Candida oleophila, Aureobasidium pullulans, Metschnikowia fructicola, Cryptococcus albidus, Saccharomyces cerevisiae) that are or have been registered for the application as biocontrol products. These examples demonstrate the potential of yeasts for commercial biocontrol usage, but this review also highlights the scarcity of fundamental studies on yeast biocontrol mechanisms and of registered yeast-based biocontrol products. Yeast biocontrol mechanisms thus represent a largely unexplored field of research and plentiful opportunities for the development of commercial, yeast-based applications for plant protection exist.Yeasts occur in all environments and have been described as potent antagonists of various plant pathogens. Due to their antagonistic ability, undemanding cultivation requirements, and limited biosafety concerns, many of these unicellular fungi have been considered for biocontrol applications. Here, we review the fundamental research on the mechanisms  by which biocontrol yeasts exert their activity as plant protection agents. In a second part, we focus on five yeast species (The online version of this article (10.1007/s11274-019-2728-4) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae, enables taking advantage of the molecular tools and plethora of data developed for these organisms for application-oriented and basic studies on biocontrol yeasts.Despite their relevance as model eukaryotes for biotechnological applications and in medical mycology, the potential use of antagonistic yeasts as biocontrol agents is still underexploited. Only a handful of yeast-based plant protection products has reached the market and even in fundamental research, antifungal yeasts have been neglected and poorly investigated with state-of-the-art technology and at the molecular level. Nevertheless, yeasts combine strong antifungal activities with advantageous properties for an application  and are thereby promising for the development of biological plant protection agents. Furthermore, the close relatedness with model yeasts, particularly Biocontrol is mostly looked at and studied in a species/isolate-centric manner: different species/isolates are tested against the target plant pathogen and the most active organism is studied with respect to its potential for biocontrol applications. However, for a successful application and improvement of biocontrol organisms, we first have to understand the biocontrol mechanisms involved and then confirm their expression under field conditions  . This may be the reason why yeasts sensu lato are generally regarded as safe and therefore applying yeasts in crops and on food products elicits less concern than applications of bacteria or filamentous fungi , which are closely related to some biocontrol species, efficient resource depletion, due to the duplication of nitrogen transporter and metabolism genes, causes priority effects  and thus acts as a driver of competitiveness among different species  was identified and shown to exhibit antibacterial activity  angusta haploid strain to identify biocontrol-minus mutant clones: while the wild-type strain proved effective in reducing brown rot lesion caused by Monilinia fructicola on apple fruit, its derivate leucine-auxotrophic mutant L1 had no significant effect in controlling the pathogen. The addition of exogenous leucine fully restored the biocontrol capability of mutant L1, whereas a leucine stand-alone treatment showed no significant biocontrol effect  has been proposed as a potential biohazard factor for biocontrol yeasts   that are released from “prey” cells, which may lead to the killing of these cells . Secreted enzymes such as chitinases, glucanases, or proteases are thus regularly reported and highlighted in antagonistic yeasts and implicated in their biocontrol activity.Aureobasidium, Candida, Debaryomyces, Metschnikowia, Meyerozyma, Pichia, Saccharomyces, Tilletiopsis, and Wickerhamomyces and in Saccharomycopsis, chitinase expression was detected in the presence of prey cells (Supplementary Table 1)  from Candida oleophila was the first gene cloned in this organism , thereby proving the involvement of glucanases in yeast biocontrol activity , the deletion of the two exo-β-glucanases (PaEXG1 and PaEXG2) significantly reduced biocontrol activity on fruits against Botrytis cinerea  exoglucanase genes was shown during the interaction with plant pathogenic fungi on infected fruits or growth with fungal cell wall preparations  transcripts were significantly enriched during predation, but neither functionally investigated . Since protease activity was only detected at later growth stages (after 6–8 days of growth in nutrient rich medium) in Pseudozyma flocculosa  produces diverse polymers ), lipids, volatiles, enzymes, and secondary metabolites. Some of these metabolites  confer antagonistic activity against bacteria or fungi  on antagonistic activity in more humid environments . Flocculosin is a low molecular weight cellobiose lipid produced by the biocontrol yeast A. pullulans proved efficient in reducing the growth and infection by B. cinerea, C. acutatum, P. expansum, P. digitatum and P. italicum both in vitro and in planta  are small  molecules with low water solubility and high vapour pressure. VOCs include a panoply of molecular classes, including hydrocarbons, alcohols, thioalcohols, aldehydes, ketones, thioesters, cyclohexanes, heterocyclic compounds, phenols and benzene derivatives   is rarely described and poorly studied in yeasts. S. cerevisiae, Rhodosporidium paludigenum, Candida saitoana, C. oleophila and Metschnikowia species induce an innate immune response and eventually cause resistance against phyllosphere pathogens in fruits  that are currently or have been registered as plant protection agents.There is a huge discrepancy between the plethora of “biocontrol yeasts” described in scientific publications and the few yeast-based plant protection products that are registered and marketed as plant protection products. A range of factors  are likely responsible for this apparent difficulty to develop yeast-based plant protection products. Here, we briefly highlight five yeast species , combinations with fungicides, different buffers , chitosan, or lysozyme were studied  and DSM 14941 (CF 40) are registered, in mixture, as active ingredients of plant protection products against the fireblight disease caused by the bacterium Erwinia amylovora and postharvest diseases (European Food Safety Authority (EFSA) A. pullulans strains were selected based on their strong inhibition towards E. amylovora in co-culture experiments at high synthetic nectar concentration (25%)  may also be involved. Specific metabolites or enzymes and their contribution to the biocontrol activity of DSM 14940 (CF 10) and DSM 14941 (CF 40) have not been identified and the strains do not seem to have been characterised genetically. In contrast to most registered plant protection products, including biological products, the original Blossom-Protect® has a rather limited range of application. The expansion to novel indications, beyond fireblight of pome fruit trees, is thus certainly also motivated by economic needs.The two registered products containing Metschnikowia comprises species of mainly phyllosphere and nectar yeasts that are globally distributed , secretion of glucanases and chitinases, and the production of volatile organic compounds  also induces an oxidative burst in the plant tissue that eventually results in the activation of plant defense responses  (European Food Safety Authority (EFSA) Originally, Cryptococcus are widespread in nature and frequently isolated from water sources, soil and decaying plant material. Studied for their potential to produce high lipid yields for biodiesel production, strains of C. albidus, C. laurentii and C. flavus have also been shown to protect peach, cherry, strawberry, tomato, citrus and pome fruits against postharvest decay  reduced the growth of plant pathogens such as A. carbonarius, A. ochraceus, A. parasiticus or Fusarium graminearum and also inhibited mycotoxin  production by these species , as well as direct transcriptional downregulation of polyketide synthesis  induces the expression of genes involved in the plant response to fungal attack  is the product Romeo® with cerevisane® as the active ingredient (European Food Safety Authority (EFSA) A number of The disparity between the number of yeast species exerting biocontrol activity against specific plant pathogens in laboratory assays and the number of yeast that are actually registered and successfully employed as plant protection products is likely caused by the lack of mechanistic understanding, the costs of registration, the lack of partners/consortia with required expertise, or a limited commercial potential. However, the general trend towards reduced pesticide use will certainly favour and create more incentives for the development of alternative plant protection solutions such as biocontrol yeasts. In the environment, these organisms interact intraspecifically, as well as with other microbes  and host plants. These complex interactions and interdependencies eventually determine whether a disease sets in or an antagonistic yeast suppresses a pathogen and supports plant health. It is impossible to manage—for plant protection applications—such complex interactions without detailed knowledge of the interacting bionts. By studying and identifying modes of action in the laboratory, a reductionist approach is thus an important first step in the development and successful application of biocontrol in general.Nowadays, the breakthrough achievements in the field of system biology, molecular biology and the related computational tools enable revealing the structural and functional peculiarities of any potential biocontrol agent. Exploiting these tools for the investigation and prediction of functional dynamics occurring between antagonists opens new avenues for the design of consortia of microbial antagonists that synergistically cooperate for the biocontrol of plant pathogens. Although strain mixtures for the biocontrol of plant pathogens are already available commercially  and described in the literature  were rarely used in biocontrol yeasts, even though these technologies have the potential to empower studying and understanding these organisms at a whole new level  biocontrol product depends on a chain of activities and disciplines, from science to industry and legislation, which have to come together, interact and build upon one another. Strengthening the exchange and interaction among these disciplines is thus essential to foster the commercialisation of biocontrol products Below is the link to the electronic supplementary material.


