocols in PD patients await further validation, preliminary data seem to suggest optimal adherence and acceptability in this particular patient population opening the field for larger research initiatives needed to define the therapeutic potential of this intervention.


Cellular senescence is a state of stable growth arrest in response to stress, which is a fundamental process of biological aging. They secrete products, the Senescence-Associated Secretory Phenotype (SASP), which consists of inflammatory cytokines, chemokines, growth factors and matrix remodeling proteins. Senescent cells accumulate with advancing age and partial elimination of senescent cells can reverse age-related dysfunction and increase mean lifespan in mice. However, it is not clear whether components of the SASP can be measured in human plasma and serve as aging biomarkers. Here we generated a candidate panel of senescence markers based on a multiplexed bead-based assay of proteins secreted by senescent preadipocytes, endothelial cells, fibroblasts, myoblasts, preadipocytes, and epithelial cells compared to non-senescent controls. The SASP undoubtedly varies by cell type; however, we observed that multiple components of the SASP are conserved. We then assessed circulating SASP components in human plasma samples from Mayo Clinic Biobank participants  using the same method. We confirmed that components of the SASP can be quantified in human plasma with the multiplexed bead-based assay and observed several SASP components robustly increase with chronological age in humans. Our study illustrates that senescence-associated secretome components are detectable in human plasma and could potentially serve as biomarkers of systemic aging and senescent cell burden.


Plant-based beverages and milk alternatives produced from cereals and legumes have grown in popularity in recent years due to a range of consumer concerns over dairy products. These plant-based products can often have undesirable physiochemical properties related to flavour, texture, and nutrient availability and/or deficiencies. Lactic acid bacteria (LAB) fermentation offers potential remediation for many of these issues, and allows consumers to retain their perception of the resultant products as natural and additive-free. Using next-generation sequencing (NGS) or omics approaches to characterize LAB isolates to find those that will improve properties of plant-based beverages is the most direct way to product improvement. Although NGS/omics approaches have been extensively used for selection of LAB for use in the dairy industry, a comparable effort has not occurred for selecting LAB for fermenting plant raw substrates, save those used in producing wine and certain types of beer. Here we review the few and recent applications of NGS/omics to profile and improve LAB fermentation of various plant-based substrates for beverage production. We also identify specific issues in the production of various LAB fermented plant-based beverages that such NGS/omics applications have the power to resolve. Wide-ranging literature review of these attributes and capacities of LAB in various food and beverage fermentations are performed with reasonable frequency, however, available data is skewed in two respects. First, the bulk of available data is physiological in nature for specific LAB strains, with many singular studies investigating metabolic characteristics of LAB fermentations under narrowly specific conditions. This results in limited understanding of the true microflora and metabolic capacities of LAB associated with these fermentations and stunted ability to identify appropriate starter strains and/or modifications to the fermentation environment in pursuit of improved product. Second, of all fermentations that LAB participate in, dairy fermentations are the most thoroughly studied, due in part to the long involvement of LAB in commercial production of liquid and solid milk products. Consequently, the dairy industry has been subject to the widest application of next-generation sequencing (NGS)/omics to the LAB involved , yieldinnatural status.Though LAB-fermented dairy products are well studied, milk alternatives produced from raw plant substrates are growing in popularity as increasing numbers of consumers now avoid dairy products for medical or ethical reasons In considering LAB in this context, two things must be remembered. First, production of beverages from grains and pulse crops starts with extracting constituents from milled raw material Interestingly, although LAB-fermented, dairy-alternative beverages are starting to have an increased omics-research focus, it is LAB involved in plant-based alcoholic beverages such as beer and wine that to date have received the most, albeit still limited, NGS/omics attention. Here we review the specific challenges related to LAB fermentation in production of different types of plant-based beverages - initially beer and wine, and then legumes, cereals, and fruit/vegetables. Additionally, where available, we present recent evidence for using NGS/omics approaches for improving the quality of the products involving LAB fermentation and indicate important future directions for such research.2.Lactobacillus brevis, Lactobacillus lindneri, and Pediococcus damnosus being the most commonly encountered bacteria that spoil beer open fermentation) and/or requires purposeful addition of