Monoclonal antibodies constitute a potent and broadly tolerable drug class, representing for some conditions the first newly approved treatment in years. As such, many are afforded “fast-track” or “breakthrough therapy” designations by the U.S. Food and Drug Administration, leading to provisional approval before Phase III clinical trials are reported. Although these drugs are usually safe, some patients experience life-threatening complications—myositis and encephalitis have led to permanent or temporary recalls. Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a hypersensitivity condition easily missed due to its long incubation period and nonspecific presentation. This minireview is primarily intended as an abbreviated guide for practitioners who may be using these powerful treatments. We searched PubMed using a string of symptoms consistent with DRESS syndrome and monoclonal antibodies approved by the FDA since 2015. Then, we excluded studies reporting dermatological complications of reactivation of nonherpetic infection, immunodeficiency-related infection, or reactions to the injection site or infusion. We searched for and accessed prior reviews and background studies via PubMed, Mendeley, and Google Scholar. Two cases of DRESS syndrome were identified in the literature, both the result of treatment with daclizumab. There was one additional case of encephalitis without cutaneous symptoms caused by daclizumab. Drug-induced hypersensitivity dermatitis was reported following treatment with nivolumab and two cases of combination treatment with ipilimumab and either nivolumab or durvalumab produced maculopapular rash and bullae in the first patient and lichenoid dermatitis and blisters in the second patient. Daclizumab was the only recently approved monoclonal antibody associated with DRESS syndrome as such. Limitations in the diagnostic reliability of DRESS syndrome as a clinical entity and the lack of negative clinical trial reporting suggest enhanced vigilance on the part of clinicians and regulators may be warranted. Monoclonal antibodies are specific and effective treatments for immune system dysfunction, skin diseases, cancers and other conditions . They caDelayed hypersensitivity reactions include DRESS syndrome, Stevens-Johnson syndrome, vasculitis, and maculopapular eruptions, among other conditions . Once thGenetic factors appear to play a role and both diuretics and anticonvulsants appear particularly likely to cause DRESS syndrome. The mainstay of treatment is discontinuation of the offending drug and systemic glucocorticoids, although treatment may be confounded by multiple relapses after discontinuation . The incAmong monoclonal antibodies, a handful has been reported to cause DRESS syndrome, including several recently approved biologic agents. One reported case presented with widespread maculopapular exanthem and lymphadenopathy 54 days after treatment of basal cell carcinoma with vismodegib (approved 2012)—lymph node biopsy showed marked eosinophilic infiltrate in the absence of cancer cells . A retroDaclizumab, a monoclonal antibody approved on a “first in class” designation, indicating a novel mechanism of action, was voluntarily and permanently recalled in March 2018 due to multiple reports of encephalitis (FDA). Daclizumab was designed first as prophylaxis for acute organ rejection but was later repurposed as a treatment for relapsing multiple sclerosis. Its hepatotoxicity led to a “black box” warning label from the FDA. In the course of postmarketing pharmacovigilance, the European Medicines Agency (EMA) found that multiple patients developed eosinophilia, skin rash, and involvement of other organs or multiorgan failure suspected to be secondary to immunomodulation. These cases were only retroactively recognized to be DRESS syndrome . More reIpilimumab is a model drug approved in 2011 to inactivate cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), a discovery whose importance was recognized by a Nobel prize in 2018. Yet the cutaneous manifestations of checkpoint inhibitors are varied and occasionally life-threatening. DRESS syndrome is one of the many adverse reactions associated with ipilimumab. Nivolumab and pembrolizumab target programmed cell death-1 (PD-1) and are known to cause adverse cutaneous reactions occurring later in treatment than CTLA-4 cutaneous reactions. Atezolizumab targets the PD-1 ligand (PD-L1) and causes similar cutaneous side effects as PD-1 inhibitors, but combination therapy presents a statistically significant increase in severe complications .The risks of missing this clinical constellation are salient—the object of this mini-review is to assess the most recently FDA-approved monoclonal antibodies for cutaneous symptoms consistent with DRESS syndrome. Case studies offer detailed clinical pictures in which the presence or absence of DRESS syndrome can be relatively cleanly ruled in or out, while clinical trials offer aggregate data to assess the proportion of patients that develop symptoms consistent with DRESS syndrome.Studies were included by searching PubMed using the following string: (dress OR erythema OR pruritus OR rash OR urticaria OR eruption) AND . Studies were then excluded by agreement between all three authors if they did not include either (a) case studies of or (b) rates of dermatological adverse effects of a recently approved biologic. Studies reporting dermatological manifestations of (a) reactivation of suspected latent infection, (b) immunodeficiency-related infection, (c) only injection-site or infusion reactions fitting the search criteria, (d) cases identified as Stevens-Johnson syndrome or toxic epidermal necrolysis, or (e) cases reporting only pooled adverse effects were also then excluded. Prior reviews and background studies were searched via PubMed, Mendeley, and Google Scholar.Total results by case report are arranged in Reports of nonsyndromic cutaneous reactions were also found following treatment with a handful of other recently approved monoclonal antibodies. Treatment with ixekizumab, an IL-17 blocker used to treat psoriasis, preceded new psoriasiform eruptions  in one pDRESS syndrome is one distinct, severe, and idiosyncratic drug reaction that is more relevant than ever due to the rapid advancements in the production of novel biologics and their widespread implementation in the treatment of rheumatic, immunologic, and neoplastic diseases. Fortunately, DRESS syndrome is a rare condition, but the difficulty in diagnosing leads to an even lower rate of identification and reporting. Severe cutaneous reactions of any type reported in clinical trials are very rare, and the only reported cases of DRESS syndrome were in clinical trials of daclizumab . DRESS sClinical manifestations are considered a hypersensitivity reaction to drugs when specific circulating antibodies and/or specifically sensitized lymphocytes are present. Pseudoallergic reactions on the other hand occur when manifestations similar to those of a hypersensitivity reaction are observed in the absence of immunological specificity , 31. AltIn nonclinical toxicity studies, type I hypersensitivity reactions are often observed following administration of biologics . Type I In contrast to an overall low incidence in clinical trials, there is a plethora of documented cases of severe cutaneous reactions, mimicking those encompassed by the clinical picture of DRESS syndrome, as a result of treatment with various biologics. Revising the literature, it is observed that some biologics caused hypersensitivity reactions independent of the dose and the duration of the therapy. Other biologics caused hypersensitivity reactions that seem to correlate with the dose, total number of cycles, and/or spacing between cycles . Interestingly the hypersensitivities accompanying mogamulizumab-kpkc administration were all categorized as acute infusion reactions. The only incidence where DRESS was namely reported is in 0.1% of patients receiving 150mg of subcutaneous daclizumab every 4 weeks for 96 weeks. The reported overall cutaneous adverse events were significantly high for necitumumab. Molecularly, necitumumab's large paratope cavities are speculated to make it less susceptible to resistance through EGFR epitope mutation , a charaWhile multiple studies have estimated the mortality rate of DRESS syndrome to be approximately 10%, the severity and extent of cutaneous involvement do not always correlate with the extent of internal organ involvement , 40. TheThe diagnostic tools, including skin testing and in vitro testing, to evaluate for immediate hypersensitivity reactions remain insufficient . SantiagDRESS syndrome as such is very rare, but may be significantly underreported due to its complicated presentation. Given that the evidence for hypersensitivity reactions and immune dysregulation from biologics is not lacking, allergists, immunologists, and dermatologists should be involved in managing these patients to achieve optimal care. Certain genetic markers have high sensitivity and specificity, providing a plausible basis for the future development of tests to identify individuals at risk for biologic hypersensitivity.