STF statistics. Here, we use a population genetic model to evaluate whether these observations could plausibly be produced by sexually antagonistic selection. To do this, we use both a single-locus model with nonrandom mate choice, and individual-based simulations to study the relationship between strength of selection, degree of between-sex divergence, and the associated genetic load. We show that selection must be exceptionally strong to create measurable divergence between the sexes and that the decrease in population fitness due to this process is correspondingly high. Individual-based simulations with selection genome-wide recapitulate these patterns and indicate that small sample sizes and sampling variance can easily generate substantial male-female divergence. We therefore conclude that caution should be taken when interpreting autosomal allelic differentiation between the sexes.Since the autosomal genome is shared between the sexes, sex-specific fitness optima present an evolutionary challenge. While sexually antagonistic selection might favor different alleles within females and males, segregation randomly reassorts alleles at autosomal loci between sexes each generation. This process of homogenization during transmission thus prevents between-sex allelic divergence generated by sexually antagonistic selection from accumulating across multiple generations. However, recent empirical studies have reported high male-female  Females and males use largely the same genome to produce distinct phenotypes and behaviors. This ubiquitous phenomenon requires an association between dimorphic phenotypes and their sexual environment . Genes rDrosophila melanogaster. Taken at face value, both the number of sexually antagonistic alleles and the degree of differentiation are striking. However, these results are difficult to evaluate as they suggest that there must be quite a large amount of selective mortality within each sex to create such high divergence within a generation  between the sexes. First, sex biases in chromosome segregation through associations with the sex determining region could distort allele frequencies between the sexes. Over time, this segregation distortion can contribute to the generation of neo-sex chromosomes . Both ofIn this paper, we will first build a model of sexually antagonistic viability selection, segregation, and transmission, extending the model of t, the genotype frequencies in females after selection will then be proportional to Consider an autosomal locus in which are found two alleles: one female-beneficial  to find the equilibria of this system and determine stability of those equilibria. The complete notebook is provided in File S1.i.e., genetic load), is generated each generation. Within each sex, the genetic load is the difference between the maximum possible fitness and the mean fitness (L) is the average of the loads for each sex , which is given by:Sex-specific viability selection creates differences in allele frequencies between the sexes each generation. We can therefore quantify the effects of sexually antagonistic selection using the male-female ss sexes :FST=.We also implemented simulations with sexually antagonistic selection acting at many loci, genome-wide with SLiM v3.1, an evolution simulation framework   conditions. Our single locus results expand upon previous the results of previous studies (s) and the ratio of selection coefficients (α), so that We will quantify the strength and degree of asymmetry between the sex-specific allelic effects using the overall strength (α in terms of the strength of selection (s), we find that for the equilibrium to be stable, α and s must satisfy the condition:s, equation (7) shows that the equilibrium is stable only if asymmetry is not larger than the strength of selection, such that Assuming random mating and additivity of allelic effects . Unless selection was very weak . However, entirely neutral simulations  resulted in the same mean and SD of male-female Our analytical results are based on a single-locus model, yet empirical studies report averages across large numbers of loci. To complement the single-locus theory, we quantified the effects of sexually antagonistic selection throughout the genome using individual-based simulations in SLiM an 0.001 . Sexuallentire population. To evaluate the effect of sampling, we calculated male-female Both the single locus model and genome-wide simulations indicate that, while theoretically possible, we would need strong sexually antagonistic viability selection to maintain high divergence between the sexes. Alternatively, the large observed ulations . Additiounbiased estimates of Empirical studies often use estimators of hod  Cheng a priori reason to expect that antagonistic mutations should be perfectly symmetrical in their effects and therefore that polymorphic loci should be stable over time. Alleles with more asymmetric effects will often sweep, producing larger but transient between-sex divergences, although again only under moderate to strong selection. Of course, other types of selection  could contribute to maintenance of more asymmetric polymorphisms, but the observations about genetic load should still hold. Here, we found that dominance has little quantitative effect on male-female divergence, particularly when selection is weak. In general, understanding what the distribution of sex-specific effects underlying antagonistic selection looks like will provide important information on the potential for sexually antagonistic loci to contribute to genetic variation and genome evolution.Second, asymmetry in the strength of selection between the sexes is critical in determining the degree of divergence generated. When the strength of selection is weak and approximately the same between the sexes, polymorphisms may be stably maintained, but between-sex divergence is small. However, there is no Thus, the theoretical predictions from our single locus model seem at odds with the empirical patterns reported to date. Taken as true measurements of sexually antagonistic selection, the empirical data could be described by two, non-exclusive genomic patterns. Divergent loci could either be stable polymorphisms or could be arising and sweeping to fixation through a constant genomic churn of antagonistic interactions. Either of these explanations require an exceptionally high genetic load. Again, there is currently no indication that mortality occurs in such a high, sex-specific manner, particularly in some of the vertebrate species that have been examined.Individual-based simulations with many linked selected loci genome-wide recapitulate the predictions of the single-locus model, finding again that even in this more complex situation, weak selection can only produce very low levels of divergence. Most importantly, however, we found that estimating male-female e.g., overall expression level). Furthermore, population substructure remains a concern even when comparing to permuted data if there is a cryptic correlation of sampling with sex. For instance, suppose that the sampled population is composed of a mixture of two diverged subpopulations, and that the sex and admixture coefficients of the sampled individuals are correlated. As noted by At the very least, studies analyzing male-female Furthermore, some studies report large numbers of loci with high average More generally, our investigation calls into question whether it is even possible, at any sample size, to identify the action of realistically strong sex-specific antagonistic selection using male-female


Assessing progression of disease or response to treatment remains a major challenge in the clinical management of nontuberculous mycobacterial (NTM) infections of the lungs. Serial assessments of validated measures of treatment response address whether the current therapeutic approach is on track toward clinical cure, which remains a fundamental question for clinicians and patients during the course of NTM disease treatment. The 2015 NTM Research Consortium Workshop, which included a patient advisory panel, identified treatment response biomarkers as a priority area for investigation. Limited progress in addressing this challenge also hampers drug development efforts. The Biomarker Qualification Program at the FDA supports the use of a validated treatment response biomarker across multiple drug development programs. Current approaches in clinical practice include microbiologic and radiographic monitoring, along with symptomatic and quality-of-life assessments. Blood-based monitoring, including assessments of humoral and cell-mediated NTM-driven immune responses, remain under investigation. Alignment of data collection schemes in prospective multicenter studies, including the support of biosample repositories, will support identification of treatment response biomarkers under standard-of-care and investigational therapeutic strategies. In this review, we outline the role of treatment monitoring biomarkers in both clinical practice and drug development frameworks. Nontuberculous mycobacteria (NTM) are ubiquitous environmental organisms capable of causing significant morbidity  and mortIn this review, we address uncertainties regarding the clinical “waypoints” of NTM treatment. Intermediate and definitive markers of treatment response provide crucial feedback to NTM patients, their clinicians, and developers of novel therapies. We outline the role of treatment monitoring biomarkers in both clinical practice and drug development. We summarize the existing knowledge base regarding biomarkers of treatment response for NTM disease and we identify areas for ongoing and potential future investigation.In his seminal framework for defining surrogate endpoints in clinical trials, Ross Prentice proposed that a valid surrogate endpoint must “capture” the underlying relationship between the intervention and the clinical endpoint, allowing for comparison of intervention groups based on the surrogate rather than the definitive clinical endpoint at any time during the follow-up period . By compThe BEST  Resource, published by the FDA-NIH Biomarker Working Group, provides descriptions and examples of disease monitoring biomarkers, which are “assessed serially over time to measure presence, status, or extent of disease or medical condition, or to provide evidence of an intervention effect or exposure, including exposure to a medical product or environmental agent” .Thus, the interpretation of a monitoring biomarker is not based on a snapshot level, as with a baseline clinical predictor of response to therapy, but rather on changes in values during serial measurements. As examples, quantification of HIV RNA in blood serves as a monitoring biomarker for the clinical efficacy of antiretroviral therapy, and the amount of air that an individual can forcefully exhale in one second (FEV1) reflects response to chronic obstructive pulmonary disease management. Ideally, a treatment monitoring biomarker could be readily adopted into existing procedures that support routine use in clinical settings .The therapeutic approach to NTM lung disease lacks the evidence base of tuberculosis, where standard-of-care combination drug regimens are based on randomized clinical trials that enrolled tens of thousands of tuberculosis patients. Unlike tuberculosis, the NTM lung disease research agenda has not been propelled by public health pressure or urgency. Consequently, much of the clinical approach to NTM disease rests on a foundation of observational data and expert opinion, supported by only a few randomized clinical trials . In geneM. abscessus complex, the optimal time point for transitioning to an all-oral regimen is uncertain. Surgical interventions may be the most appropriate approach for a subset of patients with severe M. abscessus complex lung disease, but potentially could be deferred in a subset of patients that are responsive to medical management , the use of a biomarker in the context of drug development relies on “qualification” that specifies its contextual use and interpretation. The Biomarker Qualification Program at the FDA provides a pathway for a particular biomarker to become accepted into the regulatory framework independent of approval of a specific drug . This prPresently, culture of serial sputum samples serves as the primary biomarker for monitoring the response to treatment of mycobacterial lung infections. In 2016, an expert panel composed of NTM clinicians reached consensus definitions for several endpoints related to the treatment of NTM lung disease, including cure, relapse, and re-infection . Serial M. avium complex (MAC), grouping patients into converters and non-converters based on sputum culture status after 12 months of treatment. Although baseline semi quantitative culture scores were not significantly different between converters and non-converters, the change in semi quantitative culture scores after 2 months of MAC-directed treatment was highly predictive of sputum culture conversion, with each 1-point decline in the scoring scale associated with a 20% increase in the likelihood of achieving conversion at 12 months. Importantly, early microbiologic response also correlated with symptomatic and radiographic improvements, suggesting that semi quantitative culture data can be explored as a surrogate endpoint in drug development. One limitation of this approach may be that some NTM lung disease patients do not expectorate sputum  does not use ionizing radiation this imaging modality has been studied for assessment of cystic fibrosis and sarcoidosis, performing comparably to chest CT in identifying various pathologic patterns of the disease in lung parenchyma. Chung and associates have proposed that MRI may emerge as a viable alternative to CT imaging for assessing the distribution and extent of NTM lung infection . In an e18F-fluorodeoxyglucose positron-emission tomography [18F-FDG PET] is a routine component of monitoring response to chemotherapy for lung cancer, based on the detection of increased glucose metabolism that is characteristic of malignant lesions. More recently, the use of PET modalities has been examined for diagnosis and treatment monitoring of tuberculosis, detecting the increase in glucose metabolism associated with the host inflammatory response . Furthermore, patients undergoing treatment for extra-pulmonary forms of NTM disease could potentially benefit from a validated blood-based biomarker of treatment response, for example during treatment of deep tissue infections such as osteomyelitis, where clinical response is less apparent on physical examination. Immune-based biomarkers of treatment response may also shed insight into the immunologic correlates of protection against disease in at-risk patient populations.The Capilia MAC Ab ELISA kit  developed for the diagnosis of MAC lung disease, measures serum levels of IgA antibodies against glycopeptidolipid (GPL), a major component of the MAC cell wall . Serum aCell-mediated immune responses, rather than antibody responses, could provide an alternate approach for immunologic monitoring of treatment response. Carbohydrate antigen 19-9 (CA 19-9) is a treatment monitoring biomarker for several forms of cancer. Elevated blood levels of CA 19-9 are also seen in various forms of chronic lung disease, including bronchiectasis. Recently, CA 19-9 was examined as biomarker of treatment response among 24 patients with NTM lung disease in South Korea . Among 1M. abscessus infection, early improvements in FEV1 after treatment initiation (either 30 or 60 days) were not associated with long-term microbiologic outcomes or sustained improvements in pulmonary function . In a single center study of 37 cystic fibrosis patients with pulmonary function .Clinical research centers specializing in the treatment of NTM lung disease will benefit from alignment of prospective data collection schemes that include specimen repositories for blood , sputum, and urine. The Bronchiectasis and NTM Research Registry, originally designed to enroll patients with bronchiectasis without underlying cystic fibrosis, supports serial banking of blood and deep respiratory specimens . This frM. abscessus complex. These types of large scale, multicenter studies will also be essential to the prospective evaluation of novel treatment regimens for NTM lung disease. Multiple enrollment sites will be required to provide the statistical power for comparing alternative therapeutic strategies, given the smaller number of NTM lung disease patients compared with tuberculosis on a global scale. Furthermore, clinical studies supported by the National Institutes of Health  remains a central question for NTM patients and their physicians. It is uncertain whether a single biomarker of clinical response will sufficiently capture the complexity of NTM disease presentations in a heterogeneous patient population, or whether the current “all of the above” strategy can be improved with refinement of microbiologic, radiographic, and quality-of-life assessments. Should a composite biomarker be developed under the FDA Biomarker Qualification program, it would require a “Context of Use” defining the specific algorithm for combining these individual components into a single treatment response measure. Ongoing research efforts for NTM disease should support the evaluation of biomarkers of response to treatment in parallel with studies of novel therapeutics and mechanisms of disease. Patients, clinicians, and investigators will benefit from the knowledge that the road they travel leads toward clinical cure.AM, CV, JH-F, and KH performed the literature review. CV wrote the initial draft. AM, HO, RK, JH-F, and KH critically appraised and revised.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.