LAB to sour the beer by lowering the pH through acid production The fermentation of barley, rye, or wheat grain to produce beer is arguably one of the oldest biotechnological processes carried out by humans gtf gene Given that LAB have long been viewed as beer-spoilage organisms, extensive literature is available that characterizes the spoilage characteristics of the bacteria involved. This includes detailed description of LAB-induced alterations of the sensorial profile of beer, such as off-flavour formation , unwanted acidification, and haze and sediment formation Lactobacillus backii isolates L. brevis BSO 464 Lactobacillus malefermentans KCTC 3548 P. damnosus isolates Pediococcus claussenii ATCC BAA-344TL. brevis BSO 464 (capable of growing in packaged beer with dissolved CO2 content/pressure) P. claussenii ATCC BAA-344T  L. brevis strains from non-spoiling isolates fabZ operon related to fatty acid synthesis can discriminate beer-spoilage P. damnosus strains Due to the significance of LAB as spoilage agents of beer, there has been a recent increase in sequenced brewing-related LAB genomes that are publicly available, including the genomes of five Thus far, only one metagenomic sequencing study has been done in the brewery setting; i.e., on a mixed-culture-fermented beer In an ironic twist, this recent genomics, transcriptomics, and metagenomics data has promise for application in efficiently selecting appropriate starter cultures for the craft fermentations leading to sour beers. As craft beers are emerging popular beverages, their commercial success represents an intriguing opportunity to profile LAB diversity, LAB and yeast co-fermentation, and LAB succession in beer. Further, given the general popularity of beer, opportunities exist to utilize NGS/omics approaches and available NGS/omics data to also improve the functional health  properties of beer 3.Oenococcus oeni is considered critical for quality wine production, other LAB in the genera Lactobacillus and Pediococcus can be detected throughout MLF in varying succession patterns O. oeni contribution to the taste of wine has been genomically characterized O. oeni PSU-1 genome in 2005 O. oeni isolate. Additionally, the possible stress response of O. oeni PSU-1 during growth was determined. Currently, over seventy O. oeni genomes and/or genomic projects are publicly available through NCBI . At this point, investigation has evolved beyond the genomic study of single O. oeni isolates to include the production of a partial proteome reference map for O. oeni ATCC BAA-1163 O. oeni will begin to be revealed only when coupled with transcriptomics analysis of the bacterium growing under different conditions.Although malolactic fermentation (MLF) by Metagenomic studies have also increased in frequency within the wine industry subsequent to 16S amplicon sequencing being used to determine bacterial diversity in botrytized wine Lactobacillus fabifermentans as a key organism in the breaking down of residual complex carbohydrates into fermentable sugars during storage-fermentation for the potential production of distilled spirit beverages Influence of the soil microbiome on grapevine-associated microbiota has also been recently investigated Oenococcus, Lactobacillus, Leuconostoc and Pediococcus all can contribute to the histamine synthesis occurring during wine production In addition to identified important carbohydrate, nitrogen, and enzymatic activity, which all contribute important flavour compounds to wine, undesirable metabolic activities of enological LAB have also been identified. For instance, 2 content, and limited nutrients in wine O. oeni and Pediococcus parvulus finding that some O. oeni strains have measurable immunomodulatory potential, though at a level below conventional probiotics Lactobacillus fermentum CECT5716, Lactobacillus plantarum CLC 17, and Pediococcus pentosaceus CIAL-86A Overall, genomic data has clearly aided in the understanding the complex microbial process of wine production. This data, however, has yet to be tapped to streamline and improve the selection of LAB strains with probiotic potential. As with brewing-related LAB, enological LAB must contend with harsh environmental selection pressures such as high sugar content and low pH in grape must, and high ethanol, acidity, SO4.mleA gene mleA.Legumes, particularly soy, offer a popular alternative to traditional dairy products such as milk, yogurt, and cheese; thus, it is not surprising that multiple studies have been conducted analyzing the growth and metabolic behavior of LAB in soy milk In a similar vein, it is known that the proteolytic specificities of LAB starters for cereal and legume proteins are quite diverse and differ from specificities towards milk proteins 2) biosynthesis pathway of L. fermentum has been applied to fortify B2 content in soy milk Lactobacillus reuteri strain for which a genome sequence was recently published Bifidobacterium strains improve specific health-related properties of soy, including increasing the antioxidant character Interest in soy beverage production has led to development of strategies to improve the health or nutritional value of product and, in this regard, genetic analysis of specific LAB strains is beginning to demonstrate benefits. For instance, the genetic characterization of riboflavin  designer starter culture Development of defined LAB starter cultures is of