This comprehensive review focuses on our current understanding of the proposed physiological and pathological functions of extracellular vesicles (EVs) in the developing brain. Furthermore, since EVs have attracted great interest as potential novel cell-free therapeutics, we discuss advances in the knowledge of stem cell- and astrocyte-derived EVs in relation to their potential for protection and repair following perinatal brain injury. This review identified 13 peer-reviewed studies evaluating the efficacy of EVs in animal models of perinatal brain injury; 12/13 utilized mesenchymal stem cell-derived EVs (MSC-EVs) and 1/13 utilized astrocyte-derived EVs. Animal model, method of EV isolation and size, route, timing, and dose administered varied between studies. Notwithstanding, EV treatment either improved and/or preserved perinatal brain structures both macroscopically and microscopically. Additionally, EV treatment modulated inflammatory responses and improved brain function. Collectively this suggests EVs can ameliorate, or repair damage associated with perinatal brain injury. These findings warrant further investigation to identify the optimal cell numbers, source, and dosage regimens of EVs, including long-term effects on functional outcomes. Preterm infants born <37 weeks gestational age (GA) are at a high risk of brain injury . Whilst Cerebral palsy (CP) is the most common adverse neurodevelopmental outcome encountered by this most immature group of preterm infants. CP causes a range of permanent motor disabilities ranging from mild to severe. In the general population, the incidence of CP may be either increasing, static or declining amongst preterm infants . DespiteAlthough the pathophysiological mechanisms that trigger injury to the preterm brain are multifactorial and the severity of injury is invariably a consequence of the degree of prematurity, there are several common causes. They include intrauterine infection , which is recognized as an important factor in the etiology of spontaneous preterm birth and hypoxia-ischemia (HI), including postnatal insults, such as mechanical ventilation-induced lung injury, cerebral blood flow instability, free radical imbalance, sepsis, and necrotising enterocolitis . Such peWhile improved neonatal intensive care has resulted in a decline in the incidence of focal cystic necrotic lesions and thus the severest form of injury , the incRegardless of whether neurodevelopmental impairments encountered by preterm infants are either severe, moderate, or mild, the impact to the individual is enormous. It is not only a burden to the individual, but also immediate family members, the health care system, and social institutions. The development of effective therapies will enable better outcomes for these infants and thus have significant long-term benefits that could potentially translate to individuals achieving a more productive life including reduced health care costs associated with chronic neurological disability.1 NCT01793129).Presently, there are limited options for the prevention and/or treatment of perinatal brain injury. Treatment strategies include antenatal administration of magnesium sulfate to women at risk of preterm labor and postnatal administration of erythropoietin to infants born preterm . HoweverWhilst further randomized clinical trials of hypothermia therapy are warranted amongst preterm infants, there is an ongoing need to develop standalone therapeutic interventions that can be administered to reduce perinatal brain injury or therapeutic interventions that may be administered in conjunction with hypothermia to further reduce infant morbidity and mortality. Indeed, such an approach may be advantageous if the strategy is to target different therapeutic windows and multiple mechanisms of injury. However, hypothermia has the potential to alter the pharmokinetics of adjunct therapies and thus efficacy, which requires careful consideration .Stem cells, which are self-renewing and considered the most primordial and least committed cells offer significant promise for treating perinatal brain injury  given thin-vivo animal models of perinatal brain injury.Substantial evidence suggests that stem cells exert their regenerative effects through the release of biologically active molecules that act in a paracrine manner to promote cell viability and/or proliferation and modulation of immune responses . In factThe generic term “EVs” encompasses a heterogeneous range of lipid bilayered particles that are unable to replicate but are secreted by almost every cell of the body . Histori2+ entry via oligodendroglial ionotropic glutamate receptors  is not fully ascertained those identified as being mediated by EV autocrine/paracrine signaling include neural trophic support, synaptic plasticity, regulation of myelination and intercellular communication . Neuron-C) , Extracellular signal-regulated kinases (Erk1/2) and cAMP response element-binding protein (CREB) pathway activation and generate more action potentials suggesting an enhanced potential for neuronal signaling . FurtherC) . AdditioC) . The actC)  or inhibC) . ConversC) .EVs can also play a significant role in blood-brain barrier (BBB) integrity. Platelet-derived growth factor-BB (PDGF-BB)/PDFG-receptor beta (PDGFR-β) signaling has been implicated in the release of pericyte-derived EVs that carry neuroprotective cargo . This isin-vitro conditions of oxygen and glucose can modulate neuronal firing rate and is associated with enhanced stress resistance and neuronal viability , 2′, 3′-cyclic nucleotide 3′-phosphodie- sterase (CNPase), myelin associated glycoprotein, and myelin oligodendroglial glycoprotein (MOG)] are mediators of oxidative stress through transfer of human superoxide dismutase (SOD) and catalase (CAT) . Furtheriability .+ T cell response to MOG /reoxygenation, administration of human umbilical cord Wharton’s jelly MSC-EVs not only resulted in the prevention of HI-induced apoptosis, but also promoted cell survival through EV transfer of miR-let7-5p, a known regulator of caspase-3  to identify all studies that investigated the potential use of EVs as novel therapeutic agents in animal models of perinatal brain injury  was employed  to isolate EVs ranging from 16.34 to 1000 nm . DependiBesides consideration of the optimal source of EVs, the most efficient route of EV administration is also a matter of debate. While intravenous (IV) administration is convenient, it can lead to a high accumulation of EVs in peripheral organs, such as the lung, liver, and spleen . HoweverLike the mode of delivery, the dosage and duration of delivery also varied between studies. Furthermore, some studies administered EV therapy as a single dose post-injury , whereasClinical presentation of injuries, as well as timing of the initial onset is often unclear and difficult to determine . This isPreterm infants, with prior evidence of focal and diffuse white matter lesions, invariably display atypical cortical and subcortical development, involving volumetric reductions in both total cerebral gray and myelinated white matter and larger ventricular volumes either at term or later in childhood . Further+ cells) compared to untreated HI controls , thalamus (by 12.91%), and external capsule (by 10.83%) compared to untreated animals . Similarcontrols .Studies conducted by At the microscopic level, hypomyelination is a central feature of preterm brain injury, which principally affects preoligodendrocytes (or late oligodendroglia progenitors), that predominate in the forebrain at 24–32 weeks gestation . In addi7 cell equivalents were injected 1 h and 4 days post-umbilical cord occlusion and fetuses killed 7 days thereafter (ereafter . Treatmeereafter . Howeverereafter .+Olig2+ cells) within regions of the corpus callosum following brain injury  in the subplate zone of the posterior parietal cortex as well as the CA1 region of the hippocampus -1, which are all critical for maintaining the integrity of the BBB are expressed in the germinal matrix, cortex, and white matter of the fetal human brain as early as 16 weeks GA . This su+ endothelial cells  at 1 h and 4 days following injury  in both the cortex and white matter in P3 neonatal rats 48 h post LPS injection (24 h post second dose of EVs)  . Further of EVs) .Intuitively, approaches to reduce both oxidative stress and pro-inflammatory damage would be a desirable feature of therapies. IP administration of astrocyte-derived EVs immediately prior to HI, in P7 rats, can effectively reduce brain tissue protein levels of TNF-α and IL-1β and those of oxidative stress factors, SOD, CAT, glutathione peroxidase (GPX) and malondialdehyde (MDA) . Thus, aTaken together, the data reviewed is supportive of the proven anti-inflammatory and anti-apoptotic properties of MSC-EVs , and theSeven studies have investigated whether EVs repress behavioral impairments associated with perinatal brain injury. Four were conducted in mouse models of term HI  and fourInterestingly, in the study where repeated IP injections were conducted of umbilical cord MSC-EVs to neonatal mice 14 h before, immediately before, and 3 h following HI, a significant decline in neurological severity scores for neurological assessment of movement, reflex, sensation, and balance is observed 6 h following HI . FurtherThe study by Drommelschmidt and colleagues was the only study to provide a comprehensive longitudinal follow-up assessment of long-term behavioral outcomes . The autIn later studies, More recently, Finally, neurobehavioral outcomes were also assessed in the neonatal rat model treated IP with rat astrocyte-derived EVs . AssessmIn summary, behavioral studies in rodent models of perinatal brain injury demonstrate that EV treatment not only can improve early neurological deficit scores associated with HI, but also long-term changes in performance of motor coordination (Negative Geotaxis and Rotarod test), spatial