Low-cost, high-throughput nucleic acid sequencing ushered the field of microbial ecology into a new era in which the microbial composition of nearly every conceivable environment on the planet is under examination. However, static “screenshots” derived from sequence-only approaches belie the underlying complexity of the microbe-microbe and microbe-host interactions occurring within these systems. Bacillus-Streptomyces, Aliivibrio fischeri-Hawaiian bobtail squid, and gnotobiotic mice) at various levels of taxonomic complexity and experimental control used to gain molecular insight into microbe-mediated interactions. We argue that when studying microbial communities, it is crucial to consider the scope of questions that experimental systems are suited to address, especially for researchers beginning new projects. Therefore, we highlight practical applications, limitations, and tradeoffs inherent to each model.Low-cost, high-throughput nucleic acid sequencing ushered the field of microbial ecology into a new era in which the microbial composition of nearly every conceivable environment on the planet is under examination. However, static “screenshots” derived from sequence-only approaches belie the underlying complexity of the microbe-microbe and microbe-host interactions occurring within these systems. Reductionist experimental models are essential to identify the microbes involved in interactions and to characterize the molecular mechanisms that manifest as complex host and environmental phenomena. Herein, we focus on three models ( Microbiomes shape the fundamental biology of environments and can have substantial impacts on macroscopic ecosystems. Within their hosts, microbiomes alter metabolism, behavior, and disease. Experimental insight into the molecular mechanisms underlying microbiome interactions remains elusive. High complexity, variable plasticity, and low manipulability of natural systems remain barriers to recapitulating microbiomes in the laboratory.Drosophila can mediate changes in host life span and fecundity, with some members influencing these traits only in the presence of certain other community members  on agar plates, providing macroscopic visualization of interactions. Together, the ability to perform mutagenesis screens, generate targeted gene deletions and complements, extract secondary metabolites in isolation, and easily adjust medium and plating configurations to uncover new macroscopic phenotypes all contribute to this system’s high level of experimental manipulability.Among the simplest model systems for exploring microbial interactions are pairwise interactions between culturable bacteria. Importantly, these systems intrinsically offer high experimental control to study the molecular underpinnings of interactions that occur between and within microbial communities. As an example, coculture of the soil bacteria Bacillus and Streptomyces demonstrate that secondary metabolites have multiple roles mediating competition  specifically establishes a symbiosis within the light organ of newly hatched Hawaiian bobtail squid (Euprymna scolopes). This symbiosis has proven an excellent system to investigate colonization dynamics and specificity: though the ocean harbors an incredibly complex microbial community , the relatively rare A. fischeri  specifically colonizes the light organ , but specific mechanisms may differ. For example, facultatively anaerobic pathogens exploiting inflammation-associated oxidation in the typically anaerobic mouse gut would not be readily apparent in aerobic Drosophila guts . Experimenting with multiple levels of community complexity applies to germfree hosts beyond mice (e.g., ila guts , 53. Furila guts . UltimatBy leveraging the unique features of experimental microbiome systems, important and outstanding questions can be addressed . Chief a58–As microbiome research continues, new frameworks for characterizing the interactions that occur within microbial communities will emerge from novel systems spanning the spectra of complexity and tractability and developments enabling established systems to address new questions. As examples, two particular systems that we are especially interested in are the cheese rind microbial community and the gardens of fungus-growing ants. The cheese rind microbial community is an emerging system particularly suitable for characterization of multipartite interactions and simulating ecological phenomena through control of abiotic factors –64, yet –68–In conclusion, delineating community states that contribute to emergent properties and complex interactions will require experimental models, and the ideal balance between a model’s complexity, ease of manipulation, and overall biological relevance will depend upon the scientific questions posed.


Although mortality due to critical illness has fallen over decades, the number of patients with long-term functional disabilities has increased, leading to impaired quality of life and significant healthcare costs. As an essential part of the multimodal interventions available to improve outcome of critical illness, optimal nutrition therapy should be provided during critical illness, after ICU discharge, and following hospital discharge.This narrative review summarizes the latest scientific insights and guidelines on ICU nutrition delivery. Practical guidance is given to provide optimal nutrition therapy during the three phases of the patient journey.Based on recent literature and guidelines, gradual progression to caloric and protein targets during the initial phase of ICU stay is recommended. After this phase, full caloric dose can be provided, preferably based on indirect calorimetry. Phosphate should be monitored to detect refeeding hypophosphatemia, and when occurring, caloric restriction should be instituted. For proteins, at least 1.3 g of proteins/kg/day should be targeted after the initial phase. During the chronic ICU phase, and after ICU discharge, higher protein/caloric targets should be provided preferably combined with exercise. After ICU discharge, achieving protein targets is more difficult than reaching caloric goals, in particular after removal of the feeding tube. After hospital discharge, probably very high-dose protein and calorie feeding for prolonged duration is necessary to optimize the outcome. High-protein oral nutrition supplements are likely essential in this period. Several pharmacological options are available to combine with nutrition therapy to enhance the anabolic response and stimulate muscle protein synthesis.During and after ICU care, optimal nutrition therapy is essential to improve the long-term outcome to reduce the likelihood of the patient to becoming a “victim” of critical illness. Frequently, nutrition targets are not achieved in any phase of recovery. Personalized nutrition therapy, while respecting different targets during the phases of the patient journey after critical illness, should be prescribed and monitored. Advances in ICU care allow for prolonged survival by providing life-sustaining support, making previously nonsurvivable ICU insults survivable. Innovations in ICU medicine have resulted in yearly reductions in hospital mortality . HoweverIn 2012, the post-intensive care syndrome (PICS) definition was agreed upon by Needham et al. as the recommended term to describe new or worsening problems in physical, cognitive, or mental health status arising after a critical illness and persisting beyond acute care hospitalization . Since tProper timing of nutrition therapy and optimal dosing has been suggested as critical illness and recovery metabolism changes throughout a patient’s course and energy expenditure and nitrogen losses appear to vary over time . NutritiThis narrative review provides practical guidance on nutrition therapy for the ICU, post-ICU, and long-term convalescence phases, based on recent literature and guidelines. The key role of personalizing and timing the provision of macronutrients  will be discussed.The European Society for Clinical Nutrition and Metabolism (ESPEN) recently published evidence-based guidelines on medical nutrition therapy for critically ill patients . Early eWhen to start EEN in patients in shock is a matter of debate; however, EN can be commenced after the initial phase of hemodynamic stabilization, and it is not necessary to delay EN until vasopressors have been stopped , 13. In Based on pathophysiological insights from metabolism in the early phase of critical illness, this phase is characterized by inflammation, increased energy expenditure, insulin resistance, and a catabolic response leading to generation of energy from stores such as hepatic glycogen (glucose), fat (free fatty acids), and muscle (amino acids). Feeding ICU patients is essentially different compared with feeding the healthy . The endThis U-shaped association was less clear when the results of the PERMIT trial on permissive underfeeding versus normocaloric feeding or energy-dense feeding versus normocaloric feeding in the TARGET trial are interpreted , 20. In Available data suggest that early overfeeding should be prevented and that hypocaloric or normocaloric feeding does not confer major differences in outcome when protein intake is similar. Aggressive early caloric intake leads to more episodes of hyperglycemia and need for high-dose insulin therapy, as was observed in the TARGET and EAT-ICU trials , 22. As Although refeeding syndrome (RFS) characterized by electrolyte shifts in response to reintroduction of nutrition after a period of starvation is ill-defined and many definitions are used, it can be best identified in ICU patients by refeeding hypophosphatemia (drop below 0.65 mmol/l within 72 h after the start of nutrition therapy) –25. SeveBeneficial outcomes of critical illness are positively associated with the patients’ muscle mass on ICU admission, the predominant endogenous source of amino acids . MoreoveMechanistic studies have shown beneficial effects on the loss of muscle mass and muscle protein synthesis induced by the administration of higher dosages of protein . Many obDiverging or negative results may be a result of study design, the interactions with calorie administration and overfeeding, or refeeding syndrome or due to dose, composition, and timing of the intervention . RecentlAnother explanation could be that very early high-protein intake in a post hoc analysis of the EPANIC trial, studying early versus late supplemental parenteral nutrition (SPN), was associated with negative effects on outcome . This waProteins and feeding in general are known to suppress autophagy, an important intracellular cleaning mechanism. Whether this should lead to the prevention of an autophagy-deficient state is a matter of debate . RecentlA step-wise approach to meet the protein targets during critical illness is proposed to enhance a better performance Table . This apBased on the available literature, there is no indication that pre-digested or hydrolyzed enteral feeds are better tolerated than intact protein feeds . In somep < 0.08) in ICU patients at higher nutrition risk with low and high BMIs [Early initiation of supplemental parenteral nutrition (SPN), before days 3–7, is not recommended based on a meta-analysis and recent guidelines , 50. Onligh BMIs . The rolNo studies are available comparing outcomes with monitoring versus not monitoring nutrition therapy. However, the potential for abnormal values to be associated with harm was clearly recognized by a group of international experts . LocallyFor this phase, no formal recommendations or guidelines on energy and protein intake are available. However, optimal