Biofoundries provide an integrated infrastructure to enable the rapid design, construction, and testing of genetically reprogrammed organisms for biotechnology applications and research. Many biofoundries are being built and a Global Biofoundry Alliance has recently been established to coordinate activities worldwide. At the University of Illinois at Urbana-Champaign, iBioFAB2 can build up to 1000 TALEN constructs per day at <$3 each, 0.3% of what it might otherwise cost. Moreover, iBioFAB can perform multiplex genome-scale engineering of Saccharomyces ceresvisiae in a fully automated manner3, >10-times the throughput of a single person without automation. Working with small companies, the London DNA Foundry4, Singapore SynCTI Foundry5 and US DOE Agile BioFoundry6 now provide cost-effective access to expensive equipment and the necessary expertize for product prototyping and commercial process validation, which are often required to secure additional capital investment.Over the past 5 years, research institutions around the world have been establishing biofoundries to expand their biotechnology development capacities. However, the existence of these biofoundries is not yet widely known within the biotechnology or broader biological research communities. Biofoundries aim to accelerate and enhance both academic and translational research in engineering/synthetic biology by promoting and enabling the beneficial use of automation and high-throughput equipment including process scale-up, computer-aided design software, and other new workflows and tools. Iterative Design-Build-Test-Learn biological engineering cycles Fig.  allow reTo enable global coordination of these efforts, 15 non-commercial biofoundries from four continents gathered in London in June 2018 to discuss the formation of a Global Biofoundry Alliance (GBA) that would enable the collective to share experiences and resources and work together to overcome shared challenges and unmet scientific and engineering needs. Several operational challenges reverberated through the June meeting. Participants frequently expressed concerns over sustainable biofoundry growth and development, commenting on the high costs of retaining staff, maintaining infrastructure, and replacing equipment at a predictable rate. Small user bases and a limited awareness of biofoundry capabilities among potential users currently limit the revenue streams that could eventually offset these expenditures. High experimental costs are also a significant barrier for the academic community and indicate that an extended period of public investment will be necessary to enable the benefits of biofoundries to impact the broader research community. Standards were also a key challenge with scant software, hardware, and methodological standardization in place to promote interoperability, efficiency and safety. Legal issues associated with sharing intellectual property, physical samples, and other resources across national and institutional boundaries have also proven difficult. Using BioFoundries effectively requires a paradigm shift in how we do biological engineering, and training the next generation to effectively exploit these technologies is also extremely important. Notwithstanding these challenges, it was agreed that biofoundry capabilities are a critical enabling technology for individual countries to develop capability and deliver on the significant promise of synthetic/engineering biology.Develop, promote, and support non-commercial biofoundries established around the world,Intensify collaboration and communication among biofoundries,Collectively develop responses to technological, operational, and other types of common challenges,Enhance visibility, impact and sustainability of non-commercial biofoundries, andExplore globally relevant and societally impactful grand challenge collaborative projects.The GBA will be formally launched on 9th May, 2019 in Kobe, Japan, during a meeting of the Founding Members  with each biofoundry bringing its unique strengths and capabilities to the problem. GBA members will also explore bilateral collaborations on smaller-scale projects . The benefits of the GBA are anticipated to be analogous to those experienced by the synthetic yeast genome Sc2.0


Stress and anxiety have intertwined behavioral and neural underpinnings. These commonalities are critical for understanding each state, as well as their mutual interactions. Grasping the mechanisms underlying this bidirectional relationship will have major clinical implications for managing a wide range of psychopathologies. After briefly defining key concepts for the study of stress and anxiety in pre-clinical models, we present circuit, as well as cellular and molecular mechanisms involved in either or both stress and anxiety. First, we review studies on divergent circuits of the basolateral amygdala (BLA) underlying emotional valence processing and anxiety-like behaviors, and how norepinephrine inputs from the locus coeruleus (LC) to the BLA are responsible for acute-stress induced anxiety. We then describe recent studies revealing a new role for mitochondrial function within the nucleus accumbens (NAc), defining individual trait anxiety in rodents, and participating in the link between stress and anxiety. Next, we report findings on the impact of anxiety on reward encoding through alteration of circuit dynamic synchronicity. Finally, we present work unravelling a new role for hypothalamic corticotropin-releasing hormone (CRH) neurons in controlling anxiety-like and stress-induce behaviors. Altogether, the research reviewed here reveals circuits sharing subcortical nodes and underlying the processing of both stress and anxiety. Understanding the neural overlap between these two psychobiological states, might provide alternative strategies to manage disorders such as post-traumatic stress disorder (PTSD). Indeed, after decades of research in psychology, ethology and neurophysiology, overlapping neural substrates of these two psychobiological states have been identified. However, the boundaries between stress and anxiety remain an open discussion.anticipation  , can be defined as an emergency state of an organism in response to a challenge to its homeostasis . During cipation . The concipation . Accordi (DSM-5)  “Fear isper se is not a pathological state, as it can prevent exposure to dangerous situations. However, when anxiety is sustained and/or elicited by non-threating stimuli, it becomes maladaptive  is sufficient to induce real time anxiogenic effects and conversely, inhibition of those projections causes an anxiolytic effect . Single-Altogether, the BLA is a single structure which includes neural populations that underlie processing of learned emotional valence (BLA-NAc and BLA-CeA) and a population that generates innate emotional states (BLA-vHPC). This finding suggests that BLA is a key structure to study how emotional states such as anxiety interfere with emotional valence processing.3Stressful experiences engage multiple structures to generate a coordinated physical and psychological response to a challenge. The locus coeruleus noradrenergic system (LC-NE) presents a brain-wide projection pattern  and is l+). Specifically, stimulation of CRH+ CeA-LC terminals increases activity in LC and drives anxiety-like behaviors through type 1 CRH receptor (CRH1R) activation , high tonic (3–8 Hz) and phasic activity . Acute stivation .In vivo photostimulation of these terminals modulates BLA activity, and LC-BLA stimulation is sufficient to cause conditioned place aversion as well as anxiety-like behaviors. These stimulation-induced behavioral changes require β-adrenergic receptor activity in the BLA, providing in vivo evidence that endogenous NE release from LC terminals alters BLA function and, as a consequence, modifies behavior. Additional support for β-adrenergic signaling promoting anxiety-like behaviors is provided in Acute stress also promotes anxiety and other stress related behaviors through BLA adrenergic receptor activation . Even thLC-NE neurons preferentially target neurons in the BLA that project to the ventral hippocampus (BLA-vHPC) and CeA (BLA-CeA), both downstream structures involved in negative valence and anxiety-related behaviors . This su3.1A receptor agonist during a social competition test, rats showed reduced social dominance (Even in our modern society, the necessity of positioning ourselves in a social group through social competition has an enormous impact on our daily lives. In spite of the importance that social competition has in organizing and structuring our society, the psychological characteristics that affect social competitiveness of an individual have been largely overlooked. Several brain regions such as the amygdala and the NAc have been implicated in social status and competition in both humans , and rodominance .The NAc is involved in motivation and has been implicated in the regulation of anxiety and depressive-like symptoms . The neuIn regards to the relationship between stress and anxiety, social competition itself induces an endocrine stress response . In huma4In humans, patients suffering from anxiety disorders have impaired decision making and behavioral flexibility . For exaIn rodents, anxiety levels are also related to decreased cognitive flexibility . Recent Park and Moghaddam designed and developed a task to study the dynamics of reward-based behavior while risking potential punishment . The tasAt the