learning (Morris Water Maze), adaptive memory (Barnes Water Maze) and non-spatial and non-aversive memory (Novel Object Recognition). Collectively, while this suggests MSC-EVs and astrocyte-EVs have benefits in terms of improving neurobehavioral outcome, more in-depth analyses are needed to resolve contradictory findings that exist between these various studies.Larger animal models, such as the fetal sheep model have the advantage of being able to provide measurements of clinically relevant endpoints for the therapeutic application of EVs against perinatal brain injury that would not otherwise be possible in rodent models. The larger size of fetal sheep facilitates continuous measurement of several physiological variables including electrophysiological brain activity by electroencephalogram (EEG), electrophysiological cardiac function by electrocardiogram (ECG) and hemodynamics by blood pressure and heart rate. In fetal sheep, prolonged umbilical cord occlusion is associated with long-lasting suppression of parasagittal cortical EEG activity, with delayed onset of seizures . In humaAs previously discussed, While studies identified in this review of the literature suggest MSC-EVs are a useful therapeutic candidate in the treatment of perinatal brain injury, most do not provide a potential mechanism of action through which MSC-EVs exert their effects. There are five exceptions to this, as discussed in detail below.in-vitro experiments demonstrating MSC-EVs expressing Annexin A1 or recombinant Annexin A1 improved trans-endothelial electrical resistance (a measure of BBB integrity) of primary fetal rat endothelial cells and that addition of formyl peptide receptor blockers mitigated the effect , which in turn enhances the expression of early growth response 2 (EGR2)/B-cell lymphoma (Bcl2) and prevents apoptosis.Thirdly is the elegant study by al cells . They alal cells . MoreoveFinally the fourth study, conducted by It is possible that other molecular mediators that can confer neuroprotective and neuroregenerative properties are packaged as EV cargo. However, future work is required to identify these molecules, their mechanisms of action, whether they are cell of origin specific, and whether they preferentially target specific cell types to exert their effects.The results of the current literature are promising in terms of the therapeutic use of EVs in preclinical animal models of perinatal brain injury. Despite this, the administration of any therapeutic agent can bring with it complications and side effects. The administration of EVs appears well tolerated in the rat model and deleterious side effects, such as weight loss, death or clinical illness have not been reported . Moreovein-vivo biodistribution of EVs after administration and their off-target outcomes thin 3 h . Within thin 3 h . No advethin 3 h . Neverththin 3 h . Unsurprthin 3 h . That boAs a whole, the findings suggest that from a therapeutic perspective MSC-EVs are well tolerated and are less likely to accumulate in peripheral organs when administered intranasally. Whether IV administration has similar outcomes of biodistribu- tion and tolerance remains unknown and requires investigation.The studies identified in this unbiased review investigating the potential use of EVs as a therapeutic treatment for perinatal brain injury, report an overall improvement in neurological outcomes. The studies highlight the ability of EVs to modulate inflammatory responses associated with both preterm and term acquired brain injuries. Because of these beneficial properties, they are successful in improving white matter and gray matter microstructural integrity through decreasing both gliosis and pro-inflammatory cytokine production and improving cell survival through amelioration of apoptosis. Additionally, EV therapy improved cognitive deficits  and enhanced the performance in motor coordination function. These results are comparable to that of adult experimental models of neurological brain injury, including traumatic brain injury and stroke, where EV administration imparts neuroprotective and neuroregenerative effects . This suPreclinical translation of EVs, however, is confounded by contrary publications on the effects of EVs. Contradictory results are most likely attributable to differences in animal experimental models, EV cell source, dosage of EVs, and route and timing of EV administration. This makes comparisons between studies difficult and limits interpretation of results. Although EVs represent a promising next-generation treatment for perinatal brain injury, future progress is dependent on a number of considerations. For example, while only 1/13 studied utilized astrocyte-EVs, 12/13 of the studies identified in this review utilized MSC-EVs, derived from different sources; either from the umbilical cord, umbilical cord blood, Wharton’s jelly of the human umbilical cord, or from adult bone marrow.EVs isolated from MSC cultures of Wharton’s jelly have the notable advantage of coming from the umbilical cord, an extra-embryonic tissue, which is normally discarded at birth and free of ethical donor concerns and relatively low in cost . FurtherAn important consideration will be to determine if the functional effects of MSC-EVs are reproducible using EVs from different cell sources. In this regard, it will also be crucial to establish any potential differences in mechanisms mediating EV therapeutic effects, which may exist between EV cell sources and their various small and large EV subpopulations. This is relevant, since heterogeneous populations exist and current isolation methods are relatively inadequate in resolving them . MoreoveCurrent evidence suggests EVs secreted by cells exhibit target selection and that the EV cellular source must be matched to requirements of the target application and that EVs adopt a similar homing pattern to their cell source . Thus, iin-vivo EV administration, EV cargo load is predetermined by the cellular response of cells to the culture environment.Aside from the original cell type from which EVs are isolated, the impact of cell culture parameters and microenvironment in the production of EVs, including the passage number, seeding density, cell confluence, and the frequency and time of EV collection should be taken into consideration. While considerable progress has been made in EV production methods, the passage number is of particular relevance since evidence suggests the neuroprotective effect of human umbilical cord Wharton’s jelly MSCs (and their paracrine activities) diminish with increasing passage number . FurtherUnfortunately, the lack of standardization of protocols between research groups investigating the therapeutic role of EVs in the treatment of perinatal brain injury adds a level of complexity in understanding the biochemical mechanisms at play. If future studies aim to standardise the cells from which EVs are isolated, including the methods, dosage and timing of EV administration, it may be possible to use an -omics approach to identify the mechanisms by which stem cell-derived EVs exert their functional effects. Once these biochemical mechanisms are identified we may be able to employ methods to enrich for candidate molecules in EVs as has been done in the study by in-vivo models of HI treated using EVs. However, since each study utilized different EV isolation methods, vesicle cargo likely reflects the specific subpopulation of EVs isolated  and miRNA  are potential mediators of the neuroregenerative and neuroprotective effects observed in isolated . Moreoveisolated  utilizedisolated  was encaisolated  prior toNevertheless, understanding the impact of individual mediators is critical to identifying which molecules that potentially mediate optimal neuroregenerative and neuroprotective responses from a nanotherapeutics development perspective, since synthetically manufactured EVs loaded with a specific cargo could be developed as an “off the shelf” product. However, biologically it is more likely that a combination of molecules  working synergistically will provide the greatest beneficial effect.In-vivo biodistribution of EVs is determined by the route of administration  by which EVs may exert protective effects. Such models will be crucial in establishing optimal routes and timing of administration and therapeutic effects, including any adverse effects. Furthermore, there must be appropriate negative control experiments. Surprisingly, of the perinatal studies reviewed, only two were identified where control experiments were performed using EV-depleted conditioned media  to exclude the possibility of effects through co-isolated proteins and common EV components . Others l groups .in-utero fetal sheep model  brain, with limited white matter tracts , that doep model . These sAs the therapeutic application of EVs in the treatment of perinatal brain injury is in its infancy, there still are no studies reported which have tested the benefits of EVs in combination with hypothermia or other therapeutics. It is possible that administration of EVs will work synergistically with established therapeutic interventions and enhance clinical outcomes. Consequently, further work should be done to explore the potential of combination therapies.Finally, although it is beyond the scope of this review, a unifying goal of recent and future advances in the field of EVs is to improve the therapeutic potential of EVs. More so, it is the need to find an effective way to treat newborns with brain injuries, which is one of the greatest challenges facing perinatal medicine. Achieving a promising outcome is most likely dependent on the composition of the EV cargo. As outlined previously, cargo found within EVs can include nucleic acids [miRNA , lncRNA in-vitro and in-vivo models suggesting that EVs will not only prevent, but also repair brain damage. Collectively, these studies highlight an enormous potential for future EV therapies, which requires further investigation. However, to ensure the validity of results and to advance the field of perinatal EV research, investigators should standardize future research practice by ensuring compliance to current minimal requirements for EV studies (MISEV guidelines)   and thosdelines) . EquallyTG conducted the unbiased literature search. TG and MF contributed equally to the preparation and writing of the review article. The final submitted article has been revised and approved by both authors.