caloric and protein intake is necessary to enhance recovery of functional muscle mass and to prevent further loss. It is very likely that significant calorie/protein delivery will be required to restore lost muscle mass and to improve QoL. Indirect calorimetry studies during the recovery phase demonstrate marked increase in metabolic needs, with total EE (TEE) increasing as much as ~ 1.7-fold above resting EE (REE) . In the Data on post-ICU protein targets are not available; however, considering that the average post-ICU patient is older and many of them are also frail, we may assume higher anabolic thresholds for protein synthesis (anabolic resistance). Therefore, an intake of 1.5–2.5 g/kg/day of proteins should be considered.In post-ICU patients, a recent study reported an average spontaneous oral calorie intake of 700 kcal/day and the entire population studied consumed < 50% of calorie/protein needs for the post-ICU study period .Another study evaluated 17 post-ICU patients during the hospital stay. The ward-based nutritional care showed to be of low efficacy and not in accordance with the existing recommendations. Several organizational issues were determined to be major barriers to optimal care . A somewRecent unpublished data suggest that after removing the nasogastric feeding tube from post-ICU patients on the general ward the intake of calories drops by 22% and the protein intake by 27% of target . These data suggest that prolonged tube feeding until oral nutrition intake is sufficient should be considered as an alternative to usual care.Recent data from Brussels are in line with these findings: 12 patients discharged from ICU in 2018 were followed up during the entire hospitalization. Nutritional needs, prescriptions, and delivery were objectified. Adequacy of nutrition was calculated , and possible solutions is urgently warranted. Although limited information is available, findings emphasize the importance of closely observing food intake in post-ICU patients before hospital discharge and instructing caregivers and healthcare professionals to provide optimal nutrition at home.We must continue to consider whether patients leaving the hospital following an ICU stay will be able to consume adequate oral calories and protein to optimally recover at home or in rehabilitation facilities. Further, we must all take a moment to read and revel in the defining achievement that is the Minnesota Starvation Study and learn from its landmark lessons . Even heIn patients who have lost significant strength and muscle mass following an ICU stay, a considerable period of significantly increased calorie and protein delivery is required for recovery and likely needed for months to years . Is it pp = 0.018). The number-needed-to-treat in the post-hospital discharge setting to prevent 1 death was 20.3 (95% CI 10.9–121.4) [How many of our care protocols, or our patients, will be able achieve this well-described goal without assistance from oral protein and nutrition supplementation? A large body of data demonstrates that oral nutrition supplement (ONS) must become fundamental to our post-hospital discharge care in ICU survivors. Meta-analyses in various hospitalized patients demonstrate ONS reduces mortality, reduces hospital complications, reduces hospital readmissions, shortens length of stay, and reduces hospital costs –67. A la9–121.4) . As patiICU survivors are also challenged by persistent catabolism and hypermetabolism for months to years. The HP-ONS trial and another recent review emphasize that anabolic and anticatabolic interventions, such as propranolol, oxandrolone, and other agents targeted at restoring lean muscle mass may be essential components to allow for meaningful recovery of QoL and survival post-ICU , 70. TarThe data for the routine use of anabolic/anticatabolic agents in burn care is covered by a recent review . Much caPerhaps even more compelling is the growing body of literature for the safety, clinical efficacy, and benefit of testosterone and oxandrolone in a range of patients. It is well known that oral oxandrolone, among the most anabolic of the testosterone agents, is also among the safest as it shows minimal liver enzyme use with prolonged use. Oxandrolone has been shown to reduce mortality in burn-injured patients . ConcernThis is an area in desperate need of clinical trials outside of the burn setting as a meta-analysis of these pharmacological interventions to reduce ICU-acquired weakness did not find strong signals of benefit, except for the prevention of hyperglycemia during ICU stay .The interaction of acute metabolic changes, inflammation, and nutrition in early critical illness is complex. Newer insights suggest that progressive feeding in the early phase for both proteins and calories is essential to prevent overfeeding and high caloric intake during the development of refeeding hypophosphatemia. After this phase of 4–7 days, high-protein intake and sufficient calories are essential to prevent further loss of muscle mass and function.After ICU discharge, the specific metabolic profile and nutritional needs of ICU survivors remain largely unknown and demand further research. Scarce data reveal poor nutritional practices for patients who left the ICU, are in the ward, and still have a long journey ahead of them.Following hospital discharge, we must ensure our patients are complying with high-protein targets either by prolonged tube feeding or by enhanced high-protein oral nutrition (supplement) intake. Further, nutritional and metabolic therapies such as anabolic/anticatabolic agents in the recovery need urgent study.But, to begin winning this war on long-term ICU outcomes and give our patients back the lives they came to us to restore, we must be thoughtful about optimal provision of nutrition and metabolic therapies throughout all phases of illness and ensure our patients are getting the right nutrition, in the right patient, at the right time!


Mass drug administration (MDA) campaigns are designed to empirically treat at-risk populations for neglected tropical diseases caused by infections with bacteria  and parasites  in a defined geographic region. Decisions to initiate treatment in communities are often made based on predefined prevalence thresholds, but once treatment is undertaken, it is typically provided to all individuals of certain age groups  without individual diagnosis. Such campaigns are a hallmark of neglected tropical disease control and elimination in low- and middle-income countries (LMICs) , 2. ReceA recent cluster-randomized trial conducted in Malawi, Niger, and Tanzania revealed that twice-annual MDA with azithromycin for children aged 1–59 months reduced all-cause mortality by approximately 14% . A majorHealth policy makers are now reviewing evidence for azithromycin MDA to consider whether such programs should be scaled up in settings with high childhood mortality. Although we agree with the urgency of finding effective solutions to address this public health issue, in our view, utmost caution is needed before scaling up MDA with antibacterial agents beyond existing trachoma control and elimination programs. We are convinced about the crucially important role of anthelmintic MDA programs for large-scale morbidity control of parasitic worm (helminth) infections; however, the use of antibacterial agents poses considerably greater threats both within communities where MDA is performed and more globally.A major concern when administering any antimicrobial agent on a mass scale, be it an antibacterial or anthelmintic drug, is the development and subsequent spread of drug resistance. As articulated by the O’Neill commission in 2016, antimicrobial resistance (AMR) is one of the biggest global health threats of our time . The numStreptococcus pneumoniae resistance was documented in children treated with a single dose of azithromycin as part of trachoma control programs, with 1.3% of swabs demonstrating resistance before MDA and 21.3% of samples demonstrating resistance up to 2 months following treatment [S. pneumoniae strains in children from communities treated with azithromycin twice per year (60%) compared with communities treated once per year (40%) [S. pneumoniae strains after azithromycin MDA [S. pneumoniae are at greater risk of selecting for such resistant strains in the context of azithromycin MDA [Bacteria and helminths have very different mechanisms and time courses for developing resistance. Indeed, resistance appears to emerge more readily and rapidly in bacteria, which have a shorter reproductive time and can share resistance genes horizontally, facilitating their spread . AMR wasreatment . A more ar (40%) . Althougycin MDA , some stycin MDA . It appeycin MDA .S. pneumoniae, enteric infections present another major threat [Salmonella Typhi and Paratyphi A), and invasive nontyphoidal salmonelloses, all of which have seen widespread fluoroquinolone nonsusceptibility following years of overuse and misuse [Salmonella Typhi strain [Although much of the focus in macrolide MDA is on resistant r threat , 25. Mand misuse . At presi strain , which ii strain . There ai strain , and we Salmonella and Shigella, may spread far beyond their community of origin within years [It is conceivable that the mortality benefits of azithromycin MDA  will havin years , 29 and in years . With nomcr-1) [NDM-1) [By contrast, the history of MDA campaigns against helminth infections had a different course. Indeed, MDA programs for schistosomiasis and soil-transmitted helminth infections have been in place for some two decades with praziquantel (against schistosomiasis) and albendazole or mebendazole (against soil-transmitted helminthiasis) serving as mainstays of treatment. MDA for helminth infections has also faced similar critiques. Yet, we believe that some of these critiques are misplaced, as they are largely outweighed by the benefits, as summarized in mcr-1)  and the  [NDM-1)  developiThe judicious use of all antimicrobial agents is essential to prevent the development and spread of resistance; however, much greater caution is needed when using antibacterial compared with anthelmintic agents on a mass scale. Expanding azithromycin MDA outside of trachoma programs has the potential for widespread and sustained harm secondary to AMR development and spread. More data are needed to justify the wider use of azithromycin MDA if it is to be used for survival benefit outside of trachoma control programs, and consultation with local communities, neighboring communities, and public health stakeholders should determine if the potential benefits outweigh the risks . For helEmpirical data concerning durability of mortality reduction are needed before implementing this strategy programmatically. Until such data become available, we would recommend against azithromycin MDA in regions where AMR is common or in which azithromycin is widely used for pneumonia or gastrointestinal diseases. Instead, we suggest taking a more cautious approach and seeking other methods to reduce mortality in high-risk settings rather than expand azithromycin MDA beyond trachoma programs because of the risks of long-term harms of AMR. Should these programs expand, AMR and rigorous clinical surveillance must scale up in tandem to closely monitor for the emergence and spread of resistant pathogens, which would trigger public health responses. With the increasing global burden of highly drug-resistant bacterial infections, for which azithromycin remains a critical drug in the arsenal, the stakes are high, and the evidentiary bar for its long-term safety in widespread use should be also high.