network level, the synchronicity of the theta oscillations is important to coordinate groups of neurons to complete a behavioral response . InteresThese studies revealed that synchrony between the VTA and PFC decreases with punishment probability, suggesting that VTA-PFC network encodes punishment risk C. Under 5Although the relationship between stress and anxiety is bidirectional, the influence of stress as a risk factor for anxiety disorders has been extensively studied . MappingThe standard methods to study anxiety in rodents have relied on established laboratory tests such as the elevated plus maze. However, over the last decade, an increasing number of studies have focused on developing new tools of behavioral analysis that are less invasive, require less subjective interpretations, and harken back to original ethological approaches . One of Further work on PVN-CRH has revealed that these cells are necessary and sufficient for stress-induced social investigation that is required for the transmission of stress from one individual to another . This soin vivo fiber photometry to monitor population calcium dynamics, Recent studies have shown that PVN-CRH neurons are able to respond differentially to stimuli with positive or negative valence . Using iBeyond CRH neurons, a distinct neuronal population in the PVN containing CRH1R has been recently described. This population is 90% GABAergic and has direct synaptic contact with PVN-CRH neurons, and CRH, released from PVN-CRH neurons, modulates signaling between both populations . This inAll these new data are contributing to update the role of hypothalamic neural populations in stress-related behaviors. Specifically, growing evidence implicates PVN-CRH neurons in modulating specific behaviors in a stress-related context. From controlling how an individual responds to stress exposure  to regul6trauma or stressor-related disorders. This new category requires explicitly an exposure to a traumatic or stressful event as a diagnostic criterion, and in particular, the PTSD requires a life threatening experience. Reclassifying PTSD from anxiety disorders to this newly created trauma or stressor-related disorders category has helped to move the focus away from anxiety, which is now rather considered as a comorbid pathology. The new classification in DSM-5 also emphasizes the abnormal reactivity to a stimulus. Interestingly, an altered “flight” response has been observed in patients with PTSD (The idea that stress and anxiety have segregated neurobiological substrates despite their reciprocal influence has now moved beyond the research field to reach the clinical practice, resulting in changes in diagnostic tools. Specifically, according to the DSM-5  PTSD is ith PTSD  and, in ith PTSD . Currentith PTSD . In humaith PTSD . The bal7Although the dissection of multiple brain circuits controlling anxiety- and stress-related behaviors has made substantial advances, our understanding of the neural substrates underlying the interplay between these two psychophysiological responses remains fragmented. Here, we described specific roles of neural populations in the amygdala, nucleus accumbens, prefrontal cortex, hypothalamus and locus coeruleus in emotional valence, stress and anxiety. The work described here is providing foundational knowledge, however future investigation is necessary to unravel the contribution of specific circuits to stress and/or anxiety. Specifically, future work should use activity dependent mapping and recordings of genetically or anatomically defined neural populations during stress exposure and at different levels of anxiety within the same animal. Beyond the technical advances catalyzing our understanding of the neural circuits underpinning stress and anxiety, disentangling them will require the development of new behavioral paradigms in pre-clinical models in order to finely capture the changes of neural coding in these two conditions.The authors have no conflict of interest to declare.


Dietary intervention is a challenge in clinical practice because of inter-individual variability in clinical response. Gut microbiota is mechanistically relevant for a number of disease states and consequently has been incorporated as a key variable in personalised nutrition models within the research context. This paper aims to review the evidence related to the predictive capacity of baseline microbiota for clinical response to dietary intervention in two specific health conditions, namely, obesity and irritable bowel syndrome (IBS). Clinical trials and larger predictive modelling studies were identified and critically evaluated. The findings reveal inconsistent evidence to support baseline microbiota as an accurate predictor of weight loss or glycaemic response in obesity, or as a predictor of symptom improvement in irritable bowel syndrome, in dietary intervention trials. Despite advancement in quantification methodologies, research in this area remains challenging and larger scale studies are needed until personalised nutrition is realistically achievable and can be translated to clinical practice. Diet is a modifiable risk factor for many non-communicable diseases and there is a high level of evidence supporting the efficacy of dietary interventions for both influencing disease risk and improving disease outcomes. For example, dietary intervention can reduce cardiovascular disease risk by 60%  and can Personalised nutrition essentially enables the tailoring of dietary advice to the individual level through the incorporation of data related to specific biological pathways driving that individual’s health or disease status, ultimately optimising the effectiveness of the advice. A comprehensive understanding of clinical conditions and underlying disease mechanisms is often required, including the genetic variants of the patient and the extent to which these variants interact with diet to affect disease risk and treatment in diverse populations.Personalised nutrition models integrate a variety of host-specific variables including current diet, biological or phenotypical characteristics of the individual , disease or health status) and genotypic characteristics. An understanding of epigenetics (regulation of gene expression) is also often included. Models will vary with the clinical condition and its underlying mechanisms, and with the research hypothesis, where different combinations of characteristics are possible. Most evidence supporting personalised nutrition has come from observational studies with disease risk factors as outcomes . However, there are some trials using clinical endpoints that have incorporated participant information to test prediction of responses  and largGut microbiota is one variable shown to be mechanistically relevant for a number of disease states and therefore has a potential role in the development of personalised dietary advice. Microbiota has a profound impact on our health, and alterations to its composition and its dysfunction have been associated with several chronic diseases . DespiteDiet is well known as one of the major drivers of microbiota composition  and convOverweight and obesity rates continue to rise worldwide. In 2013, among adults (age ≥20 years), 37% of men and 30% of women were considered overweight (BMI 25–29.9) or obese (BMI ≥30) . ObesityIrritable bowel syndrome (IBS), a chronic functional bowel disorder characterised by abdominal pain and altered bowel habit , affectsLifestyle advice, including healthy diet and exercise, is usually considered as first line therapy, followed by symptom-directed pharmacotherapies  which have varying efficacy and safety profile . The lowRecent research suggests that dietary advice could be revolutionised towards a more personalised approach for a spectrum of disease states. Accurate prediction of clinical response, such as weight loss in obesity or symptom improvement in IBS, may not only improve short-term clinical response but also long-term treatment efficacy and overall health outcomes in response to dietary intervention. This paper aims to review the current state of evidence related to how knowledge of gut microbiota may facilitate personalised dietary treatments in obesity and in IBS, with a focus on human dietary intervention trials. In addition, the article aims to identify the knowledge gaps and address the implications of research to date. Studies were selected for inclusion if they assessed baseline microbiota composition as a prediction tool for the clinical response after dietary intervention in human cohorts with IBS and obesity. Can gut microbiota composition predict response to dietary treatments?Bacteroidetes abundance, an increase in Firmicutes [Akkermansia muciniphila [Bacteroides and more Firmicutes [Bifidobacteria [Several lines of evidence support a role for microbiota in the pathophysiology of obesity. The obese mouse model is characterised by a 50% decrease in rmicutes  and a loiniphila  comparedrmicutes , as wellbacteria  comparedbacteria . Efficacbacteria  and faecbacteria   in regulating gut hormones and influencing energy harvest . Gut micBacteroidetes correlates with percentage loss of body weight [Acinetobacter. Furthermore, a six-week energy-restricted high-protein diet in 38 obese adults improved low gene richness  and increased the abundance of most gene clusters [Prevotella and decreased Roseburia genera from baseline, whereas the Mediterranean diet led to the reverse, a decreased abundance of Prevotella