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The cerebrovasculature has become increasingly recognized as a major player in overall brain health and many brain disorders. Although there have been several landmark studies to understand details of these crucially important structures in an anatomically defined area, brain-wide examination of the whole cerebrovasculature, including microvessels, has been challenging. However, emerging techniques, including tissue processing and three-dimensional (3D) microscopy imaging, enable neuroscientists to examine the total vasculature in the entire mouse brain.Here, we aim to highlight advances in these high-resolution 3D mapping methods including block-face imaging and light sheet fluorescent microscopy.We summarize latest mapping tools to understand detailed anatomical arrangement of the cerebrovascular network and the organizing principles of the neurovascular unit (NVU) as a whole. We discuss biological insights gained from studies using these imaging methods and how these tools can be used to advance our understanding of the cerebrovascular network and related cell types in the entire brain.This review article will help to understand recent advance in high-resolution NVU mapping in mice and provide perspective on future studies. This type of approach is not feasible using current ex vivo imaging methods that can be loosely categorized into block-face imaging with serial sectioning and light sheet fluorescence microscopy (LSFM). We will also discuss the sample processing and vessel labeling strategies that contribute to an integral part of the imaging process. These imaging modalities provide the means to study details of the cerebrovasculature, and each has its own set of strengths and limitations. Importantly, these studies are technically challenging, not only in terms of imaging, but also in terms of analysis pipelines that require high-level computational skill. Given this, as well as the rapid expansion of these modalities and our understanding regarding the importance of the brain vasculature, it has become necessary to synthesize the knowledge and resources gained from recent research efforts.Fortunately, technological innovation has paved the way for studies of cerebrovascular mapping in the whole mouse brain. Although the list of methods continues to grow, the focus here will include a current adaptation of several cellular resolution 2,,,,,Several serial sectioning-based methods for mesoscale whole-brain mapping have been developed and extensively utilized for neuronal cell type mapping.,,–,,,There are several variations of block-face imaging techniques including, micro-optical sectioning tomography (MOST), knife-edge scanning microscopy (KESM), serial two-photon tomography (STPT), and block-face serial microscopy tomography (FAST). MOST and KESM are two variations of similar technologies that consist of a microtome paired with a light microscope.2.1,,,,,,,,in vivo two-photon imaging and ex vivo imaging in the same animal showed that perfusion-based vessel labeling preserves the overall geometry of cerebrovasculature .,To study the vasculature at cellular resolution, several labeling methods for block-face microscopy have been developed. Some of the first vasculature-focused studies, using both MOST and KESM, were paired with the gelatin-Indian Ink perfusion method.2.2,,,,However, there are drawbacks for perfusion-based labeling and sectioning-based imaging methods for studying the vasculature. For example, the FITC-fill and Indian-ink perfusion techniques can result in incomplete filling of the vessel lumen, which is also the case for other methods that rely on vessel filling.3,,,,,,,Recently, the optimization of brain clearing methods has paved the way for use of LSFM to examine the cerebrovasculature in a whole mouse brain. LSFM consists of a light microscope, in which the cleared sample is illuminated with a two-dimensional (2D) plane of thin light with a detector positioned at an orthogonal angle .11,32,333.1,,––,,,,,To conduct LSFM for vascular imaging, tissue samples must be optically cleared with optional immunolabeling. Several clearing methods have been developed and adapted for studying various animal models and cellular markers. A commonly used clearing method is iDISCO, a variation of solvent-based clearing, which is useful for vascular labeling but is associated with a slight volumetric shrinkage of the tissue and quenching of endogenous fluorescence.In fact, two recent labeling methods highlighted the capabilities of whole-brain immunolabeling by capturing each compartment of the vessel tree within the same sample. One study utilized modified iDISCO clearing, followed by a combination of antibodies to uniquely label arteries, veins, and capillaries with three different colors.3.2,,,in vivo two-photon imaging) and accordingly adjusting measurements. Additionally, while LSFM imaging itself is rather quick (a few hours per mouse brain), the sample processing time required for most brain clearing methods takes several weeks.,,,As with other modalities, brain clearing methods and LSFM do have disadvantages. One major issue is the volumetric swelling or shrinkage associated with most brain clearing methods, which significantly impacts volumetric and vessel parameter analysis.4,,,To properly analyze the sheer amount of data produced by 3D vascular imaging, there are several computational steps. For example, algorithms and machine learning pipelines are required to perform 3D image processing , segmentation, binarization, skeletonization, tracing, and finally registration to a reference brain atlas, all of which have been major technical hurdles in furthering understanding of the vasculature in different brain regions.5These various imaging technologies have provided the means to study the macro- and microvasculature at cellular resolution and uncover the anatomy, organization, geometry, and intricate characteristics of this complex network. One major step forward involved the 3D reconstructed rendering of pial surface vessels and annotation of the first whole-brain vascular atlas with stereotaxic coordinates at ,,The real power of high-resolution 3D vascular mapping is its ability to image and resolve the entire network of capillaries in the brain. Microvascular networks in the adult mouse brain prevent rarefaction through redundancy and low tortuosity.,,,,,,Recent studies also highlight regional differences in cerebrovascular organization.,in vivo conditions.High-resolution cerebrovascular datasets provide crucial structural information to simulate blood flow using in silico modeling.6,,Dysfunction in the cerebrovasculature has been highly implicated in many brain disorders including cerebral ischemia and neurodegenerative diseases.Recently, there is increased interest in cerebrovascular development and its roles in developmental disorders, such as autism spectrum disorders.,Finally, mounting evidence suggests these important structures may also play a major role in neurodegenerative diseases. For instance, recent evidence suggests that AD is heavily influenced by vascular impairment.Together, these studies demonstrate how the power of high-resolution 3D mapping methods reveals the crucial roles of the brain vasculature throughout the lifespan and in various pathologies.7–––3D mapping methods also provide means to examine how cerebrovasculature is structurally organized in the context of other NVU cell types including neurons, glia, and perivascular cells. Neurons expressing neuronal nitric oxide synthase (nNOS) are particularly important for increasing blood flow upon activation to mediate neurovascular coupling.7.1,,Each of these imaging modalities presents with their own strengths and problems and often provide complementary findings that further improve this field of study. Importantly, these methods also allow for the study of hard-to-reach subcortical structures at micron and submicron resolution, which have been largely understudied. For example, in only a few years these studies have identified striking differences between cortical and subcortical regions of the brain, beginning to unveil the detailed structure and organizing principles of the cerebrovasculature. These findings, some of which are summarized in