Microfluidics and microfabrication have recently been established as promising tools for developing a new generation of in vitro cell culture microdevices. The reduced amounts of reagents employed within cell culture microdevices make them particularly appealing to drug screening processes. In addition, latest advancements in recreating physiologically relevant cell culture conditions within microfabricated devices encourage the idea of using such advanced biological models in improving the screening of drug candidates prior to in vivo testing. In this review, we discuss microfluidics-based models employed for chemical/drug screening and the strategies to mimic various physiological conditions: fine control of 3D extra-cellular matrix environment, physical and chemical cues provided to cells and organization of co-cultures. We also envision future directions for achieving multi-organ microfluidic devices. Discovery of new therapeutics is an essential endeavor, encompassing several branches of science from chemistry to biology and representing a remarkable economic burden worldwide. The process of taking a candidate drug compound from discovery to the market is estimated to cost from hundreds of millions to billions of US dollars to the industry ,2. A keyThe information obtained in pre-clinical studies provides data on the effects that candidate drug compounds have on cellular-scale systems or organism-scale systems. In vitro studies are conducted by supplying candidate compounds to relevant cell culture models  , howeverIdeally, an optimal screening system should allow identification of most failures during early-stage experiments. Nevertheless, the drug development process is currently troubled by high attrition rates as most candidate compounds fail in late-stage screening steps, denoting a greater economic burden for the process . Indeed,One of the strategies that attracted interest and expectations is increasing the efficiency of pre-clinical in vitro studies by employing culture systems that better recapitulate physiological microenvironments. Mimicking specific characteristics  of drug-targeted in vivo districts  may indeed hold potential for performing in vitro drug testing that is more specifically tailored to the target physiological structures, with superior efficiency in predicting drug behavior. Among technical solutions for engineering advanced, physiologically relevant in vitro culture models, microfabrication and microfluidics have emerged in the last decade as promising techniques for basic and translational research ,14. IndeThe last decade saw the rise of bio-inspired microfabrication technologies, with a large number of studies reporting advanced biological models cultured by means of microdevices. The current literature on microdevices and microfabrication techniques dedicated to the culturing of advance models of in vivo biological structures has been widely reviewed elsewhere ,18,19,20The liver is the first organ perfused by compounds absorbed in the digestive tract and the main metabolizing organ in humans. Liver cells, specifically hepatocytes, are organized into functional units named lobules and are responsible for breakdown of drug compounds. Hepatocytes therefore represent a fundamental in vitro model largely employed in pre-clinical screenings for evaluating drug toxicity, metabolic behavior and drug transporters function. Typical assays for in vitro pre-clinical screenings of hepatic drug response involve massive parallelization of standard hepatocytes cell cultures (high-throughput screening) and analysis of metabolic responses and hepatotoxic effects . For insThe dramatically different environmental conditions occurring in in vitro systems are likely responsible for these discrepancies. For instance, microenvironmental cell–cell and cell–extracellular matrix (ECM) interactions are more precisely reconstructed within 3D hydrogel-based or spheroid-based cellular constructs, rather than in bidimensional culture platforms ,25. Alth50  and modulation of CYP activity.Mimicking the architecture of the liver lobules in terms of cell types contributions is another valuable strategy towards engineering an in vivo-like liver model for drug screening. Indeed, co-culturing hepatocytes with other relevant liver cell types increases the performance of in vitro hepatic models ,35. The Another key physiological aspect that is often overlooked in standard cell cultures is the constant perfusion of cultured cells. Blood flow constantly perfuses hepatocytes in vivo, supplying nutrients and removing waste. Nevertheless, standard cell cultures fail to reproduce this aspect in vitro: the static volume of culture medium typically supplied to cells in standard culture vessels leads to nonsteady-state conditions with nutrients diminishing and waste increasing over time in an uncontrolled manner. Constant perfusion cell culture systems, on the other hand, are able to precisely control steady concentration profiles of nutrients and drug molecules to cultured cells. High resolutions obtained with standard microfabrication techniques allow a remarkable control over fluidic quantities within microfabricated devices. In the field of liver models for drug screening, several microdevice-based liver models employed flow-based systems ,48,49,50Central Nervous System (CNS) disorders are known to represent a severe threat to the organism mainly due to the limited regenerative potential of brain tissue, limited immune response and difficulty in CNS targeting by drug molecules. Due to the complexity of the disorders, the largely unknown anatomy and biology of the brain and the physiological barriers protecting the CNS (blood–brain barrier), fewer approved drugs are targeted at the CNS compared to other therapeutic areas. To date, major CNS disorders such as Parkinson’s disease, Huntington’s disease or multiple sclerosis still lack efficient pharmacological treatment .In order to evaluate drug efficacy, safety and mechanism of action, it is important to recreate aspects of neuronal physiology in vitro such as the formation of neurites or axonal protrusions connecting neurons with each other in a functional network. This phenomenon is key for reproducing in vivo-like brain cultures and results regulated by complex interplays of chemical and physical cues. Microfluidic devices have been employed in literature to assess neurite and axonal formation under chemical stimulation in the form of controlled gradients of neurotrophic factors ,60,61,62Another crucial aspect of neuronal physiology is the electrical activity of neuronal networks. Direct reading of electrophysiological signals generated by cultured neurons is relevant for studying responses and mechanism of action of potential CNS drugs . While mPre-clinical drug screenings of CNS-targeting drug formulation are not only addressed at evaluating safety and efficacy in restoring physiological neuronal functions and synaptic activity. An even greater challenge exists in the effective delivery of candidate drugs to the pathological CNS site of interest. While peripheral blood vessels or capillaries normally exchange nutrients and small molecules with surrounding tissues, CNS endothelial cells form tighter junctions in vessel walls and exhibit an overall reduced permeability to small molecules. This peculiar structure of CNS vessels is named blood–brain barrier (BBB) and has tremendous implications for CNS drugs in terms of targeting efficiency as 98% of small molecules do not cross the BBB ,75. SincAlongside microdevices based on bidimensional cultures of endothelial BBB monolayers, strategies have recently emerged for culturing 3D structures resembling the organization of the physiological BBB within microfabricated platforms and performing drug screening experiments. Brown et al., described a multi-layer PDMS microplatform with a microporous membrane sandwiched between two microfabricated channels: the bottom channel was dedicated to endothelial cell culture and the top channel to the injection of a collagen-based 3D hydrogel embedding neurons and astrocytes . The forCardiotoxicity is a main issue in drug development, frequently undetected during initial phases of drug screening and showing great differences depending on cardiac pathology and genetic variants . MicroflMicrofluidic systems were firstly used to characterize single cardiomyocytes ,94,95, hDespite the advancements in assessing cardiomyocytes contractility, MTF do not recapitulate in vivo 3D myocardial environment. In the healthy myocardium of the adult heart, cardiomyocytes are embedded in ECM and organized in a 3D brick wall-like structure, crucial to sustain a proper action potential propagation and synchronous contraction. Nunes et al.  made furMathur et al.  developeMarsano et al.  made fur2). The model proved that shear-activated nanotherapeutics disruption increase in stenotic sites and, consequently, maximal endothelial cells nanoparticles uptake is achieved in the stenotic region. These findings illustrate in vitro biomimetic systems as valuable, high throughput surrogate models at the first stage of drug development.Due to the high control on microchannels geometrical features, the applications of microfluidics in the development of micro-vascular models has long been a subject of interest. Contrarily to in vivo models and macro-scale approaches, microfluidic channels with obstructing regions are relatively easy to produce and proved to be a high-throughput, accurate and reproducible vascular stenosis models ,105,106.Blood vessels barrier function is responsible for controlling the selective passage of macromolecules between blood stream and neighboring tissues in the whole body. Therefore, abnormal vascular permeability threatens homeostasis and impairs organ function and screening vasculature responses to drugs is of paramount importance. In vivo models in small animals rely on surgery-based time consuming protocols, while non-invasive techniques do not provide microscale resolution. Standard in vitro models for vascular reactivity assessment fail in mimicking anatomical structural properties and are not suitable for studies that require luminal flow. Chrobak et al.  providedFinally, Günther et al.  combinedThe lungs are vital organs, responsible for gas exchanges from air to blood . Since pIn the past, standard 2D static culture exploiting primary human tracheal-bronchial cells and lung primary AECs  seeded on multiwell or Transwell plates represented the gold standard to perform drug toxicity tests ,115. TheIn this perspective, great efforts have been made to reconstruct the microarchitecture of the lung alveolar-capillary interface. A pivotal example of this effort is represented by the lung-on-chip . This weSimilar asthmatic conditions were also reproduced in the previously described MTF-based device . In partAside from platforms reproducing alveolar-capillary interfaces ,124,129 The kidney is a highly organized organ containing more than ten types of renal cells structured in a 3D network around a complex vasculature system . The renNephrotoxicity and drug-induced kidney injury (DIKI) represent important dose-limiting factor in pharmacotherapy often detected only during later phases of drug development  ,141,142.Microfluidic technologies are candidate for precisely tailoring the in vivo-like microenvironmental cues including FSS and to reconstitute the original renal cell phenotype, leading to increased sensitivity to toxic agents and more reliable screenings . We repo2 applied for 5 h on primary rat inner medullary collecting duct (IMCD) cells cultured on a porous membrane within a double-layer microfluidic device enhanced cellular conformation rearrangement (highly polarized columnar morphology