key interest for cereal-based fermentation as it allows for improved product quality, as noted for L. brevis too early in the fermentation xaj-pitha  has assisted the development of a suitable fermentation starter culture for this beverage L. plantarum isolate has been monitored during fermentation of pearl millet slurry Recent successful applications in this regard include the metagenomic characterization of the yeast-LAB population for improved production of Chinese rice wine, with the study also demonstrating that product spoilage resulted from the rapid growth of L. plantarum Lp90 Weisella cibaria MG1 in production of barley- and soybean-based products to improve both mouth-feel and structural stability While metagenomic analysis gains traction within the field of cereal-based beverages, an emerging area of interest is application of this technology to profile the capacity of LAB to produce exopolysaccharides which can be used to improve product texture and stability, as has been studied extensively at the genetic level for sourdough 6.functional beverages. Importantly, raw fruit and vegetable substrates contain beneficial nutrients such as minerals, vitamins, dietary fibers, and antioxidants, and do not contain dairy allergens Fermented fruit and vegetable juices have favourability and popularity with consumers of all ages and thus make ideal Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus paracaseiL. plantarum; tomato juice Lactobacillus acidophilus, L. casei, L. delbrueckii, and L. plantarum; red beets with L. acidophilus and L. plantarumL. delbrueckii and L. plantarumL. plantarum in carrot and pineapple juices was studied via micro- and macro-array platforms to understand physiological processes involved in adaptation to the specific juice environment Leuconostoc mesenteroides strain isolated from the Mexican fermented beverage Pulque Lactobacillus farcimis, the key organism in the Japanese fermented health beverage kôso, has recently been released, underpinning future studies of this bacterium's probiotic and interesting proteolytic capabilities kôso fermentation As with cereal-based beverages, fruit and vegetable juices substrates are heterogeneous and diverse, with properties differing across geographic location and fermentation/storage conditions; nonetheless, the general microbial profile of various fruit and vegetable juices have been characterized and reviewed L. fermentum, L. plantarum, and spoilage fungi in tomato fruit While metagenomics has begun to reveal specific characteristics of LAB fermentation, it was noted previously that the microflora of juice (and other products) establish in the following sequence as fermentation progresses and growth inhibitory qualities increase in strength: non-fermentative psychrotrophic Gram-negative bacteria → fermentative Gram-negative bacteria → LAB → yeasts → filamentous fungi 7.designer LAB starter cultures for such fermentations can only be done with detailed knowledge of attributes individual LAB possess. In this context, the identification and basic characterization of the native microbial flora involved in spontaneously fermented foods and then determining their individual isolate metabolic capacity through traditional microbiology research is a useful starting point. Available nucleotide sequencing technologies then have the power to rapidly reveal important genetic detail for the LAB involved in these fermentations; to date, however, there has been limited output of such research. Nonetheless, in the few instances where NGS/omics have been applied, the data obtained clearly reveal the limitations of traditional metabolic and physiological studies to fully describe complexity of LAB metabolic processes taking place during the fermentations used to produce plant-based beverages. This is clearly exemplified within the fields of brewing and oenology, wherein application of genomic and transcriptomic techniques has demonstrated benefit for increasing the ability to not only rapidly screen for spoilage LAB organisms, but importantly to also provide genetic information that can be used for product innovation through LAB fermentation. NGS/omics data obtained for brewing and enological LAB thus has the potential to not only assist in increasing the functional attributes of beer and wine, but also the expanding assortment of plant-based beverages in general. Through increased application of currently available genomics, transcriptomics, and metagenomics technologies to the LAB used in the production of diverse plant-based beverages, better product outcomes for LAB fermentation can be achieved, regardless of the raw plant substrate involved.Plant-based beverages produced via LAB fermentation can consist of a nearly infinite number of compositions and associated issues. This means the LAB involved must possess specific capacities to confer desirable (improved) properties to these beverages. Selecting appropriate LAB and/or developing