Cutaneous wound healing is a complex, multi-stage process involving direct and indirect cell communication events with the aim of efficiently restoring the barrier function of the skin. One key aspect in cutaneous wound healing is associated with cell movement and migration into the physically, chemically, and biologically injured area, resulting in wound closure. Understanding the conditions under which cell migration is impaired and elucidating the cellular and molecular mechanisms that improve healing dynamics are therefore crucial in devising novel therapeutic strategies to elevate patient suffering, reduce scaring, and eliminate chronic wounds. Following the global trend towards the automation, miniaturization, and integration of cell-based assays into microphysiological systems, conventional wound healing assays such as the scratch assay and cell exclusion assay have recently been translated and improved using microfluidics and lab-on-a-chip technologies. These miniaturized cell analysis systems allow for precise spatial and temporal control over a range of dynamic microenvironmental factors including shear stress, biochemical and oxygen gradients to create more reliable in vitro models that resemble the in vivo microenvironment of a wound more closely on a molecular, cellular, and tissue level. The current review provides (a) an overview on the main molecular and cellular processes that take place during wound healing, (b) a brief introduction into conventional in vitro wound healing assays, and (c) a perspective on future cutaneous and vascular wound healing research using microfluidic technology. Mechanical injuries, burns, and illnesses are, among others, the leading causes of external or internal tissue damage or lesion, generally referred to as wounds. Breaks in the epithelial barrier, known as the cutaneous wound, disturb the skin’s primary function, which is the protection of the body against the external environment . FollowiConsequently, this review focuses on the current state of established microfluidic wound healing assays that assess the role of cell migration in the overall wound healing process including methods for wound generation, wound healing analysis schemes, and the influence of molecular stimuli and/or inhibitors  in mechanistic studies on cell migration and wound healing. A series of activated intracellular and intercellular pathways initiate the wound healing and repair process, as shown in The first stage of wound healing is inflammation, which occurs as an immediate response to a break in the epithelial barrier. In this stage, inflammatory pathways prevent further bleeding and infection . NeutropIn the second stage of wound healing, known as the proliferative stage, re-epithelialization and vascularization occurs to fill and cover the wound . The migIn the remodeling stage, anti-inflammatory cytokines such as interleukin 10 (IL-10) start to regulate immune cell infiltration , as well as collagen type I synthesis, through the regulation of cell proliferation and ECM remodeling ,10. AfteIt is important to note that dermal fibroblasts play a vital role in all stages of cutaneous wound healing and connective tissue regeneration , and theIn summary, the complex signaling cascades and pathway activations that take place during the wound healing process govern the cell-to-cell and cell-to-matrix interactions that lead to wound clearance, rebuilding, and maturation. The failure to progress in any of the three stages of wound healing can therefore lead to impaired healing, chronic wounds, and excessive scar formation. The ability of mammalian cells to migrate represents a fundamental aspect in biology that is essential for embryonic development, immune response, cancer metastasis, and wound healing . TherefoA scratch assay is still the most common method used for cell migration assessment, despite its many shortcomings such as a lack of standardization, high variability, and low reproducibility. Scratch assays are widely used in different research fields such as fundamental biology, drug screening, cancer metastasis, immunology, and wound healing . The maiIn summary, conventional migration and wound healing assays based on cell exclusion or removal feature a range of limitations and are associated with endpoint detection, have non-linear or uncontrolled gradients, lack reproducibility, are not automation-friendly, require the manual removal of inserts, damage matrix coatings, and have variability between control and experiment scratching. Consequently, next generation wound healing assays need to address variability, show flexibility in application, permit live cell imaging, perform high-content analysis, and feature simple on step protocol (automation-friendly). To address the above-outlined shortcomings, various microfluidics and lab-on-a-chip systems have been developed to improve standard wound healing assays with various potential applications such as drug discovery, diagnostics, and basic research. The general principle of microfluidic chip technology is to create a platform for miniaturized and automated bioassays . The smaOver the last two decades, many on-chip wound healing assays based on microfluidics have been reported using various microchannel designs that create cell-free areas by either cell exclusion or cell depletion , as showInstead, the remaining sections focus on how chip-based assays  improve In addition to mechanical depletion or exclusion methods, enzymatic detachment is another frequently used wounding method based on the depletion of cell-free areas by streaming enzymatic detachment solution over portions of confluent cell monolayers see . Nie et al. reported an NIH-3T3 fibroblast cell migration assay using enzymatic wound creation by the hydrostatic laminar flow patterning of trypsin/EDTA on one side of the microfluidic channels . SimilarSimilarly, van der Meer et al. used an enzymatic wound-healing chip for HUVEC endothelial monolayers to show that VEGF gradients, as well as fluid shear, improve endothelial cell migration speed. However, shear severely impacts the directionality of migration along the fluid flow direction see D,E 46]..46]. FurLike the actuated pillar approaches mentioned earlier, Sticker et al. developed two microdevices for performing automated cell migration assays based on both the cell exclusion and cell depletion methods for wound creation see A–C 53]..53]. Bot2), highlighting how one can improve the reproducibility and comparability of wound-depletion approaches.The authors applied their microsystem to investigate the effect of medium supplements such as growth factors and proliferation inhibitory drugs on human dermal fibroblast (HDF) cell migration see D. For inWound healing assays using microfluidic technologies are based on cell migration into cell-free areas created using various physical and biochemical methods. The most common strategy to create cell-free areas (wounds) in cell monolayers is enzymatic detachment using laminar flow patterning, which influences the cell dynamics on the edges due to the enzyme interaction with cells. Mechanical approaches, however, present more realistic strategies for on-chip wounding. However, these methods often require a more complex microdevice design regarding the number of layers and channels and additional controllers of actuators that deplete or exclude cells. On the one hand, laminar flow patterning is most often used and requires syringe pumps, while hydrostatic fluid handling can obviate pumps and improves scalability for drug-screening applications. On the other hand, applying pressure via pneumatic actuators requires pressure lines and pressure controllers to automate the wounding process via cell exclusion or cell depletion. Once the wounding strategy is selected, the challenge is using these microdevice solutions for the biological read-out of wound healing processes. Microfluidic systems are used for wounding to frequently investigate either human endothelial cells (HUVECs) or mouse embryonic fibroblasts (3T3 fibroblasts). The selection of HUVECs is relevant for the investigation of microlesions and ruptures that occur throughout the human vascular system because they are well-established, easy to handle, and commercially available. Embryonic fibroblasts are a questionable choice as a relevant model for dermal wound healing processes that can be predictive for human wounds and should be considered for very early stage and proof-of-principle studies. Keratinocytes, combined with dermal human fibroblast cells and other more volatile cell populations , would be the best option for wound models capable of predicting the complex processes of wound healing in general. These cells can be eventually integrated into state-of-the-art, commercial, full-thickness dermal and epidermal skin models for wound healing assessment. In line with the embryonic tissue problem, cancer cell lines are frequently used for cell migration and wound healing studies even though these models are more relevant for research fields such as cancer cell migration processes in metastasis, such as tumor cell extravasation and invasion. Microfluidic technologies have outperformed conventional wound healing assay because of the precise control of vital microenvironmental culture parameters, including oxygen and fluid shear. The automation of on-chip wound healing has further improved the reproducibility of wound shape and size by eliminating human errors during the wounding process. Finally, the miniaturization of wound healing assays using chip technology has the main advantage of using smaller amounts of reagent and cell materials, which is an essential aspect for the scalability of pharmaceutical wound healing investigation for personalized medicine in preclinical research fields such as a chronic wound healing.In order to use microfluidic wound-healing assays as state-of-the-art tools for wound healing research, 3D tissue models using keratinocytes, fibroblasts, endothelial, and immune cells should be further advanced to better recapitulate anatomical and pathophysiological processes at the cellular and molecular levels. Such complex wound models would have the capacity to be used as skin disease models for research on and the drug-screening of pathologies including diabetic wounds and skin fibrosis. The establishment of full-thickness pathological wound models would provide a more scalable and cost-efficient technology platform for drug screening and medicine development when combined with automated and quantitative analysis schemes . Combing wound healing microdevices with dynamic cultures by including patient-derived cells/stem cells would lead to the development of personalized medicine based on a patient’s unique genetic background. This approach could significantly reduce the need for animal testing and could be used to develop patient-specific devices for drug screening or cosmetic testing due to the small number of cells and drugs needed for each wound healing assay.