vs. flattened form), cytoskeletal reorganization and cell junctions [In addition to perfusion-based systems, pertinent blood-kidney barrier models are fundamental to better mimic cellular filtering capacity and to better investigate molecular transport phenomena. Traditional Transwell systems have thus been fully exploited to study cross-epithelial transport ,155, butunctions . With thunctions . The plaunctions  exploiteunctions  and is munctions ,156,160.unctions  poorly punctions  and inte2) restored normal columnar form, increased expression of Na/K-ATPase, aquaporin 1 (AQP1) water channels and cross epithelial transporters fundamental for the proximal tubule functionality. In addition, FSS enhanced the number of cells expressing primary cilia, key component in mechanosensing and tubular morphology regulation. The authors exploited this model to study nephrotoxic effects of the chemotherapeutic cisplatin, demonstrating lower cellular injury and more incisive recovery effect of cimetidine, compared to Transwell culture systems.Although these microfluidic devices recapitulated key features of the kidneys, they are limited by the use of animal-derived cells. Significant differences indeed exist between animals and humans in terms of substrate specificity, expression, relative abundance and tissue distribution of clinically relevant cellular drug transporters in the kidney ,164. ThiHuman in vitro models therefore represent potent tools for nephrotoxicity screening, reproducing relevant kidney functions thanks to the combination of suitable cell systems and microfluidic culturing technologies. Furthermore, kidney-on-chip devices can also be exploited to establish new quantitative, sensitive and clinically relevant DIKI biomarkers to predict cellular toxicity after drug exposure. As demonstrated by Adler et al. , HPTEC cIn this review, we highlighted microfabricated culture systems employed for drug-screening applications. We focused on studies that, by applying a variety of microfabrication strategies for improving the mimicry of organ-level or tissue-level environments in vitro, envision a more rational and physiologically relevant pre-clinical drug screening in vitro testing. Current literature is not limited to the biological structures covered in the previous paragraph, and it is worth mentioning that studies also report microfluidics-based drug screening for skeletal muscle tissue , cartilaIn this scenario, it is also tempting to devise modular approaches where multiple biological structures are cultured on a single device and challenged with a candidate drug compound in a single experiment. Body-on-chip devices would represent unique tools, created by incrementally include interactions between multiple organs or tissue units in one device, to predict organism-level pharmacokinetics and pharmacodynamics of candidate drug compounds before human clinical trials. Indeed, pioneering works and perspective papers are present in current literature discussing and implementing modular solutions ,180,181.This perspective class of microdevices faces many challenges, of which the definition of accurate design criteria is the initial and paramount task. In order for body-on-a-chip devices to accurately predict organism-level drug response, pharmacokinetics and/or pharmacodynamics models need to be defined and adapted to candidate chip designs . In thisIn summary, with more than a hundred reported drug/chemical screening applications, microfabricated strategies represent a potentially relevant approach for directing in vitro pre-clinical studies. Nevertheless, most of the papers here reviewed employ drug screening as a validation step for newly developed microplatforms. There is a need for in-depth studies performed with standard procedures and providing novel insights into in vitro drug safety, efficacy and mechanisms of action to further prove capability of microfluidic-based biological models and to effectively give rise to a new generation of drug screening microdevices.


Understanding person-environment fit, or how well older adults’ environment suits their needs, is complex. While all methodological approaches to gain deeper knowledge about fit have great value, the combination of methodologies provides a layered account of the experience of aging in a place, including the people older adults find important. This research found a clear benefit of combining results of 2 focus groups, 85 in-depth qualitative interviews, and 100 web-based surveys to assess how older adults discuss who and what is meaningful to them. Results show the combined benefit of using a qualitative and mixed-method process during the psychometric testing of the Person-Place Fit Measure for Older Adults. Qualitative focus groups and interviews offered additional domains and exact language older adults used to discuss place. Early qualitative phases improved later stages of this research, namely, online testing, reduction of survey items, and psychometric testing of the final quantitative instrument.


Bone metastases are common complications of solid tumors, particularly those of the prostate, breast, and lungs. Bone metastases can lead to painful and devastating skeletal-related events (SREs), such as pathological fractures and nerve compressions. Despite advances in treatment for cancers in general, options for bone metastases remain inadequate and generally palliative. Anticancer drugs  do not achieve therapeutic concentrations in the bone and are associated with dose-limiting side effects to healthy tissues. Nanomedicines, with their tunable characteristics, have the potential to improve drug targeting to bone metastases while decreasing side effects for their effective treatment. In this review, we present the current state of the art for nanomedicines to treat bone metastases. We also discuss new treatment modalities enhanced by nanomedicine and their effects on SREs and disease progression. Cancer metastasis is a complex and multistep process in which tumor cells spread from their primary site to distant organs. This process involves the loss of intercellular adhesion, cell migration, angiogenesis, access to systemic circulation, survival in circulation, evasion of the immune response, and growth at a distant organ . In soliThe symptoms of bone metastases often are the first sign of disseminated disease in breast and prostate cancer ,7. Bone Recently, nanotechnology has emerged as a tool to enable early disease diagnosis and for drug delivery to improve disease response and patient quality of life. Nanomedicines approved for clinical use such as Abraxane, an albumin nanoparticle (NP) delivering paclitaxel (PTX), and Doxil, a liposomal formulation of doxorubicin (Dox), improve clinical outcomes and decrease toxicity compared to their corresponding free drugs ,10,11. AMetastasis to bone is a multistep process, with each step required for the initiation of distant metastatic sites. To metastasize, cancer cells must detach from the primary tumor, (1) invade the surrounding tissue, (2) intravasate into the circulatory and lymphatic systems, evade attack from the immune system, (3) survive and translocate through the bloodstream to bones, (4) extravasate and survive in the bone marrow, and (5) eventually colonize to form a macroscopic secondary tumor in the bone  19,20,2,220,21. The transdifferentiation of cancer cells from an epithelial phenotype to a more motile and invasive mesenchymal phenotype in the EMT process is an important step in metastasis  and is a+) myeloid cells and, subsequently, cancer cells to initiate metastasis [The bone microenvironment is actively modified to support metastatic spread by the primary tumor. Tumors produce soluble factors, including vascular endothelial growth factor (VEGF) and TGFβ, to generate a premetastasis niche (PMN) to support cancer-cell localization and growth, and initiation of metastatic sites ,34. Tumotastasis ,39,40. Atastasis . AlthougAlthough there are considerable preclinical efforts to prevent metastasis, patients usually present with metastatic disease at diagnosis. The crosstalk between the tumor cells and bone cells can create an environment that supports osteoblastic or osteolytic metastases . MetastaSurgical treatment is an option for localized bone metastasis, but is not applicable for advanced disease with multiple metastatic sites. Surgery for bone metastasis is palliative and aims to alleviate pain  or to seSystemic anticancer chemotherapy helps manage primary tumors and bone metastases. Taxanes (docetaxel and cabazitaxel), which are mitotic inhibitors, remain the only chemotherapeutic options for patients with advanced-stage prostate cancer . TreatmeRadiotherapy for bone metastases uses external-beam radiotherapy or radiopharmaceuticals to induce DNA damage and cause apoptosis of tumor cells . Externa32P), strontium-89 (89Sr), and rhenium-186 (186Re) etidronate are bone-seeking radiopharmaceuticals that accumulate preferentially in osteoblastic bone metastases compared to normal bone due to an increased rate of bone formation [Radiopharmaceuticals are used to treat patients with extensive bone metastases where external beam therapy is not practical. Phosphorous-32 , orchiectomy, and castration alleviates pain in 70% of treated patients and these remain vital tools for managing bone-only or bone-predominate metastases . UltimatBisphosphonates are analogs of pyrophosphates present in the bone matrix and they have a high affinity and selectivity to hydroxyapatite (HAp) ,66,67. BDenosumab is a fully human, monoclonal, synthetic antibody that inhibits receptor activator of nuclear factor-κβ ligand (RANKL) with high affinity, thereby blocking the development of osteoclasts . SolubleDenosumab is indicated for the prevention of SREs and is just as effective as Zol in delaying the time to first SRE by 8.2 months, reducing the risk of a first SRE by 17%, and reducing the risk of multiple SREs by 18% . DenosumAdvances in the treatment of bone metastasis have reduced the pain and incidence of fractures associated with the disease . UnfortuNanoparticles (NPs) serve as carriers for small-molecule drugs, proteins, and nucleic acids to treat bone metastasis . Due to Nanoparticles also improve the efficacy of other modalities currently in practice to treat bone metastasis. Particles made from metals and metal oxides can generate heat when stimulated by external stimuli, such as ultrasound, radiofrequency, and magnetic fields, leading to the thermal ablation of tumors . NanoparThe effectiveness of NPs is dependent on their accumulation in vascularized solid tumors via the enhanced permeability and retention (EPR) effect. Fast-growing tumors develop poorly organized vasculature with large endothelial fenestrations that allows NPs to accumulate in the tumor interstitium. Tumors also have impaired lymphatic drainage that enhances NP retention in the tumor ,89. AlthTargeting of therapeutics to bone metastases or any other tissue can utilize the unique physical characteristics of the tissue (passive targeting) to improve drug delivery. In the bone, passive targeting takes advantage of the fenestrations in bone-marrow capillaries. Active targeting exploits specific receptors upregulated on bone-marrow endothelial cells or specific interactions with the bone matrix to improve drug delivery. In this section, we discuss the different methods of targeting, the ligands used, and the NPs utilized .The bone marrow contains highly fenestrated capillaries with pore sizes up to 170 nm in diameter . These cThe use of monodisperse polystyrene NPs has provided further insight into the effect of the physical characteristics of NPs on their localization in bone marrow. Less than 1% of the injected dose of bare polystyrene