and increased abundances of the Roseburia and Oscillospira genera from baseline [Energy restriction is the staple dietary intervention in obesity. When obese humans are assigned to either a fat-restricted or carbohydrate-restricted diet, the resulting increase in abundance of y weight . Others y weight . The indclusters . Anotherbaseline . These dBifidobacteria compared with controls who received maltodextrin [Bifidobacterium spp. Structural changes at the individual bacterial genome level were also significantly associated with improvements in host metabolic health  alongside the 9.5% loss of initial bodyweight [In addition to energy restriction, dietary interventions designed to target gut microbiota have used a range of potential modulators and have assessed various obesity risk factors. For example, one prebiotic supplementation study showed that a 16-week intervention of oligofructose-enriched inulin in overweight or obese children (7–12 years) led to a greater abundance of odextrin . Many otodextrin . Microbidyweight .In the last five years, there have been four relatively small dietary trials that assessed the association of baseline microbiota with either weight response in obese cohorts or the response of postprandial hyperglycaemia in healthy individuals, an independent risk factor for obesity. Furthermore, there have been two studies that used microbiota as a prediction tool to model weight response in obesity cohorts and two to predict glycaemic response in healthy individuals .n = 38) [Akkermansia muciniphila was associated with greater improvement in insulin sensitivity and body fat distribution after a six-week energy-restricted diet (n = 49) [n = 50) [Lactobacillus, Leuconostoc and Pediococcus genera. The overall microbiota composition at baseline was not identified by the framework as a predictor for weight loss. In another modelling study, the likelihood of weight loss in 78 obese adults undertaking high-fibre dietary interventions was related to the abundance of Firmicutes at baseline [First, a summary of the studies assessing weight response in obesity is presented. Two unrandomised trials have demonstrated the utility of baseline microbiota in predicting bodyweight response to dietary intervention. One showed that a higher gene richness at baseline was associated with a greater reduction in adipose tissue and systemic inflammation after a six-week energy-restricted high-protein diet n = 38) . The oth(n = 49) . The fir . The otbaseline .Prevotella/Bacteroides ratio, higher relative abundance of Dorea and greater microbial potential to ferment complex oligosaccharides at baseline compared with non-responders [Key studies in the personalised nutrition field have investigated postprandial glycaemic responses (PPGR) to dietary intervention. One crossover randomised clinical trial (RCT) randomised 39 healthy participants to a three-day intervention of barley kernel-based bread or white wheat flour bread (100 g starch/day). The 10 participants demonstrating the most pronounced improvement in glucose and insulin response after a standardised breakfast following the barley kernel-based bread intervention were classified as responders. Responders were characterised by a higher sponders . Also imsponders .n = 800) blood glucose was continuously monitored for one week whilst they recorded daily activities and dietary intake [The most impressive study that has investigated predictors of biological responses to diet to date is a large predictive modelling study. Participants’ , the type and intensity of intervention  and the number of taxa analysed. Most studies defined their subject cohort, including presence of comorbidity and medication use; however, many did not report on other external factors that could influence microbiota composition , which may have contributed to the heterogeneity of findings.Firmicutes/Bacteroidetes ratio [Bifidobacteria abundance [Evidence for microbiota in the development and/or as a driving force of symptom severity in IBS has been accumulating for some 35 years. The line of evidence that is most well supported in the literature is the observation that the faecal microbiota composition of patients differs from that of healthy controls, although there is little consistency in findings across studies ,44. Repoes ratio ,47,48,49bundance ,53,54,55bundance  in IBS cbundance ,51,57. Dbundance  or presebundance ,59 suppoA number of other lines of evidence supporting the important role of microbiota in the pathophysiology of IBS come from animal models. The presence of transplanted microbiota from individuals with IBS in germ-free mice leads to the transfer of the disease state, including altered microbiota composition, visceral hypersensitivity, altered transit, immune activation and behavioural manifestations of the condition ,61. OtheBifidobacteria compared with controls and/or pre-intervention [Bifidobacteria abundance at baseline exhibit the greatest depletion in response to FODMAP restriction [Bifidobacteria and symptom severity [Bifidobacteria-containing probiotic supplements in IBS [Over the past 10 years, clinical trials of the low FODMAP diet have vastly outnumbered studies of other ”whole diet” interventions in IBS. Response rates of 50–80% are reported in RCTs in which the advice is dietitian-led . The imprvention ,69,70,71triction . Alteredtriction ,71. Whetseverity ,57 and ts in IBS .The ability to predict symptomatic response to the low FODMAP diet has been of recent interest, particularly considering the diet is intensive to implement and requires dietetic supervision , and thaBacteroides and Bacteroidales was predictive of dietary response [Bacteroides and Faecalibacterium prausnitzii in responders compared with non-responders [Five studies have investigated whether baseline microbiota could predict symptomatic response to the low FODMAP diet . One fouresponse . This wasponders . Howeversponders ,70.There are obvious challenges in interpreting the evidence from these trials. Pre-intervention environmental factors  that could impact on baseline microbiota composition are not always reported or controlled. Heterogeneity in baseline stool consistency and psychological comorbidity, both associated with altered microbiota composition ,80,81, aBased on the literature thus far, there is inconsistent evidence to support the use of one baseline microbiota signature to accurately predict response to a low FODMAP diet. Evidence that a baseline bacterial volatile organic compound profile may very accurately select responders  suggestsFew human studies  and quantification methodologies applied across studies thus far have been inconsistent. The increasing power and sensitivity of modern sequencing techniques has led to the rapid development of high-throughput methods for assessing genome-wide genetic variations. However, the approaches used to characterise the human microbiota still vary widely. Furthermore, technical accuracy is crucial throughout processing and analysis. For example, the suboptimal mechanical lysis during extraction of the microbiota DNA from faecal or biopsy samples, a key step in the analysis pipeline , will diSecond, there are several shortcomings in the predictive modelling analysis methods utilised. Therefore, it is important that consistent analysis pipelines be adopted worldwide enabling comparison of data between studies. Studies may be limited to exploratory statistical analysis until clinical studies can be adequately designed and powered for primary analysis.Third, there are many problematic confounding factors that can impact on baseline microbiota composition. These factors include, but are not limited to, the host genetic makeup, long-term dietary habits, ethnicity, sanitation, geographical location, exercise and lifestyle habits, and antibiotic use. This further highlights the conclusion that any personalised predictive model incorporating gut microbial composition must consider multiple additional relevant individual datapoints, which may vary with disease state. It is also acknowledged that some chronic diseases, although benefiting from dietary intervention, may never be amenable to a microbiota-based personalised nutrition approach due to inherent heterogeneity in microbiota composition across individuals.Finally, for ultimate translation into clinical practice, there is a need to understand if the results gained from short-term studies predicting host response can be translated into durable responses over time, leading to long-term positive health outcomes. Longer duration of studies and intervention periods are also needed.Diet is one of the most important determinants of the gut microbiota composition. However, the relationship between diet and microbiota is complex and not completely understood. Consequently, personalised nutrition models that predict clinical response to dietary treatment based on the microbial composition are still extremely challenging to test in the research context. Some evidence of associations between gut microbiota and response to dietary treatments for both obesity and IBS suggests that links exist between microbiota composition and inter-individuality in host response to diet. However, personalised nutrition research is in its infancy and specific microbiota signatures that predict individualised responses to dietary treatment are still elusive; advancements in analysis technologies and consistent bioinformatic approaches will be important for progress.