Drosophila). This rather narrow focus has hampered the establishment of a comprehensive view of CHC genetics across wider phylogenetic boundaries. This review attempts to integrate new insights and recent knowledge gained in the genetics of CHC biosynthesis, which is just beginning to incorporate work on more insect taxa beyond Drosophila. It is intended to provide a stepping stone towards a wider and more general understanding of the genetic mechanisms that gave rise to the astonishing diversity of CHC compounds across different insect taxa. Further research in this field is encouraged to aim at better discriminating conserved versus taxon-specific genetic elements underlying CHC variation. This will be instrumental in greatly expanding our knowledge of the origins and variation of genes governing the biosynthesis of these crucial phenotypic traits that have greatly impacted insect behavior, physiology, and evolution.Cuticular hydrocarbons (CHCs) have two fundamental functions in insects. They protect terrestrial insects against desiccation and serve as signaling molecules in a wide variety of chemical communication systems. It has been hypothesized that these pivotal dual traits for adaptation to both desiccation and signaling have contributed to the considerable evolutionary success of insects. CHCs have been extensively studied concerning their variation, behavioral impact, physiological properties, and chemical compositions. However, our understanding of the genetic underpinnings of CHC biosynthesis has remained limited and mostly biased towards one particular model organism ( One main contributing factor to the remarkable evolutionary success of insects, eventually becoming the most diverse and species-rich class in the animal kingdom  are one of the major CHC compound classes  have been found to occur quite frequently in a number of insect taxa as well , or unsaturated CHCs , which is mainly derived from the citric acid cycle, is converted into malonyl-coenzyme A . This constitutes the main rate-limiting step for the general production of fatty acids and hydrocarbons  and one in the fat body (FASN1)  with similar expression patterns  has been revealed to be mainly responsible for affecting ratio and production of the female sex pheromone components 7,11- and 5,9-heptacosadiene in D. melanogaster by introducing a second double bond  CHC compounds , also expressed in males, showed no chain-length specificity. However, knockdown of these elongases affected abundances of several CHCs with no particular compound class specificities  whose oenocyte-specific knockdowns resulted in moderate to strong decreases of several CHCs from all compound classes  have been shown to be directly associated with intraspecific CHC variation . Just as target specificity for different chain lengths of LCF acyl-CoAs has been demonstrated for eloF . FarO is specifically expressed in larval oenocytes and regulates oenocyte growth, with no demonstrated effect on CHCs in adults , providing further evidence that chain-length-specificity potentially constitutes a common trait in FARs . This would also suggest that CYP4Gs constitute highly conserved, evolutionary stable elements in the CHC biosynthesis pathway, essential for catalyzing its last steps. However, clearly more direct experimental validation is required to demonstrate the importance and universality of CYP4Gs in CHC biosynthesis.It has long been assumed that FARs directly reduce VLC acyl-CoAs to VLC aldehydes , as their knockdown not only resulted in down-, but also upregulation of CHC compounds. These changes were sex-specific to some extent, but not for any particular CHC compound class  and one for a palmitoylation enzyme (App) that can modify cytoplasmic proteins by adding palmitic acid residues  remains unclear. Intriguingly however, the observed changes in CHC compositions are again reminiscent of the chain-lengthspecific effects hinted at for FARs and eloF mentioned above.Recently, a surprising correlation between ss Table . FurtherCG5599 and CG8680, had an upregulatory effect on Drosophila CHC production  has the potential to greatly help complementing insights from correlative gene expression data, as has already been successfully demonstrated in a few case studies (e.g., Finet et al. Together this raises the question whether most differences in CHC profiles can be explained through differential regulation of the underlying genetic networks rather than presence or absence of biosynthetic genes. It becomes increasingly clear that fine-tuning gene expression, e.g., through transcription factors, post-translational as well as epigenetic modifications, very likely plays a fundamental role in generating CHC variation. To the best of our knowledge, however, the influence of particularly epigenetic factors on CHC profiles has not been investigated at all so far. Furthermore, quantitative genetic studies hint at additive effects of many genes with the potential to cumulatively impact CHC variation, complementing studies on heritability and genetic correlations between individual CHC compounds (Foley and Telonis-Scott Another confounding factor in studying CHC genetics is the apparently large degree of phenotypic plasticity characterizing CHC profiles (Otte et al. Finally, changes in the microbiome can alter CHC profiles via different mechanisms, such as direct degradation of CHCs (Napolitano and Juárez Drosophila genus and are not necessarily representative of CHC biosynthesis across wider phylogenetic boundaries. For instance, in other insect taxa, particularly Hymenoptera, mb-CHCs tend to occur in far higher and more dominant quantities than in Drosophila (e.g., Buellesbach et al. Drosophila-specific n-alkenes and alkadienes (Jallon and David Drosophila have contributed to the comparably limited understanding about the genetic factors governing variation in this particular compound class (Blomquist and Bagnères Drosophila. It will be very helpful to greatly expand on these findings in future studies, incorporate more insect taxa and implement more comparative approaches to shed light on the conserved blueprint of CHC biosynthesis on a larger phylogenetic scale.As evident from this review, most of the genetic investigations undertaken so far have remained very specific to the Through an even wider evolutionary lens, the cuticles of insects and plants share remarkable structural similarities in their hydrocarbon compositions, even down to generally similar biosynthetic pathways (Kunst and Samuels Drosophila, we have not yet resolved individual genetic factors exclusively governing the synthesis of particular CHC compound classes, let alone single compounds. This is partially due to the continuing lack of insight into differences between conserved versus more taxon-specific genes regulating CHC biosynthesis, in turn preventing us from developing a more general blueprint of this fundamental biological process. Therefore, the most promising avenues of research lie in incorporating an increasing number of insect taxa with a larger focus on integrating comparative genome-wide, phylogenetic and functional genetic studies. Ultimately, this will not only help us to unravel the more general aspects of CHC genetics but also potentially answer interesting evolutionary questions about the origins, diversifications, and expansions of these essential phenotypic traits.We have come a long way from uncovering the first genetic factors underlying sex- and species-specific CHC variation to now being able to elucidate the contributions of individual genes to minute variations in CHC compositions. However, we are still far from fully and conclusively understanding the extent of the genetic framework underlying CHC biosynthesis. Specifically, apart from a few well documented case studies of unsaturated CHCs in Table S1


Current staging systems of cancer are mainly based on the anatomical extent of disease. They need refinement by biological parameters to improve stratification of patients for tumor therapy or surveillance strategies. Thanks to developments in genomic, transcriptomic, and big-data technologies, we are now able to explore molecular characteristics of tumors in detail and determine their clinical relevance. This has led to numerous prognostic and predictive gene expression signatures that have the potential to establish a classification of tumor subgroups by biological determinants. However, only a few gene signatures have reached the stage of clinical implementation so far. In this review article, we summarize the current status, and present and future challenges of prognostic gene signatures in three relevant cancer entities: breast cancer, colorectal cancer, and hepatocellular carcinoma. Tumor development involves many genetic alterations, especially in cell growth and proliferation. Since the advent of gene expression analysis, we can detect and classify these changes. By definition, a gene expression signature is a particular group of genes correlating genetic alterations with specific clinical variables, such as diagnosis or prognosis . One of Predicting the prognosis of a disease is crucial for cancer management and is still a challenge for many malignancies due to the limitations of clinicopathologic parameters. Prognostic gene expression signatures can help improve patients’ therapy by classifying tumors into different groups, thus providing guidance for a personalized treatment-decision. The first example was developed in 2002, when Bernards et al. validated the prognostic power of a 70-gene signature in 295 breast cancer (BC) patients by successfully classifying them into good or poor prognosis groups . This teHowever, despite the significant number of prognostic gene signatures developed, very few have been introduced into clinical routine and even fewer have been thoroughly validated. As breast cancer, colorectal cancer (CRC), and hepatocellular carcinoma (HCC) are ranking among the most common cancers and leading causes of cancer-related deaths on a global scale , we aim Fueled by high-throughput technologies, today HCC molecular changes can be investigated in multiple dimensions, including chromosomal structure, genetic polymorphisms, epigenetic changes but also gene expression profiling . AnalysiThe feasibility of using archived specimens, including Formalin-Fixed Paraffin-Embedded (FFPE) samples, in microarray technology to further develop prognostic biomarkers has been demonstrated ,23. MostAmong the first