NPs (diameters of 60 nm and 150 nm) localized to the bone marrow . CoatingActive targeting of therapeutics to bone metastases takes two main forms: direct targeting to the bone matrix using bisphosphonates or targeting therapeutics to the bone marrow by using ligands that interact with the receptors of endothelial cells lining the capillaries. Once nanoparticles accumulate within the bone, they may be further targeted to specific cells for drug delivery. Depending on the type of treatment, cellular targets can include the cancer or stromal cells  .Although bone metastasis initially starts in the bone marrow and associates with the mineralized bone in later stages, most NP design strategies for treating bone metastasis have targeted the mineralized bone. Mineralized bone is composed of HAp, which accounts for 50% of bone volume and 70% of bone weight ; HAp is The high selectivity and affinity of bisphosphonates to bone make them useful for drug delivery to bone metastases. In addition to binding to the bone, bisphosphonates have demonstrated induction of apoptosis in cancer cells by indirect inhibition of adenine nucleotide translocase via intracellular accumulation of isopentenyl pyrophosphate . BisphosVarious bisphosphonates, particularly Aln and Zol, have been explored for active targeting of nanoparticles to the bone. The presence of a primary amine in Aln and the modifiable nitrogen group in Zol makes them amenable to common conjugation chemistries used with drug-delivery systems ,67. As sConjugating, anchoring, or coating of NPs with bisphosphonates increased the accumulation and retention of polymeric biodegradable NPs in bone tumors . ConjugaBisphosphonates also readily bind onto inorganic NPs for drug delivery and imaging applications. For example, Zol-anchored mesoporous silica NPs demonstrated four-fold greater binding to bone compared to plain silica NPs. Additionally, Zol-coated mesoporous silica NPs showed more significant interactions with cancer cells, resulting in higher cell death . N,N-carbonyldiimidazole, to ensure the release of Zol after cellular endocytosis of the NPs, to improve its action intracellularly [The addition of imaging functionality to the NPs can improve their clinical application by improving diagnostic techniques or enabling tracking of therapeutic particles in the body. Zoledronate-anchored mesoporous silica NPs covered with gadolinium have been employed for imaging bone metastases, in addition to treating them . In thesllularly .Similarly, self-assembling pH-sensitive micelles functionalized with PEG showed increased accumulation into bone metastasis after conjugation to Aln. Once in the acidic tumor microenvironment, the NPs degrade to release their therapeutic load . Tetracyclines (TETs) are natural or semisynthetic broad-spectrum antibiotics (acting on both gram-positive and gram-negative bacteria) that inhibit protein synthesis in bacteria . The affThe high affinity of TET to HAp can be exploited to target NPs to the bone, while the innate fluorescence of TET under ultraviolet light allows for the rapid assessment of the targeting efficiency of TET-conjugated NPs . The conHydroxyapatite-binding peptides provide another strategy for localizing therapeutics to the bone. Early studies mimicked the repeating acidic amino acid sequences of non-collagenous HAp-binding proteins by using repeating units of aspartate  or glutaDrug delivery to the bone marrow is a promising approach to treat early-stage bone metastasis when the secondary tumor in the bone has not ossified and remains in the marrow space. Strategies for bone-marrow localization of NPs take advantage of the unique characteristics of the endothelial cells of bone-marrow capillaries. Endothelial cells that line the bone-marrow capillaries constitutively express E-selectin and vascular cell adhesion molecule-1 that enables cell homing . ConjugaCancer cells within the bone are target options because of their upregulation of certain cell-surface markers, including cadherin-11 and αvβ3 integrin. Taking advantage of the upregulated expression of αvβ3 integrin in breast-cancer cells in the bone microenvironment, one group developed micelle-based NPs functionalized with a targeting ligand for αvβ3 integrin and loaded the particle with docetaxel. These NPs localized to the bone metastases in a metastatic breast-cancer murine model, reducing the tumor burden with less bone destruction and toxicity compared to the free docetaxel . Nanomedicine has improved the efficacy of chemotherapeutic drugs for bone metastasis while decreasing toxicity. The application of nanotechnology has also introduced new therapeutic regimens based on nucleic acid delivery and photothermal therapy that hitherto may not have been possible.N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer to deliver PTX to treat bone metastases, tumor growth and microvessel density were significantly reduced compared to untargeted controls, without systemic toxicity. Critically, bone preservation was achieved by Aln-conjugated HPMA-mediated delivery of PTX [Nanoparticle-mediated delivery of chemotherapy improves tumor targeting while minimizing toxicity. Doxorubicin (Dox) is a potent first-line therapy for bone metastasis, but it induces cardiotoxicity. Loading Dox into PLGA NPs decreases cardiotoxicity and improves drug pharmacokinetics, resulting in better response to therapy ,119. Doxy of PTX ,123. The potential for NPs to provide combination drug therapy, such that multiple drugs that target different pathways in cancer progression are loaded into a single NP system, is appealing for the treatment of bone metastasis. For example, codelivery of curcumin (an inhibitor of osteoclasts) and bortezomib (a protease inhibitor) by PLGA NPs inhibited the progression of bone metastases and protected against bone loss due to osteoclast activity . In thisInorganic NPs have also been employed to deliver chemotherapeutics to bone metastases with the goal of decreasing osteoclast function. Inorganic mesoporous silica NPs targeted to the bone with Zol efficiently deliver plumbagin, a natural compound that targets the JNK/ERK pathway, to decrease cancer-mediated osteoclast formation, and hence reduce bone loss . To imprDysregulation and lack of epigenetic control of essential genes is a hallmark of cancer progression and metastasis. As such, RNA interference is a powerful tool for suppressing the expression of oncogenes with high specificity to improve patient survival. Similarly, reactivation of tumor-suppressor genes in cancer cells promotes their response to other therapies. A key challenge to the application of nucleic acids as therapy is their rapid degradation by nucleases or their stimulation of the immune system ,127. DueNanoparticle-mediated delivery of short interfering RNA (siRNA) can inhibit different aspects of the bone metastasis process after both local and systemic injection. Local injection of PLGA NPs loaded with siRNA against osteopontin and bone sialoprotein decreased the incidence of bone metastasis and bone lesion size compared to free siRNA delivered via an osmotic pump in an intraosseous bone-metastasis model. Interestingly, even at a lower dose, siRNA in NPs showed greater uptake and results in greater knockdown of the osteopontin and bone sialoprotein genes compared to free siRNA . In addiIn addition to siRNAs, NPs can be used for the delivery of tumor-suppressor microRNAs (miR) to downregulate oncogenes. Chitosan NP-mediated delivery of miR-34a downregulates the expression of the proto-oncogenes MYC, MET, and AXL, and induces autophagy in cancer cells . NanoparNanoparticles have versatile physicochemical and tumor-selective properties that make them useful for thermal tumor-ablation therapy. In this treatment modality, sufficiently high temperatures are generated within tumors to induce apoptosis by denaturing proteins, fragmenting lipid membranes, and increasing oxidative stress. Nanotechnology-mediated thermal ablation of bone metastasis can be classified as photothermal or magnetic field induced heating—each with its own advantages ,87,134 Gold NPs, carbon nanotubes, and graphene with high photothermal conversion efficiencies, when illuminated with near-infrared light, decreased tumor growth and increased overall survival of tumor-bearing mice ,136. AddHyperthermia cancer therapy with magnetic NPs in a magnetic field enables treatment of deep-lying tumors. These therapies may be crucial for prostate cancer where the incidence of metastasis in the vertebrae and pelvic bones is high and not easily treated with photothermal therapy. Hyperthermia therapy in bone is feasible with ferromagnetic bone cement ,138 or m64CuS) NPs accumulate in tumors and irradiate cells to decrease growth and metastasis [64CuS NPs is increased by the incorporation of 131Iodine into the particle, which magnifies the amount of ionizing radiation the tumors experience [64CuS NPs take advantage of ionization radiation from 64Cu and the plasmonic properties of the CuS NPs to enable both radiotherapy and photothermal therapy [Nanomedicines can also decrease the toxicity associated with radiopharmaceutical-mediated radiotherapy. Nanocarriers increase radioisotope localization into tumors and decrease nonspecific accumulation in healthy tissue. Copper sulfide  are a precursor to metastasis at distant sites, so their detection is important. Studies of nanobased approaches to identify CTC in peripheral blood that use antibody-conjugated surface-enhanced Raman spectroscopy (SERS) have demonstrated encouraging results ,147,148.The unique characteristics of nanomedicines give the opportunity to target different aspects of the metastasis process, not just the established metastasis.The inherent bioactive properties of nanomaterials can be therapeutic on their own. Unmodified gold NPs reversed EMT in ovarian cancer cell lines by downregulating the expression of mesenchymal markers N-cadherin and vimentin as well as Snail, a transcription factor associated with EMT. The gold NPs reduced the expression of several cytokines, including TGFβ, a potent initiator of EMT ,154. ImpRemoving CTCs from blood and lymph has the potential to decrease the incidence of metastasis. In a proof-of-concept study, magnetic NPs (MNPs) functionalized with ephrin-A1 mimetic peptides removed ovarian cancer cells overexpressing the EphA2 receptor from peritoneal fluid in a murine model of ovarian cancer. This treatment reduced CTCs with corresponding decrease in metastasis . These tNanomedicine has advanced therapeutic modalities for bone metastases. Nanoparticles enable tumor-ablation therapy and improve the efficacy of photothermal therapy even in deep bone lesions ,142. VarEven with advances in drug-delivery systems, treatments for advanced multifocal metastases remain a challenge and are mostly palliative. As such, early disease diagnosis is needed to ensure prompt treatment. Advances in the nanotechnology-mediated detection of primary tumors or metastatic lesions via different imaging modalities have enabled early disease detection. The use of NPs in liquid biopsies to detect CTCs also allows for prompt disease diagnosis. However, a gap remains in our ability to diagnose micrometastases that do not exhibit the EPR effect used by NPs for tumor accumulation. Moreover, micrometastases may be beyond the detection limits of clinical-grade MRIs. NPs coupled to high-resolution imaging modalities, such as photoacoustic tomography, could enable the detection of micrometastases resulting in early treatment. Overall, preclinical studies for treating bone metastases with nanomedicine are encouraging, and show that nanomedicines could provide better treatment options for cancer patients to improve survival and quality of life.