The question of whether anesthetic, analgesic or other perioperative intervention during cancer resection surgery might influence long-term oncologic outcomes has generated much attention over the past 13 years. A wealth of experimental and observational clinical data have been published, but the results of prospective, randomized clinical trials are awaited. The European Union supports a pan-European network of researchers, clinicians and industry partners engaged in this question (COST Action 15204: Euro-Periscope). In this narrative review, members of the Euro-Periscope network briefly summarize the current state of evidence pertaining to the potential effects of the most commonly deployed anesthetic and analgesic techniques and other non-surgical interventions during cancer resection surgery on tumor recurrence or metastasis. Surgery (under anesthesia) is an important part of modern health care, helping millions of people lead healthier, more productive lives . More thPreclinical and clinical studies support the hypothesis that anesthesia influences cancer biology and outcomes. Although there is plenty of retrospective published data and some prospective randomized studies available and ongoing, these are not sufficiently conclusive to suggest changes in clinical practice. The strongest support for a role of anesthetics on cancer biology comes from preclinical studies. However, the literature is sparse, often conflicting, and preclinical studies cannot fully reproduce the complexity of human physiology during the perioperative period. A previous consensus statement was published in 2014 but has not been updated [Globally, research into the impact of anesthesia on cancer during the last twenty years has focused either at the pre-clinical or clinical level. This disconnected approach has led to inconsistent selection of endpoints and a limited perspective in a poorly understood field. A similar problem has been highlighted in perioperative pain research . RecentlInevitably, in some cases, the quality of biological studies could be improved by ensuring an adequate number of experiments to provide more robust statistical analysis. Likewise, for translational studies ideally testing a single primary hypothesis, the sample size should be calculated according to the true variance of the data and the minimum biological effect.p-values. Therefore, it is recommended that sample size should be calculated and data analysis performed according to the tolerable False Discovery Rate  and False Negative Rate  [Transcriptomic and genomic studies testing multiple biological hypotheses cannot be interpreted only from itivity) . As FDR itivity) .Ideally, translational projects should combine omics approaches in conjunction with epidemiological confirmation of the disease as well as clinical trials, when appropriate. Preferably, this should be done in combination with analyses of clinically relevant endpoints and biologically promising biomarkers. This may permit identification of specific mechanisms that could be addressed, such as perioperative angiogenesis and neuroimmune interactions. Last but not least, these interventions may take place in multi-arm/platform trials, particularly in complex perioperative settings.Catecholamines, prostaglandins, and angiogenic factors are secreted abundantly during the perioperative period in response to stress and surgery and were shown by translational studies as possible promoters of tumor metastasis. Circulating tumor cells are seen in all cancers and transient systemic inflammation accompanying surgery can enhance capillary leakage facilitating angiogenesis of dormant micrometastasis, the proliferation of dormant cells and seeding of circulating cancer stem cells resulting in early relapse.Epidemiologic studies suggest that the choice of anesthetics may affect recurrence in breast cancer through changes in angiogenesis. Studies in a xenograft model showed that antiangiogenic therapy in the perioperative or direct postoperative period following primary breast tumor resection reduces progression of bone metastasis .Perioperative administration of the NSAID ketorolac  has been associated with a significant reduction in early recurrence following breast cancer surgery . NSAID mComparing general anesthesia with sevoflurane-opioids versus propofol-paravertebral block showed that the latter inhibits cancer cell proliferation, reduces the stress response, as well as metalloproteinases and transforming growth factor β . OpioidsHowever, using analgesic doses of morphine and other opioids in preclinical studies in mouse models for HER2+ breast cancer found no adverse effect on tumor metastatic dissemination, angiogenesis and the concentration of tumor-infiltrating immune cells after surgery, suggesting that opioid analgesics can be used safely .In summary, there are no conclusive studies relating to angiogenesis in breast cancer patients that should change our clinical practice. The nervous system is a primary actor in early and late cancer development. Many cancers e.g., colon and prostate present a specific growth pattern called “perineural invasion” which is driven by neurons. The growth of some cancers like prostate, pancreas, and breast cancer, is associated with “neurogenesis” and an abundant infiltration of the tumor by the autonomic nervous system. The stress response, results from stimulation of the sympathetic system, which enhances tumor growth through immunosuppression and beta-adrenergic receptor stimulation, expressed on tumor cells . In animSurgery evokes an acute-phase response, described above as the stress response that includes a transient immunosuppression and alterations in gastrointestinal function and there is clear evidence that sarcopenia, malnutrition, arginine bioavailability, and a high arginase activity state contributes to adverse