to perform gene expression analysis in HCC, particularly to predict the prognosis of those patients were Iizuka and co-workers in 2003. They analyzed gene expression on 33 HCC quantifying 6000 genes. This first prognostic signature was postulated to predicted early intrahepatic recurrence or nonrecurrence with a positive predictive value of 88% . ThroughSince unsupervised signatures failed to be independently confirmed and composition of an ideal signature remained (and remains) undetermined but also due to the technological advance subsequently making full genome expression screens available, unsupervised approaches became increasingly common. Among the first to apply unsupervised approaches were Lee and Thorgeirsson, who published several potentially prognostic signatures ,28. In aIn the following years, gene expression was used to define not just two but more groups in such a way that they could define survival subgroups of hepatocellular carcinoma. Yamashita and co-workers defined 70 EpCAM co-expressed genes to construct a predictive model and define four HCC subgroups: HCC EpCAM positive with either AFP positive or negative and EpCAM negative with either AFP positive or negative. Hoshida et al. performed a meta-analysis to report three distinct HCC subclasses  correlated with different clinical parameters . MeanwhiAmong the most promising signatures, the 5-gene signature established on 314 HCC samples from France used a coefficient and regression formula of a multivariate Cox model to demonstrate an association of HN1, RAN, RAMP3, KRT19, and TAF9 with survival times. These data were further validated in patients from Europe, the United States, and China. The same group furthermore reported a significant predictor of survival in patients treated by resection and ablation and in advanced HCC . HoweverIn recent years gene expression profiles also became part of larger efforts to integrate genomic, epigenomic, and expression data to reveal prognostic subtypes of HCC. These strategies are highly complex, laborious and certainly need to be proven in independent validation .Besides the above-mentioned HCC signatures, there are also some prognostic signatures of specific subtypes of HCC. Fibrolamellar HCC (FLC) is a rare cancer of the liver that grows in teens and young adults who have healthy livers. Cornella et al. found three molecular classes by using unsupervised clustering, and generated a prognostic 8-gene signature which can predict the survival time of FLC patients . In 2010Unfortunately, unlike breast cancer, none of the HCC signature-based biomarkers are in routine clinical use. There are many factors accounting for this status quo, mainly involves biological, technological, statistical, and informatics challenges . First, A large fraction of CRC is diagnosed in a locally advanced or metastatic stage, therefore prognostic biomarkers are valuable for the selection of specific treatment or surveillance strategies. Several expression signatures have been evaluated in tumor tissues from clinical trials, yielding promising results. However, similar to HCC, multi-gene expression tests are so far not routinely used in the management of CRC ,44.p = 0.004). Interestingly, in the subgroup of T3 microsatellite stable tumors, only ColoPrint, but not other clinicopathological features (such as the NCCN guideline risk factors) could predict the risk of relapse. ColDx  is a prognostic test relying on a 634 gene signature that was tested in several independent cohorts [p < 0.01). In contrast, only borderline significance was found for age and mismatch repair status. Of note, a high rate of assay failure was observed for this ColDx, which was partly due to the age of the tissue samples. Other gene expression signatures developed for the early-stage CRC include the 5-gene signature Oncodefender [Prognostic markers are particularly valuable for Stage II and III CRC, since patients might benefit from adjuvant therapy after surgery. However, the selection of patients is currently suboptimal due to a lack of established biomarkers (except for mismatch repair deficiency), leading to either over- or undertreatment . Differe cohorts ,52, incldefender , the Stadefender  and Stagdefender . HoweverBeyond the commercial multi-gene expression tests for early and intermediate-stage colorectal cancer, transcriptome-based analyses have been published in recent years that not only aimed to improve clinical patient stratification, but also to establish new biological classification system of CRC. For instance, reported gene expression signatures specific for adult intestinal stem cells and their derivates in the intestinal crypt, which they established using FACS-purified mouse intestinal stem cells . This clWith respect to prognosis, CMS4 was associated with the worst overall survival and relapse-free survival in early and intermediate-stage cancer, while CMS1 cancers showed the worst survival rates after relapse . AdditioIn metastatic disease, the impact of CMS is different than in intermediate stages. First, CMS1 (not CMS4) is associated with the worst prognosis in this stage, while CMS2-4 have better overall outcomes ,70, a fiThus, CMS represents a transcriptome-based classifier of CRC that has prognostic value in both intermediate and advanced stages of CRC. It also appears to have predictive value for specific drugs, although associations of CMS with outcome upon treatment with currently used combination therapies have been difficult and in part conflicting, demanding prospective trials to determine the true value of this classification in clinical practice.In summary, CMS subtyping of CRC is not routinely used in clinical practice, lacks standardization, and requires bioinformatics resources, which currently limits a more widespread use. On the other hand, commercially available and more easy-to-use prognostic expression-panels for early and intermediate-stage prognostication lack a clear biological interpretability, have no predictive value, and should only be used in specific clinical situations.Breast cancer is, to date, the most successful field regarding the clinical use of validated multi-gene prognostic tools, and the impact of genomic panels in BC management is noticeable. In the 8th cancer staging manual by the American Joint Committee on Cancer 2017 update, 5 multi-gene panels , investigating the predictive potential of Oncotype DX RS in patients with HR-positive, HER2-negative breast cancer and 1 to 3 involved axillary lymph nodes were presented [Oncotype DX  uses RNA isolation of FFPE breast cancer tissue followed by RT-PCR. It analyses a 21-gene signature, 16 cancer-related and 5 reference genes , selecteresented . Here, aTherefore, the Oncotype DX RS delivers important prognostic information but also predictive information on the benefit from adjuvant chemotherapy or the lack thereof in HR-positive, HER2-negative early invasive breast cancer with up to three involved axillary lymph nodes . It is cMammaPrint  requires FFPE or fresh breast cancer tissue to perform an RNA microarray analysis. Its 70-gene signature  is basedFinally, the prospective randomized Phase III MINDACT study investigated if patients considered to be clinically high-risk of recurrence as defined by the Adjuvant! Online tool but low genomic risk according to MammaPrint, could safely forego adjuvant chemotherapy. The study included 6693 women with early breast cancer and up to 3 metastatic axillary lymph nodes, and showed that chemotherapy could be avoided in such patients .However, unlike Oncotype DX, it has not shown an ability to directly determine the benefits of CT in any of its risk groups . MammaPrTo date, three commercially available signatures  are recommended by several guidelines as prognostic tools in HR_positive, HER2-negative breast cancer with up to 3 positive nodes, including the NCCN algorithm with a level of evidence 2A , the ASCEndopredict  is a 12-gene signature  that useProsigna/PAM50  is a 50 genes signature  that conIts use as a prognostic tool was validated in the ABCG8  and TranThe Breast Cancer Index  analyses 7 genes , 5 knownFewer studies have focused on triple-negative (TNBC) or HER2-positive disease, presumably because the indication for adjuvant chemotherapy is less uncertain in most of these cases. However, several gene expression studies on TNBC, have helped to increase the understanding of this heterogenous disease, which is not characterized by common features but rather by the common absence of the three markers. Initially, Lehmann et al. identified six biologically distinct subgroups within triple-negative breast cancer, basal-like 1, basal-like 2, luminal androgen receptor (LAR), mesenchymal, mesenchymal stem-like and immunomodulatory subtypes . The claCurrently, the commercially available BC genomic signatures help clinicians define the treatment in two specific clinical situations: supporting adjuvant chemotherapy in early ER+ HER2− BC patients and supporting adjuvant extended hormone therapy in post-menopausal ER+ BC patients.In early-stage estrogen-receptor positive and HER2 negative breast cancer patients with node-negative disease or with up to 3 positive lymph nodes, the use of the 21-gene Recurrence Score (Oncotype DX) is currently recommended to predict the benefit of adjuvant chemotherapy and is the only test that has been prospectively validated for this purpose. Patients 50 years of age or older with HR-positive, HER2-negative disease, and 0–3 involved lymph nodes can safely be spared adjuvant chemotherapy. In patients younger than 50 year with node-negative disease and a RS of >15, a small benefit from chemotherapy cannot be excluded and need a discussion on an individual basis. Patients younger than 50 with 1 to 3 involved lymph nodes should be offered adjuvant chemotherapy as they derive a small but statistically significant benefit regardless of the Recurrence score. Adjuvant chemotherapy should also be offered to all patients with a RS of >25. The 70-gene signature (MammaPrint) can also help to identify patients who are deemed at high clinical risk but are classified as genomic low-risk who have been demonstrated to have a favorable prognosis even without chemotherapy. Other multi-gene signatures offer important prognostic information and if they predict a low ROR are used to select patients for adjuvant endocrine therapy alone in routine clinical practice and based on guidelines, even though they have strictly not prospectively been demonstrated