Unmitigated UV radiation (UVR) induces skin photoaging and multiple forms of cutaneous carcinoma by complex pathways that include those mediated by UV-induced reactive oxygen species (ROS). Upon UVR exposure, a cascade of events is induced that overwhelms the skin’s natural antioxidant defenses and results in DNA damage, intracellular lipid and protein peroxidation, and the dysregulation of pathways that modulate inflammatory and apoptotic responses. To this end, natural products with potent antioxidant properties have been developed to prevent, mitigate, or reverse this damage with varying degrees of success. Mitochondria are particularly susceptible to ROS and subsequent DNA damage as they are a major intracellular source of oxidants. Therefore, the development of mitochondrially targeted agents to mitigate mitochondrial oxidative stress and resulting DNA damage is a logical approach to prevent and treat UV-induced skin damage. We summarize evidence that some existing natural products may reduce mitochondrial oxidative stress and support for synthetically generated mitochondrial targeted cyclic nitroxides as potential alternatives for the prevention and mitigation of UVR-induced skin damage. Figure 1A).Ultraviolet radiation (UVR) is a primary risk factor for both melanoma and non-melanoma skin cancer, and the principal cause of photoaging. It is both a complete carcinogen and an immune suppressor. Of the three subcategories of UVR, only UVB (290–320 nm) and UVA (320–400 nm) reach the surface of the earth in substantial quantities and are thus responsible for most UVR-related health effects. While UVB typically comprises less than 10% of UVR, it is a high energy component that is mainly absorbed by epidermal cells, while UVA, the more prevalent component, is weaker but penetrates into the dermis . Within The skin has evolved multiple protective mechanisms to prevent extensive damage from UVR. Chromophores are a primary component of UVR skin protection pathways. They include a group of bioactive molecules, which absorb UVR energy through conformational and chemical changes, allowing them to serve as the first line of defense against UV radiation . When thDNA is the most abundant chromophore present in the epidermis and the major chromophore implicated in UV-induced erythema . Excess Nucleotide excision repair is the primary DNA repair system capable of fixing these direct UVR-induced photolesions and is also critical for mending ROS-induced base modifications. DNA corrections by the NER pathway occurs through either transcription coupled repair (TCR) or global genome repair (GGR). In the former, lesions are specifically removed from the transcribed strand of active genes, while the latter system removes lesions throughout the genome . GGR begb] carbazole (FICZ), acts as a UVA chromophore. Combined with UVA, FICZ generates ROS and induces oxidative DNA damage while inhibiting the removal of DNA photolesions by interfering with NER  is produced by the action of L-histidine ammonia lyase on histidine. Upon UVR exposure, the chromophore trans-UCA undergoes a photoisomerization to its cis-isomer (Cis-UCA). The conversion leads to intracellular ROS generation and oxidative DNA damage, PAF release and altered cytokine release, resulting in cell growth arrest and antigen presentation impairment  by keratinocytes upon UVR stimulation. UV-irradiation of keratinocytes activates the generation of ROS that promote the photoperoxidation of polyunsaturated cellular phospholipids. Oxidation of esterified fatty acyl residues introduces oxy functions, rearranges bonds and fragments carbon–carbon bonds by β-scission generating PAF and PAF-like lipids. PAF contributes to cancer progression by acting as a potent immune-suppressor, disrupting DNA repair, inducing apoptosis, and activating pro-tumorigenic epigenetic machinery . PAF-R as-isomer   and vitamin C, which are abundant cytosolic antioxidants, while α-tocopherol (vitamin E) is localized in cell membranes . GSH is Mitochondria are particularly sensitive to excessive UVR generated ROS, as they are a major source of cellular oxidants. Since mitochondrial DNA (mtDNA) is located in the matrix attached to the inner mitochondrial membrane, it is directly exposed to mitochondria generated ROS. mtDNA is very susceptible to damage as a result of a combination of factors, including the absence of introns that results in more than 95% coding sequences, the absence of protective histones, and few nucleotide excision repair mechanisms compared to nuclear DNA .Figure 1B). Given these increasingly apparent susceptibilities, it is not surprising that there is increasing interest in the development and identification of agents capable of preventing or mitigating ROS-induced mitochondrial damage. Strategies to protect mitochondria from UV induced ROS damage hold particular promise for the prevention and treatment of UVR-induced carcinogenesis and photoaging.UV radiation induced mitochondrial dysfunction directly contributes to photoaging . Even viMany ongoing efforts to develop UVR skin protectants leverage naturally occurring plant-derived compounds with potent antioxidant and anti-inflammatory properties. Their effectiveness in preventing UVR-induced skin damage in animal models and in clinical trials, by either oral or topical application, is the subject of considerable research and has been extensively reviewed . These pOf the many natural products developed to prevent or mitigate UVR-induced skin damage, some have demonstrated a direct impact on mitochondrial function. Clinical trials with ingestion of the dietary carotenoid lycopene, derived from tomato paste, demonstrate protection from UVR that is associated with reductions in mtDNA 3895-bp deletions, and reduced expression of matrix metalloproteinase (MMP-1), an enzyme responsible for the degradation of the extracellular matrix proteins associated with photoaging . LycopenPolyphenols are the most common natural products that have been observed to improve the function of stressed mitochondria. Of the polyphenols, resveratrol has been the most extensively studied. Resveratrol has been shown to directly impact mitochondria by modulating the oxidative phosphorylation system. It can act as both a ROS scavenger by inducing gene expression of antioxidant enzymes such as MnSOD , and by Similarly, the polyphenol EGCG has been shown to protect mitochondria exposed to radiation. EGCG maintains mitochondrial mass and has been shown to have anti-apoptotic effects through suppression of pro-apoptotic Bax and inducing the anti-apoptotic BCL-2 proteins. Additionally, EGCG helps stabilize irradiated cells by inducing the mitochondrial antioxidant SOD2 . EGCG isin vivo is a critical challenge to preserving mitochondrial function in the face of UVR induced oxidative stress. As an alternative to gene therapy approaches and biological therapeutics to counteract radiation damage, we and others have synthesized and assessed small organic molecules as radiation mitigators that have the potential to react with mitochondrial ROS and impede intrinsic apoptosis and necrotic cell death pathways . The best characterized mitochondria-targeted antioxidant, MitoQ [decyl)triphenylphosphonium], shuttles a ubiquinone moiety to mitochondria as a function of negative membrane potential and is effectively recycled to the active ubiquinol by complex II in the respiratory chain . The latter compound is a prodrug. The phosphate group is rapidly cleaved in solution, releasing a highly positively charged thiol that can scavenge a range of electrophilic species   , traumatase (HD) , ischemiase (HD) , and hemase (HD) . In addiase (HD) , revealiase (HD) . Moreovease (HD) . Thus, tTopically delivered and orally ingested natural products have been incorporated as preventative agents to delay skin photoaging and prevent UV-induced skin cancers with mixed success. Given the importance of UV-induced mitochondrial dysfunction in the development of both skin photoaging and skin cancer, it follows that agents capable of directly preventing this mitochondrial damage are more likely to be successful. To date, few natural products have been shown to directly reach mitochondria concentrations sufficient for protection. In this context, synthetic antioxidants, and in particular cyclic nitroxides, that can be designed to specifically localize in mitochondria have considerable promise. Early studies suggest that they may contribute significantly to developing strategies to reduce the incidence of UV induced skin cancers and prevent or delay photoaging.RB, PW, AD, and LF wrote and/or edited a portion of the review article. ME and JG contributed to the concepts discussed.PW, ME, JG, and LF are inventors of related intellectual property covered by patents. The remaining 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.