surgical outcomes. Approaches to improve this will be discussed later. In short, approaches include pharmaconutrition, aimed at improving immune function (termed immunonutrition) and both parenteral and enteral approaches including amino acids (arginine and glutamine), lipids (omega-3 fatty acids), micronutrients (vitamins C and E), and nucleotides . The benAs the scientific investigation of the potential effects of perioperative agents on cancer evolves from retrospective analysis of human trials to potential prospective work, animal investigations can play an important role in refining potential therapeutic strategies for such studies. Animal models of tumors have numerous advantages and drawbacks when compared with human research. Animal models frequently have better control of variables and deliver faster results than human studies. There is, however, no perfect non-human cancer model; they may lack many of the biological steps of tumor development and metastasis as well as not necessarily reflecting the complex immune system and tumor interplay seen in human disease. Although there have been limited animal studies to date, lidocaine infusion has been implicated in having a beneficial role in the perioperative setting and as a chemotherapeutic agent in the non-operative setting . PropofoMetastases account for the great majority of cancer-associated deaths, yet this complex process remains the least understood aspect of cancer biology. The dissemination of cancer cells from primary tumors and subsequent seeding of new tumor colonies in distant tissues involves a multistep process known as the invasion-metastasis cascade, in which the Src system seems to play an important role . LidocaiAmong several perioperative factors, some anesthetic agents may promote tumorigenesis and metastatic recurrence. Indeed, it has been shown that isoflurane can increase hypoxia inducible factor (HIF) , promotiRetrospective clinical studies evaluated postoperative outcome after total intravenous anesthesia (TIVA) versus inhalation anesthesia in cancer patients. In their study including approximately 5,200 patients undergoing surgery for different types of cancer, Wigmore et al. found that inhalational (volatile) anesthesia was associated with a hazard ratio of 1.46 after multivariate analysis when compared with TIVA . RecentlSeveral prospective clinical studies comparing the outcome after TIVA versus inhalation anesthesia for cancer surgery are currently under development or in progress. The effects of regional analgesia/anesthesia and of local anesthetics are also being assessed in some RCTs. Most prospective RCTs compare TIVA ± regional anesthesia with inhalation anesthesia for breast and colon cancer surgery . Other types of cancer are also being studied e.g., pancreatic cancer (NCT2335151) . There aFinally, we will consider the possible role of opioids, in negative outcomes for patients undergoing cancer surgery.Most experimental research, consisting of both cancer cell culture studies and in vivo animal models of cancer, suggest that opioids facilitate cancer cell proliferation  and therefore could be detrimental in cancer patients ,60,61. HThere are several observational studies with conflicting results, the majority favoring an association between opioid use during cancer surgery and increased risk of recurrence . Also noOpioid-Free Anesthesia (OFA) based on the pre-, intra-, and postoperative administration of analgesics known to prolong disease-free survival e.g., NSAIDs and LA might be a promising therapeutic strategy for surgical cancer patients. OFA strategies for this group of patients may include the use of propofol as a hypnotic, analgesia based on neuroaxial or locoregional techniques to reduce/avoid the use of opioids, intravenous infusion of lidocaine from anesthesia induction to the immediate postoperative period, use of beta-blockers and pre- and postoperative administration of NSAID . The effThe role of epidural analgesia (EDA) in improving long-term outcomes following colorectal cancer surgery has been discussed . One stuRetrospective data on patients operated for rectal cancer surgery points to favorable outcome in patients having epidural analgesia. No prospective study with an adequate number of patients has yet been completed but important findings are awaited. Another promising adjunct to analgesia is the NSAIDs, especially in the context of inflammation and COX-2 dependent mechanisms. The story of the NSAIDs in this context began with the identification of a bimodal pattern of hazard of relapse among early stage breast cancer patients, in multiple databases. Using computer simulation and access to a high-quality database from Milan for patients treated with mastectomy only, it was proposed that relapses within 3 years of surgery are stimulated somehow by the surgical procedure. Retrospective breast cancer data suggested a plausible mechanism. Use of ketorolac, a common NSAID analgesic, was associated with superior disease-free survival ,70. The The findings have recently been confirmed in a mouse-model by Krall et al.  and in aHowever, the Ketorolac in Breast Cancer trial (NCT01806259), a multicenter, placebo-controlled, randomized phase III trial in high-risk breast cancer patients, has recently shown that a single dose of ketorolac tromethamine versus placebo 30 min before surgery, does not increase disease-free survival in high