to predict the benefit of adjuvant chemotherapy.Prosigna, BCI, and EPclin have been demonstrated to predict late recurrences in patients with HR-positive, HER2-negative breast cancer after 5 years of endocrine therapy. BCI has also demonstrated to predict the benefit of extended endocrine therapy in the retrospective analysis of two clinical trials addressing this question  and is the only test recommended specifically for this purpose.However, these two questions are rarely regarded independently, so in clinical routine testing mostly is initiated after surgery to help the decision about adjuvant chemotherapy. These results will then influence the decision about extended endocrine therapy in addition to clinicopathologic features  Oncotype DX and MammaPrint have not provided similar evidence to predict late recurrences. In the case of MammaPrint, this is quite comprehensible, as it was specifically trained to identify patients with recurrences during the first 5 years after diagnosis. With respect to late recurrences, BCI offers the highest level of evidence .The development of high-throughput gene expression testing, addressing relevant clinical questions, has allowed an optimized use of the current therapeutic resources by reducing the rates of over/under treatment and avoiding unnecessary side-effects in cancer patients. To date, the field of breast cancer research has a lead in the area. Due to several factors including the significant interest in the disease, high incidence, mortality, and social burden, the identification of different biomarkers robustly linked to the patient’s prognosis and treatment response, and the identification of different molecular subtypes of the disease, each one with clearly distinguished medical treatments, was rapidly progressing. However, there remain open questions, and there is still some debate about the use of gene signatures in early BC with positive lymph nodes. The rXPONDER trial has recently answered this important aspect, but still reimbursement remains an issue in the setting and these signatures are globally not accessible to all patients, despite the fact that their use to avoid chemotherapy appears to be cost-effective ,115 and Hepatocellular carcinoma remains probably the most challenging entity. Arising mostly on the cirrhotic liver, comparison of diseased samples against non-tumorous tissue is difficult as the control tissue itself may have significant changes in gene expression. The analysis is even more complicated by a clearly heterogeneous tumor entity. Against this background, algorithms were developed that using a large number of random signatures and applying “swarm intelligence”  but alsoIn CRC, the classification in CMS partly using transcriptomic profiles also remains difficult. CMS were well evaluated in CRC studying two comparable Phase III trials. However, these studies led to completely different results. Difficulties here may be to classify all patient samples in one of four subtypes, characterized by multiple molecular features. Some samples may have mixed results characteristics which may not fit easily in one of the proposed categories. Instead of forcing those samples into one specific subgroup it may be more beneficial to the overall result to exclude some samples which are not easily classifiable. Although this will result in no prognosis prediction for some patients, CMS classification effectiveness and quality of the prediction for the remaining samples may improve and be more robust.Potential solutions for improving the gene expression analysis in particular hepatocellular carcinoma but also CRC and BC will certainly arise from a better definition of standard procedures for sample collection and preservation but also from standardized use of the diagnostic algorithms leading to the classification in gene expression analysis. Various publications have demonstrated the potential of integrative OMICS analyzes incorporating different biological levels in which the transcriptome may play a valuable role. This appearance of new integrative tools including multi-omics analysis could push the field forward and improve the clinical outcomes in these devastating diseases.


Microbes have an arsenal of virulence factors that contribute to their pathogenicity. A number of challenges remain to fully understand disease transmission, fitness landscape, antimicrobial resistance and host heterogeneity. A variety of tools have been used to address diverse aspects of pathogenicity, from molecular host-pathogen interactions to the mechanisms of disease acquisition and transmission. Current gaps in our knowledge include a more direct understanding of host-pathogen interactions, including signaling at interfaces, and direct phenotypic confirmation of pathogenicity. Correlative microscopy has been gaining traction to address the many challenges currently faced in biomedicine, in particular the combination of optical and atomic force microscopy (AFM). AFM, generates high-resolution surface topographical images, and quantifies mechanical properties at the pN scale under physiologically relevant conditions. When combined with optical microscopy, AFM probes pathogen surfaces and their physical and molecular interaction with host cells, while the various modes of optical microscopy view internal cellular responses of the pathogen and host. Here we review the most recent advances in our understanding of pathogens, recent applications of AFM to the field, how correlative AFM-optical microspectroscopy and microscopy have been used to illuminate pathogenicity and how these methods can reach their full potential for studying host-pathogen interactions. Despite tremendous progress in global health initiatives, the world continues to be confronted by infectious disease, underscoring the need for their survey, prevention, containment and treatment . HumanitVast technological developments over the past-decade in next-generation sequencing and proteomics have helped delineate the genetics of pathogenicity and mechanisms of host-pathogen interactions at the molecular level, respectively . Howeverin vitro methods  was originally developed  to charaAFM has become a powerful technique for probing live biological systems, providing information on diverse parameters such as morphology, surface ultrastructure and nano-mechanical properties (force nanoscopy) at the sub-molecular level . NotableEven though AFM is a powerful technique offering exceptionally high resolution, it has many inherent drawbacks; it is a surface scanning technique therefore providing minimal information on internal structures, samples need to be immobilized, and it has a small field of view with limited vertical range . AFM is Escherichia coli, and many other bacteria, surface sensing and attachment is mediated by long appendages called pili and fimbriae  between the two, originating from bacterial surface adhesins and specific ligands on the corneocyte surface. Using a combination of AFM, Raman micro-spectroscopy and scanning EM, S. aureus and Pseudomonas aeruginosa isolated from infected chronic wounds, and their adhesion forces to glass surfaces. The inverse relationship between viability and adhesion for the two species in the context of the multi-species biofilm, and the shift from a single to multilayer biofilm for the mixed-species underscores the complexity of clinical biofilms, which must be considered when developing treatment options. Pathogen adhesion to abiotic surfaces such as catheters and other medical instruments is a major source of secondary infections in hospitals  ranges or probing different cellular regions. AFM, with its unprecedented surface resolution, when combined with optical microscopy expands the FOV range from nm to mm to view the smallest molecules to the entire cell Figure 1Aspergillus nidulans and its mutants lacking the sugar galactofuranose, involved in pathogenicity and cell wall function, showed changes in cell wall biochemistry and surface roughness, best attributed to the cell wall integrity pathway  to illuminate an AFM tip for the dual purpose of nanoscale imaging while using near-field infrared spectroscopy to report on local chemical groups just nanometers below the tip . SINS of pathway . Kochan ctioning . Since tctioning  to SINS C. albicans, E. coli and HEK cells during induced oxidative stress , larger than the size of most subcellular structures, and only achievable under optimal conditions. Fluorescence was added to the optical microscopy suite in the early 1900s, offering enhanced contrast, single protein specificity and sensitivity . Wide fie stress . This ste stress Figure 2rescence . El-KiraC. albicans biofilms during exposure to the antimicrobial defensin Psd1, using AFM to follow membrane disruptions and leakage in tandem with confocal to track C. albicans viability within the biofilms. K. pneumoniae ATCC13882 and clinical isolates, using AFM to characterize the biofilm surface ultrastructure and confirm nanoparticle morphology while fluorescence microscopy visualized biofilm eradication during the chlorohexidine treatment. Such research questions are well served using a correlative microscopy approach.Both AFM and confocal microscopy have been instrumental in understanding biofilm phenotype, ultrastructure and behavior. Control and treatment of biofilm forming pathogens is a serious clinical challenge based on their ability to resist commonly prescribed antimicrobials . GonçalvYersinia pseudotuberculosis adhesion to HeLa cells to examine the recruitment of glycosylphosphatidylinositol (GPI)-anchored protein domains to the binding site at the bacterial cell surface, with concomitant cytoskeletal re-arrangements. Bacteria-functionalized AFM cantilevers delivered bacteria to the HeLa surface where a suite of fluorescence microscopy techniques  were used to view GPI dynamics during adhesion and entry, comparing the concomitant actin remodeling and cell signaling activated during pathogenic adhesion versus that during internalization , photoactivated localization microscopy (PALM), and other forms of single-molecule localization microscopy (SMLM) can be used to visualize individual proteins in live cells by virtue of highly specific dyes that can be switched on and off . The asslization .Yersinia pseudotuberculosis infection in Ptk2 cells, revealing its precise location along with that of host organelles, and the mechanical properties of each .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.