Perforator-free flaps, in autologous breast reconstruction, have expanded to exploit tissue available at smaller donor sites while retaining high success and low risk rates. Abdominal based flaps, such as the deep inferior epigastric perforator, remain the most common; however, when the abdomen is not an appropriate donor site, lower extremity flaps are options. The profunda artery perforator has the benefit of hiding unsightly scar in the gluteal crease but has the drawback of poor donor site volume. Our mosaic fleur-de-profunda artery perforator flap technique for breast reconstruction has shown to increase volume with the addition of a vertical limb, include full angiosome of perforators, and exhibit donor site morbidity equivalent to a medial thigh lift. The fleur-de-lis technique has been discussed in a number of different flap locations including: deep inferior epigastric perforator (DIEP), LD, and TUG. This expansion technique, to include both a horizontal and vertical limbs, has shown to increase volume, add vascular territories, and allow for a more favorable breast shape.5 For those who are not candidates for a DIEP flap and do not desire the unsightly scar of a LD or TUG flap, a PAP scar can be well hidden in the inferior gluteal crease. In addition, in comparison to the TUG, the PAP has shown to have longer pedicle length, larger volume, and does not sacrifice muscle.6 Literature review shows how a vertical PAP flap can expand vascular territories and increase donor volume but there remains an absence of discussion regarding the PAP with fleur-de-lis technique.7 The mosaic flap, 2 adjacent flaps with separate pedicles from a single vascular source, is advantageous for increasing volume while avoiding addition of separate donor vasculature. Further, it provides enhanced vascularity to avoid potential fat necrosis with the increased harvested volume. The purpose of this case series is to present an innovative surgical technique for breast reconstruction called the fleur-de-PAP, which incorporates mosaic flap properties, and would include both the vertical and horizontal perforator angiosomes.The expansion in variety of perforator-free flaps in autologous breast reconstruction has provided reconstructive microsurgeons with increased options when the abdomen is not available for autologous reconstruction. Traditional secondary autologous tissue transfer options include latissimus dorsi (LD), superior gluteal artery perforator, inferior gluteal artery perforator, profunda artery perforator (PAP), or transverse upper gracilis (TUG) muscle flap.A 63-year-old nonsmoking woman, with left breast cancer, opted for bilateral autologous breast reconstruction Fig. . IntraopAt follow-up appointments, the only complication was minimal dehiscence at the T-junction of the thigh incisions bilaterally which was treated with silver nitrate applications. She subsequently underwent second-stage esthetic procedures for contouring by excising the skin paddle, fat grafting for volume in the superior pole (140 mL to right breast and 80 mL to the left breast), and nipple tattooing, without complications  and longest pedicle (average of 10 cm), of all posterior thigh perforators. The PAP has an average of 3 perforators, to supply the medial and posterior thigh skin and subcutaneous tissues, approximately 6 cm below the buttock crease. Computed tomography angiogram scans show that perforators have an equal chance of being medial or lateral to the midline of gluteal crease in vertical distribution.When the abdomen is not a viable donor site, the mosaic fleur-de-PAP flap technique for breast reconstruction has shown to increase volume with the addition of a vertical limb, include full angiosome of perforators, and proposed donor site morbidity equivalent to a medial thigh lift.


Exosomes are small nano-sized vesicles that deliver biologically active RNA molecules and proteins to recipient cells through binding, fusion or endocytosis. There is emerging evidence that endogenous exosomes released by cardiovascular cells and progenitor cells impact cell survival and proliferation, thus regulating angiogenesis, cardiac protection and repair. These cardioprotective and regenerative traits have the potential to translate in to novel therapeutic options for post-ischaemic cardiac regeneration, thus potentially delaying the progression to ischaemic heart failure. Cellular stressors influence exosomes' secretion and the molecular composition of the exosome cargo, thus impacting on the above processes. Evidences are emerging that loading of proteins and RNAs in the exosomes cargos can be manipulated. Similarly, manipulation of exosomes surface proteins' expression to target exosomes to specific cells and tissues is doable. In addition, nature-inspired synthetic exosomes can be assembled to deliver specific clues to the recipient cells, including proliferative and differentiation stimuli, or shed paracrine signals enabling to reconstructing the heart homeostatic micro-environment. This review will describe exosome biogenesis and emerging evidence of exosome-mediated regenerative cell-to-cell communications and will conclude discussing possibilities of using exosomes to treat ischemic heart disease. Exosomes have been extracted from either conditioned cell culture media or biological fluids and then transplanted in animal disease models, showing to induce functional effects  is the most frequent cause of heart failure (HF) , microvesicles (100 nm−1 μm) and exosomes  with the plasma membrane , circular RNA (circRNA), long non-coding RNA (lncRNAs), and microRNAs . Interestingly, this was the case for miR-223 which was packaged by a RNA binding protein called Y-box Protein I (YBX1) , smooth muscle cells, neuronal cells, resident stem cells and fibroblasts  and progenitor cells, such as mesenchymal SC (MSCs), embryonic SC (ESCs), hematopoietic SC and cardiac progenitors have shown the capability to differentiate toward cardiomyocytes (or at least cardiomyocyte-like cells) and vascular cells, at least Exosomes' natural function to protect, target and deliver cellular components to recipient cells brings great interest to use them as novel tools for regenerative medicine. Moreover, it may be possible to artificially engineer exosomes  therapeutic cargo  that needs to be delivered to the diseased organ and (2) synthesize clinically effective synthetic exosomes containing this cargo (García-Manrique et al., via embryonic stem cell filtering (Jeong et al., in vivo (Li K. et al., via western blot, flow cytometry and cellular uptake in HeLa cells with confocal laser scanning microscopy (Sato et al., In designing naturally occurring exosomes or exosomes from modified cell populations as therapeutic tools, researchers face many hurdles with isolation, purification and production on a large scale and at a suitable clinical grade (Li S. P. et al., in vitro binding assay, Denzer et al. has observed that exosomes isolated from B cell specifically bind to follicular dendritic cells (Denzer et al., in vivo studies in mice showed that the fluorescently labeled exosomes did target the cancer affected area, however was not highly specific due to immunogenicity. Exosomes have been engineered to target several receptors for therapeutic applications through peptide fusion with the N-terminus of exosome membrane proteins. However, these complexes can be degraded or cleaved as well, resulting in loss of homing capability (Xitong and Xiaorong, The role of integrins have been shown to play an important role in exosome cellular homing and indeed integrins have been even shown to guide homing of bioinspired synthetic exosomes (Ben-Arie et al., Endogenous exosomes and laboratory engineered synthetic exosomes could bring a whole new era of therapeutic approaches for multiple diseases. The latter are particularly attractive. Such nature-inspired nanoparticles could represent paracrine cargo deliveries homing to their specific target location. In this review, we have discussed the role of endogenous exosomes in ischaemic heart disease as well as past attempts and new possibilities to engineered exosomes by manipulating their cargo with various types of molecules, such as miRNAs, proteins, peptides, and synthetic drugs. When designing exosomes, careful consideration of justifiable surface markers to express externally on the exosome membrane and cargo components has to be investigated to prevent possible off-target adverse effects. As discussed above, in the ischemic heart it is important to address the whole micro environment in a controlled matter. Hence, precision medicine approaches should consider the parallel and serial use of multiple exosome types to maximize the therapeutic responses. In ischemic heart disease, engineered exosomes could prove able to replace and surpass the first-generation stem cell therapies that have been shown to work via paracrine actions. Moreover, exosomes could play in concert with improved cell-based therapies and tissue engineering to deliver transformative therapeutic solutions.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer VB and handling Editor declared their shared affiliation.