risk breast cancer patients ,74. OverPerioperative interventions that may have a positive effect on the outcome also include other factors than anesthetic/analgesic techniques. Proper, patient-tailored nutrition plays a major role in all kinds of cancer surgery. Its beneficial effect on short- and long-term outcomes is multifactorial. Not only does it help the patient to recover faster from surgical trauma, but also decreases the number and severity of postoperative complications . Non-comWell-planned preoperative nutrition also helps to prepare a malnourished patient for surgery and oncological treatment. Whenever possible, enteral or oral route is preferred as it helps to maintain gastrointestinal physiology and mucosal integrity. For patients in whom enteral route is not adequate, supplemental parenteral nutrition may be used. Patients without mechanical obstruction of gastrointestinal tract should be prescribed oral nutritional supplements (ONS) as standard part of their preoperative nutritional preparation. Standard ONS use has been shown to be effective in both malnourished and non-malnourished, weight losing patients . The Enhanced Recovery After Surgery (ERAS) paradigm has been proven safe and beneficial in most surgical fields, including surgical oncology. The paradigm should be applied in all surgical cancer patients, with early oral feeding where applicable and fluid restriction therapy ,80.Briefly, a modern, patient-tailored nutritional approach aimed at reducing nutritional deficiencies and minimization of the fasting period should be a part of oncological treatment plan. The nutritional status of the patient significantly influences the outcome of the treatment, whether it is obesity or malnutrition with loss of muscle mass . InadequSome experiences show that it is possible to evaluate the patients multiple times by a nurse dealing with nutrition in consultation with the anesthesiologist. For example, the first appointment may be directly after the surgery counselling when the surgeon sets the date of surgery , on the day of surgery, upon leaving the intensive care unit (3rd day), on discharge from the hospital and the 15th and 30th postoperative day. The nurse can take bioimpedance measurements in addition to patient history on nutritional habits and NSR 2002. Preliminary results show that the data gained through NSR 2002, bioimpedance and primary laboratory results can be effectively used to tailor individual nutrition plan either enteral or parenteral. Bioimpedance proved to be a useful additional surveillance tool as it added objectiveness to muscle loss evaluation which is often an underestimated marker of malnutrition, even if loss of muscle mass due to cancer cachexia would not be easily differentiated . This caThe individual’s level of physical fitness prior to an operation may be predictive of postoperative outcome: physical fitness is an overall term encompassing aerobic capacity, endurance, muscular strength, and body composition ,88,89,90A recent meta-analysis by Moran et al. has shown that prehabilitation, consisting of inspiratory muscle training, aerobic exercise, and resistance training, appears to decrease the incidence of all postoperative complications in patients undergoing intra-abdominal operations  . This efThe main obstacle to the spreading of nutritional therapy as of prehabilitation is compliance. Current standards in cancer care demand minimal delay between cancer diagnosis and treatment, thus incorporating these interventions into a time-restricted clinical pathway may be challenging.To overcome this limit, prehabilitation and ERAS, both aimed to minimize operative stress and complications, can be integrated. However, the literature specific to ERAS and prehabilitation is limited, and the two programs are seldom combined . The desThe aging population has an increased prevalence of cancers in the setting of a progressive state of vulnerability (frailty). Homeostenosis is the narrowing of the homeostatic functional capacity of an organism. Deconditioning is the decreased ability to adapt to normal conditions, which can also occur in patients with cancer. Therefore, under these conditions, morbidity and mortality after cancer surgery is progressively increased and impaired because of the cancer itself. The individual’s level of physical fitness prior to an operation may be predictive of postoperative outcome: physical fitness is an overall term encompassing aerobic capacity, endurance, muscular strength and body composition .Physical activity is known to reduce the risk for cancer development. Moreover, physical activity performed both before surgery (prehabilitation), or after cancer surgery (rehabilitation), improves immune function and metabolism, reduces length of stay, disability, complications, early retirement, all-cause mortality, and even recurrence and mortality from some cancer types ,96,97. IIn conclusion, the hypothesis that anesthesia, analgesia and other perioperative interventions such as optimizing nutrition and prehabilitation might influence cancer biology and therefore patient outcomes is supported by some preclinical and clinical studies. The conflicting results highlight the need to establish a large consortium, using innovative approaches, while linking data from multiple sources and multiple techniques, and to undertake prospective, randomized, multicenter clinical trials.