Ample evidence hasindicated that reduced food-intake significantly delays the onset of age dependentdeterioration in neural, metabolic and cardiovascular systems and extend life-span fromyeast, fly, rodents to non-human primates. Indeed, highly conserved nutrient sensingmolecules and key metabolic regulators, such as mTOR, IGF1 and AMPK, have beenimplicated as the key players in the interaction between diet and health span. An exciting progress in the field ofaging studies is the emerging recognition that dietary restriction mediated benefits maybe not due to the reduction of total calorie intake but rather resulted from restrictedintake of specific macronutrient components. More specifically, protein restriction isshown to be responsible for the life-span extension effect of food/caloricrestriction. However, theprecise components in dietary protein responsible for the life-span modulatory effectremain to be clearly defined. Recently, two studies from Lamming’s laboratoryoffered further evidence that branched-chain amino acids  may hold the key to protein restriction mediated health benefitsduring aging,6.In our ongoing quest to unlock the secrets for longevity and healthy aging, oneof the most conserved and reproducible interventions proven to extend life-span acrossthe evolutionary tree is dietary restrictionet al., two models of mutant mice with acceleratedaging phenotype were first utilized. One is theLmnaG609G/G609G mouse which carriesthe same disease-causing mutation in the Lmna gene forHutchinson-Gilford Progeria Syndrome. Another isLmna−/− completeknockout mouse with complete deletion of the Lmna gene. Both mousemodels developed progressive cardiomyopathy associated with accelerated aging. Authorstreated these mice from weaning with either a low BCAA or a low total amino acids diet.The low BCAA diet treatment showed a significant extension in life span similar to theeffect of low amino acid diet observed in femaleLmna−/− mice. Asignificant extension in life span was also made in both male and femaleLmnaG609G/G609G mice. When implementedin wildtype mice, life-time feeding of a low BCAA diet extended healthy span (asmeasured by age-dependent changes in obesity and frailty index) in both male and femalemice, but significantly extended life span only in male mice. In contrast, when low BCAAdiet was initiated in mid-aged mice (16 months of age), aging related frailty wasimproved but no significant extension of life span was observed. These findings offeredfirst in vivo evidence that a BCAA restricted diet might be sufficientto confer beneficial effect on healthy aging in mammals. Considering the knowncomplications associated with low-protein diet in causing frailty and sarcopenia,results from Richardson et al. suggests that low-BCAA diet is able to prolong life-span whileimproving frailty and metabolic health in aged mice. This may have significantimplications in future clinical translation. However, the study is still preliminary,and the benefits of low-BCAA diet are not yet fully characterized at organ physiologylevels, including cardiovascular, respiratory, neural and metabolic systems. Moreimportantly, the study also reveals the potential complications and limitations of BCAAtargeted dietary manipulation. The low-BCAA diet mediated modulation on life-spanappears to be influenced by sex but the mechanism is unknown. In addition, low-BCAA diet failed to improvecardiac deterioration in the Lmna−/− micedespite of a positive effect on life-span, suggesting that low-BCAA diet may affectother organs vital to longevity. Finally, none of the dietary manipulation was performedin mice with pre-existing conditions, such as cardiometabolic disorders. Therefore,low-BCAA mediated anti-aging effect is only demonstrated in a prophylactic manner andits application as a therapeutic strategy to halt aging process, particularly amongvulnerable populations with pre-existing mortality risks remain to be demonstrated.In the study by Richardson . BCAA is known to link nutrient environment withcellular growth and metabolism through well established mTOR and other signalingpathways. Therefore, it is actually quite assuring that through unbiased transcriptomeprofiling in skeletal muscle tissue, authors indeed found that the molecular signaturesassociated with different BCAA treatment groups are highly enriched with genes involvedin mTOR mediated downstream signaling pathways, such as phagosome function (forautophagy) and PI3K-AKT pathway.At mechanistic level, the anti-aging effect of low-BCAA diet highlights theimportance of BCAA as critical nutrients as well as potent nutrient signal molecules fororgan function and homeostasis. Indeed, BCAA have unique contribution to nutrientsignaling relative to all other essential amino acidset al. further delineated the specific role of each individual BCAA indietary impact on metabolic health.Giving the potential impact of BCAA restricted diet in aging modulation, and nextobvious question is which of the three BCAAs are responsible for the observed effect.BCAA are essential amino acid with three members, valine, leucine and isoleucine.Because they share the same rate-limiting enzymes as part of their individual catabolicpathways, the homeostatic regulation of the three BCAA is often intricately correlated.However, extensive evidence suggests each of the BCAA may have unique and distinctfunction in metabolic regulation and nutrient signaling. In a subsequent study by thesame group, Yu et al. systematically measured the impact oflowering individual BCAA intake on the metabolic health in mice. While lowering BCAA asa group showed potent and unique effect to improve metabolic status comparing to otheressential non-BCAA amino acids, lowering individual BCAA in food intake revealed astrong effect of valine and isoleucine on glucose control, insulin resistance andhepatic gluconeogenesis. In contrast, lowering leucine showed no effect on the samemetabolic indexes. Conversely, when individual BCAA was added back to the low-amino aciddiet, isoleucine and valine were able to at least in part abolish the beneficial effectsconferred by low-amino acid diet. Again, adding back leucine did not have such effect.These results suggest that isoleucine and valine are potentially responsible for themetabolic outcome from BCAA manipulation, instead of leucine. Such a revelation issomewhat unexpected, but consistent with the observation made in liver-specificTsc1 knockout and Gcn2 knockout mice by theauthors. Yu et al.demonstrated that isoleucine restriction mediated beneficial effects were not affectedin these mice, implicating an mTOR and Gcn2 independent mechanism, both pathways areknown mediators of leucine induced signaling in cells. Instead, from transcriptomeanalysis in liver and adipose tissues, authors identified a significant perturbation inFGF21 expression and energy expenditure following isoleucinerestriction, which was necessary for low-isoleucine diet mediated metabolic protectionin mice. Therefore, as nutrient signaling molecules, individual BCAA exerts distinctdownstream activity in metabolically critical tissues, such as liver, muscle andfat.Using a series of amino acid defined diets, Yu vs. deleterious effects of BCAA in aging process. These insightswill not only provide vital information for future development of anti-aging mimics tocombat age related diseases, but may also raise necessary awareness to the dietaryintake of BCAA from our daily meals.Although many questions still remain to be answered for the physiologicalimportance and molecular mechanisms in BCAA mediated modulation in aging, these recentfindings continue to foster the increasing recognition that BCAA homeostasis andregulation are central to health span and longevity . BCAA is