Endemic and emerging respiratory viruses are a threat to public health, and a robust public health laboratory system is essential to ensure global health security.This program sought to expand molecular laboratory testing capacity to detect a broad range of respiratory pathogens in clinical respiratory specimens collected during disease surveillance and outbreak investigations.As a part of the Global Health Security Agenda (GHSA), the United States Centers for Disease Control and Prevention utilised the equipment and training infrastructure already in place at the World Health Organization National Influenza Centers to expand testing capacity for respiratory viruses in laboratories in GHSA partner countries. This was done through the provision of quality assured reagents, including multiplex platforms and technical guidance for laboratory staff, as well as the assessment of laboratory testing accuracy.Early findings illustrated that GHSA laboratories have been able to expand testing capacity using specimens from routine surveillance, as well as from outbreak situations. One of GHSA’s primary areas of focus, known as an ‘action package’, is an emphasis on strengthening national laboratory systems (Action Package: Detect 1).9 Through this action package, GHSA enables countries to enhance real-time bio-surveillance and ensure that laboratories are capable of safe, accurate detection and characterisation of epidemic-prone pathogens, including both known and novel threats.9 In order for public health laboratories to prepare for outbreaks and emerging pandemic threats, they should demonstrate ongoing capacities for routine diagnostics.The Global Health Security Agenda (GHSA) . The NICs often have standardised, state-of-the-art equipment, in addition to well-trained molecular biologists. Specimens for routine testing at NICs are received from influenza surveillance sites that include regional hospitals and clinics using the severe acute respiratory infection (SARI) or influenza-like illness case definitions. Frequently, SARI and influenza-like illness sentinel systems are models of successful surveillance in resource-limited countries where epidemiologic data are linked to specimen collection and laboratory testing.10 An opportunity exists to expand the existing surveillance systems at NICs in GHSA countries by expanding testing capacity for other priority agents.For almost a decade, the United States Centers for Disease Control and Prevention (CDC) Influenza Division has developed laboratory capacity for seasonal influenza virus and pandemic influenza preparedness. As a WHO collaborating center, CDC provides technical assistance for influenza testing in a network of global laboratories, known as National Influenza Centers (NICs), which have functional links to their assigned WHO reference laboratory and access to quality assured reagents from the CDC Influenza Division through the International Reagent Resource (IRR)  was distributed as a kit for SARI surveillance and outbreak investigations. All CDC in-house assays utilise Taqman® probes  with a 5’ FAM fluorophore and either an internally linked BHQ-1®  or 3’ terminal BHQ-1®  quencher. The RNP control allows laboratory scientists to know if the quality of nucleic acid has been compromised, potentially confounding any rRT-PCR results. These assays have been previously validated to work with several commercial one-step rRT-PCR enzyme kits such as the AgPath-IDTM One-Step RT-PCR kit  and the qScript® One-Step qRT-PCR kit, low ROX . Real-time PCR instruments evaluated by CDC to be compatible with this reagent panel include the 7500 Fast DxTM , Mx3000P® and Mx3005P® , CFX96TM and CFX96TM Touch , and the ViiaTM 7 in both TaqManTM Array Card and 96-well PCR plate formats .16In order to expand its laboratory capacity to test for respiratory viruses beyond influenza, CDC’s Division of Viral Diseases deployed a panel of in-house singleplex real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays .11,12,13® , a commercially available multiplex assay that can detect up to 33 common respiratory viruses and bacteria. The FTD33 comes with its own proprietary primers, probes, PCR buffer, enzyme mix and controls needed to perform the tests. A previous version of the FTD33 kit, the FTD21 Respiratory Pathogens Kit, was validated against the CDC respiratory virus singleplex assay panel.17When encountering an outbreak of unknown etiology, a multiplex platform may enable laboratories to rapidly identify the potential pathogen causing illness; therefore, CDC provided countries access to a multiplex assay kit known as FTD33 Respiratory PathogensThis article followed all ethical standards for a research without direct contact with human or animal subjects.Quality assured reagents are essential for accuracy in diagnostic testing. In addition, a reliable method for procurement and distribution of available reagents is necessary to order and receive laboratory materials, particularly when many of the reagents are temperature sensitive. Utilising GHSA funds, the online reagent portal, the IRR, was expanded to include 14 new custom products including the FTD33 kit, the CDC-developed respiratory virus rRT-PCR assay kits and controls, as well as ancillary commercial products such as rRT-PCR enzyme kits and specimen extraction kits required to run the CDC respiratory virus rRT-PCR assays. The IRR is a programme established by CDC to provide registered users with various reagents and information for the study and detection of influenza virus. It was chosen as the delivery mechanism for these reagents, because the selected laboratories for GHSA were already using or in the process of being approved to use the IRR for the purpose of WHO influenza surveillance, eliminating the need to establish a new or duplicative reagent distribution plan.In addition to enhancing laboratory capacity through the availability of quality reagents, CDC has enhanced its technical capacity through partnerships to conduct workforce training. Working with the Association of Public Health Laboratories, laboratory training took place in Atlanta in July of 2016. Nine countries participated and were trained in the expanded respiratory viral pathogen detection, including Bangladesh, Burkina Faso, Cameroon, Côte d’Ivoire, Pakistan, Senegal, Uganda, Tanzania and Vietnam. The Pasteur Institute of Paris, France, conducted similar laboratory training in Yaoundé, Cameroon, in April 2017 with representatives from Benin, Guinea-Bissau, Mali, Mauritania, the Democratic Republic of Congo and Togo. The training curriculum included lectures from subject matter experts and hands-on laboratory exercises in order to familiarise attendees with the CDC rRT-PCR assays and the use of the FTD33. Didactic lectures covered a range of topics including an overview of respiratory viruses, quality assurance and biosafety guidelines, setting up a molecular diagnostics laboratory, and performing rRT-PCR assays on human clinical respiratory specimens. The hands-on exercises covered total nucleic acid isolation from clinical respiratory specimens, setting up rRT-PCR reactions for CDC singleplex and FTD33 multiplex assays, as well as analysis of the rRT-PCR data. Laboratory subject matter experts from CDC and the Association of Public Health Laboratories guided participants through the exercises ensuring everyone followed proper techniques. The specimens used for training were mock respiratory specimens spiked with inactivated respiratory viruses. By pre-characterising these specimens with the CDC respiratory virus panel, the participants’ results could be used as a metric for successful learning.http://www.qcmd.org), enable laboratories to process and test proficiency samples using the same protocol they use for clinical specimens. The results will be reported back to Quality Control for Molecular Diagnostics for scoring and the compilation of performance reports. Confidential individual reports will be returned to the respective laboratories. Based on their individual performances, CDC may recommend additional training and technical support, if necessary. For laboratories expanding their viral testing capacity, the availability of external quality assessment panels ensures quality performance.In order to test each country’s laboratory capabilities and availability of reagents following the training, CDC is preparing to provide laboratories with external quality assessment panels. These panels, produced by Quality Control for Molecular Diagnostics (From February 2016 to September 2017, the IRR shipped 502 individual products, supporting respiratory virus rRT-PCR testing, to 25 different laboratories in 19 countries, and scientists from 15 countries received training . Prelimi18 In the arena of national laboratory systems, two of these indicators include the capacity for the detection of priority diseases, as well as for effective and modern diagnostics. The training and materials provided through CDC’s engagement in GHSA laboratory strengthening has provided tools and building blocks to more than one-third of all GHSA countries to more readily achieve their goals in laboratory capacity. Data generated from expanded testing will allow for a more complete understanding of the burden of disease in their country. A routine system of collection, transportation, testing and results sharing in a quality assured environment will enable the public health system to respond to outbreaks when they occur.The GHSA measures its success according to metrics defined by the WHO’s Joint External Evaluation tool.Lessons learnedThe IRR is an important tool in the rapid expansion of testing capacities for the GHSA countries. Introducing new laboratory testing reagents through the IRR is a way of supporting countries that do not normally perform routine surveillance testing beyond influenza virus detection, and enables them to generate baseline data for additional pathogens. Centralising the distribution of custom CDC testing reagents along with commercial products through the IRR ensured that laboratories had a streamlined, single-stop source for vital, quality-assured laboratory reagents. This eliminates the need to get separate quotes for the same products specific to all the different laboratories. Additionally, by using the IRR as a reagent distribution mechanism, it is easy and fast to deploy new reagents for emergency situations, such as outbreaks of newly emergent disease.Developing and deploying training programmes with standardised curricula enables multiple laboratories and countries to practise uniform testing procedures, thereby allowing for better direct comparisons between countries and regions, and ultimately offering a better resolution in understanding the behaviour of respiratory viruses around the globe.In combination with strong epidemiological data, multiplex platforms such as FTD33 or other comparable products may provide valuable diagnostic information to better understand possible etiologies of respiratory disease, and ultimately improve detection capacities. Additionally, some multipathogen detection assays and platforms may still be cost prohibitive for many countries and public health systems, especially when used as a surveillance tool where the quantity of specimens needing testing can be quite high.Through the experiences described above, we identified key challenges and lessons learned where greater attention and investment may be warranted for future laboratory capacity building.


Mitochondria are best known as the powerhouses of the cells but their cellular role goes far beyond energy production; among others, they have a pivotal function in cellular calcium and redox homeostasis. Mitochondrial dysfunction is often associated with severe and relatively rare disorders with an unmet therapeutic need. Given their central integrating role in multiple cellular pathways, mitochondrial dysfunction is also relevant in the pathogenesis of various other, more common, human pathologies. Here we discuss how live-cell high content microscopy can be used for image-based phenotypic profiling to assess mitochondrial (dys) function. From this perspective, we discuss a selection of live-cell fluorescent reporters and imaging strategies and discuss the pros/cons of human cell models in mitochondrial research. We also present an overview of live-cell high content microscopy applications used to detect disease-associated cellular phenotypes and perform cell-based drug screening. Mitochondrial diseases can result from mutations in the nuclear (nDNA) or mitochondrial DNA (mtDNA). These mutations often lead to disruption of mitochondrial function and/or (ultra) structure leading to “primary mitochondrial disorders”  that areoF1-ATP synthase (or CV) converts food-derived metabolites into ATP. This process consumes oxygen (O2) and establishes a membrane potential (Δψ) across the mitochondrial inner membrane  of the electron transport chain (ETC) and the Fmembrane . But mitmembrane .In this review, we summarize how live cell analysis using fluorescent reporter molecules and (semi)quantitative microscopy can be used to analyze mitochondrial phenotypes. We also discuss various cellular models of mitochondrial disease with respect to their benefits, disadvantages, technical applicability and appropriateness as a disease model. Finally, we present a collection of high-content microscopy strategies to evaluate mitochondrial contribution to disease and to perform drug toxicity and efficacy screening.in situ in living cells. Moreover, microscopy uniquely allows for simultaneous time-lapse monitoring  and (semi)quantitative measurements of multiple parameters by multispectral imaging . In particular, developments in fluorescent reporter technology tremendously boosted the use of light microscopy for cell biology studies quantitative change in fluorescence upon target binding. A drawback of the ratiometric dyes is related to their portability to high-throughput where doubling data dimension can create acquisition, storage and processing issues. Implementing ratiometric dyes in multispectral assays can be also inconvenient because of the wavelength limitation.When mitochondrial contribution to disease is evaluated in living cells, we consider mitochondrial morphology and membrane potential, ROS, ATP and mitochondrial respiration crucial indicators of mitochondrial health status. Their compatibility with fluorescence microscopy assays will be presented in the next paragraphs and is summarized in ® family. Among these molecules, TMRM was described to be the least toxic, the fastest in equilibrating across membranes, and displaying the lowest non-specific localization  and membrane potential (Δψ). Fluorescence live-cell imaging is the most direct method for assessing their spatiotemporal dynamics . Differelization . Therefolization . The cellization  and are lization . To techlization . Althouglization  attemptslization .Protein-based probes targeted to the mitochondria, such as mito-GFP, are also a valid tool to study mitochondrial morphology and dynamics , howeverReactive oxygen species (ROS) is a general term that includes both oxygen radicals and non-radical agents that can be easily converted into radicals . ROS areSeveral non-microscopy based approaches are available  to indirectly study ROS via the quantification of the accumulated reaction products  . Due to 2DCFDA)   and dihy2DCFDA) . While COS types . DespiteMitoSOX, which is DHE linked to a TPP moiety for rapid accumulation in mitochondria, would ideally allow detection of mitochondria specific ROS production . HoweverLipid peroxidation of the mitochondrial inner membrane represents a major cause of mitochondria disruption  and ferr2O2 probe HyPer , redox-sensitive green fluorescent proteins (roGFP family) and the Hbe HyPer . The worbe HyPer . HyPer i protein . These g protein .ATP plays a central role in bioenergetics and intra/inter-cellular signaling. It can be considered an indicator of cellular and mitochondrial health status . A numbeCurrently, luciferin-luciferase bioluminescence assays using plate readers represent still the gold standard to measure ATP in cells and robust kits are commercially available from different providers.oF1-ATP synthase combined with fluorescent proteins of different colors, were developed to differentially target cytosol, nucleus or mitochondrial matrix and have been validated in several studies  sensors of the ATeam family  that con studies . BTeam t studies .+, have been recently developed for real-time imaging of mitochondrial ATP in living cells . Quantifying intracellular O2 consumption (respirometry) is therefore a direct estimate of the mitochondrial respiratory activity and as such another crucial readout for the mitochondrial and cellular health status  are generated by fusing nuclear-depleted cells derived from patients carrying mtDNA mutations with cell lines in which the mtDNA has been removed . The cybrids model has been traditionally used to study mtDNA mutations and dissect the relationship between mtDNA and phenotype alterations . By intrUnfortunately, the patient-specific interplay between mitochondrial and nuclear genomes, which may play a contributing role in the OXPHOS dysfunction manifestation , is lostHuman induced pluripotent stem cells (iPSCs) are obtained from somatic cells through the process of cellular reprogramming . iPSCs cOn the other hand, iPSCs also hold disadvantages. Some studies reported that mtDNA MELAS mutations impair cellular reprogramming to iPSCs . CellulaHigh-content screening (HCS) is defined as a cell-based phenotypic approach where readouts are imaged by multiplexed and automated microscopy ; this isBecause of the fast developments of technologies, probes and applications and the upcoming field of iPSCs technology generating faithful cell disease models, the field of cellomics is now on the brink of catching up with the other–omics approaches.Already in 2007 an HCS method was developed combining Δψ analysis with other cellular parameters measured in human liver carcinoma cells (HepG2) grown in a microfluidics device . Also peAll these studies show the applicability of mitochondrial morphofunction and ROS analysis as robust HCS/cellomics applications. These will likely in the near future be complemented with fluorescent imaging technologies to detect ATP and oxygen consumption, which, to the best of our knowledge, have not been explored yet.Cellomics represents a powerful technology, already established in many research labs, to study in an unbiased manner mitochondrial functions monitoring multiple readouts, portable to different cells models and able to provide valuable insight in mitochondrial phenotypes and their contribution to disease.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.www.khondrion.com). JS is the founding CEO of Khondrion. WK is a scientific advisor of Khondrion, Mitoconix Bio Ltd.  and Fortify Therapeutics, Inc. . The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.EI, JB, and HR are full-time employees of the SME Khondrion (


Despite a growing number of middle-aged employees in the workforce, Hong Kong still lags behind other developed countries in implementing age-friendly policies to protect older workers from being discriminated in recruitment, performance appraisal, and other personnel decisions. This paper therefore aims to investigate the effects of age discrimination on work-related outcomes and well-being. A total of 333 Hong Kong Chinese employees aged 40 years and above  completed an online survey on work experiences. Self-perceived age discrimination in the workplace , perception of occupational future time , work engagement , work stress , and psychological distress  were measured. Almost 53% of the participants reported that older workers were less likely to get the same training opportunity for new technology as their younger peers, and 46.8% perceived that older workers did not get the same promotion opportunity as their younger peers. Regression analyses further revealed that employees’ perceived age discrimination in the workplace indirectly influenced their levels of work engagement , work stress , and psychological distress  through perception of occupational future time. The findings of this study unveil the severity of workplace age discrimination in Hong Kong and the negative impacts of age discrimination on work-related outcomes. Recommendations for organizational practices will be discussed.


Despite decades of formidable exploration, multi-organ ischemia-reperfusion injury (IRI) encountered, particularly amongst elderly patients with clinical scenarios, such as age-related arteriosclerotic vascular disease, heart surgery and organ transplantation, is still an unsettled conundrum that besets clinicians. Remote ischemic conditioning (RIC), delivered via transient, repetitive noninvasive IR interventions to distant organs or tissues, is regarded as an innovative approach against IRI. Based on the available evidence, RIC holds the potential of affording protection to multiple organs or tissues, which include not only the heart and brain, but also others that are likely susceptible to IRI, such as the kidney, lung, liver and skin. Neuronal and humoral signaling pathways appear to play requisite roles in the mechanisms of RIC-related beneficial effects, and these pathways also display inseparable interactions with each other. So far, several hurdles lying ahead of clinical translation that remain to be settled, such as establishment of biomarkers, modification of RIC regimen, and deep understanding of underlying minutiae through which RIC exerts its powerful function. As this approach has garnered an increasing interest, herein, we aim to encapsulate an overview of the basic concept and postulated protective mechanisms of RIC, highlight the main findings from proof-of-concept clinical studies in various clinical scenarios, and also to discuss potential obstacles that remain to be conquered. More well designed and comprehensive experimental work or clinical trials are warranted in future research to confirm whether RIC could be utilized as a non-invasive, inexpensive and efficient adjunct therapeutic intervention method for multi-organ protection. Early restoration of blood flow has been demonstrated to bring substantial benefits via salvaging viable tissues from ischemic injury . HoweverRecently, attention has been focused on an innovative approach, termed as ischemic conditioning (IC), particularly remote ischemic conditioning (RIC), knowing that repetitive, transient and sublethal series of IR bursts can trigger endogenous protection and tolerance against subsequent ischemic threats . RIC mayIn this regard, this article encapsulates an overview of IC on multi-organ protection, including a brief developmental history of IC, intervention forms, underlying mechanisms and current clinical evidence as a promising option to attenuate IRI, . It alsoBased on the sites of intervention, IC strategies can be classified into two forms, that is, in situ and in remote organs or tissues.IC in situ involves repeated IR intervention applied locally to the organ or tissue itself, thus offering subsequent protection against IRI-induced lethal injury. The concept of IC in situ was first demonstrated by Murry et al. in 1986, who reported that 4 cycles of 5-min occlusion of the circumflex coronary artery interspersed with 5-min reperfusion could significantly reduce the myocardial infarct size induced by a subsequent longer period occlusion of the same vessel ..3].RIPreC. A landmark study for the breakthrough of IC in situ in the possible clinical application was conducted by Przyklenk et al. in 1993, who used 4 cycles of 5-min IR of the circumflex coronary artery prior to the left anterior descending coronary artery (LADCA) occlusion to reduce myocardial infarction volume at the territory of LADCA in a dog model [og model . This prog model . A furthog model . In 2002og model . The resRIPerC. Another subtype of RIC is RIPerC, which is referred to as the induction of sublethal conditioning stimuli during an episode of ischemic attacks. Schmidt et al. first demonstrated RIPerC by applying cyclical hindlimb IR with a tourniquet in experimental pigs during LADCA occlusion, with a satisfactory result [y result . In two y result ,13. One y result .RIPostC. The history of RIPostC dates back to the first report aiming to investigate the cardioprotection of ischemic postconditioning in 2003, showing that brief repetitive ischemia before fully establishment of flow in a canine model of coronary occlusion-reperfusion could attenuate reperfusion-associated myocardial injury [l injury . Two yeal injury . Subsequl injury –19.Selective meta-analyses published up to December 2014 illustrated the potential salutary effects of postconditioning on cardioprotection in subjects with STEMI scheduled for percutaneous coronary intervention (PCI), although some results might be disappointing or even contradictory . UnfortuMurry et al. reported that the positive effect of IC diminished or even run out after a few hours, suggesting the existence of a time window for the protection . SubsequThe protective mechanisms of RIC through which the protection is transferred to remote targeted organs against IRI are quite sophisticated and have not been fully explored. Current data reveal that the possible underlying mechanisms are correlated with several aspects, mainly including neuronal and humoral pathways, both of which also display inseparable interactions with each other, and the dominant one may depend on the applied stimuli and specific circumstances . AlthougThe neuronal mechanism has been proposed to play a requisite role in RIC, whereby protective signals from the periphery limbs can be transferred to ischemic organs. A growing body of evidence supports this neuronal hypothetic notion of RIC. For instance, the use of ganglionic blockers such as hexamethonium and trimetaphan could abrogate the RIC-mediated protection in rat models or humans ,27,34. CHumoral mediators. The humoral pathway hypothesis refers to repeated cycles of IC at a distant site (for example at limbs) may stimulate the release of certain substances that travel into the blood circulation and then reach the prolonged ischemic organs/tissues to produce a protective effect [e effect . This hye effect . In a poe effect . Shimizue effect . The rese effect ,50. Notae effect . A recene effect . Taken tDespite the clinical importance, actual identification of humoral factors following conditioning response faces great challenges and remains ambiguous. Many proteomic analyses using plasma harvested from both animals and humans undergoing RIC have yielded inconsistent or even paradoxical results. A variety of circulating factors have been proposed as contributors, such as hypoxia inducible factor-1α –60, connImmuno-inflammatory responses. Emerging evidence plunks for the fact that induction of endogenous protection via RIC is partially attributed to the modulation of immuno-inflammatory responses. Konstantinov et al. initially discovered that RIC in healthy humans was capable of downregulating proinflammatory genes while upregulating anti-inflammatory genes, around 30 of which were supposed to be involved in leucocyte adhesion, chemotaxis, cell adherence/migration, apoptosis, TNF-α signaling pathway, and Toll-like receptor pathway [ pathway . Of part pathway . On the  pathway ,115. How pathway . Similar pathway ,115,116. pathway ,118. Add pathway ,120. Imp pathway . Cai et  pathway . A more  pathway . They fo pathway . HoweverAn increasing number of researchers have explored the association between RIC and anti-oxidative activity, suggesting that limb RIC could markedly attenuate IRI through downregulating the expression of free radicals while upregulating the expression of antioxidant proteins. For instance, the level of malondialdehyde (MDA), which is frequently used as an indicator to assess the extent of oxidative stress, was significantly reduced following the application of RIC in combination with or without local IC in different animal models, such as cerebral, myocardial, hepatic and renal ischemic models –128. In Recently, several investigations have reported that the neuroprotective effects afforded by RIC are closely associated with autophagy activation and the attenuation of mitochondrial injury following cerebral IRI in rat or mice model –133. TheEndothelial dysfunction is a common finding in patients with atherosclerotic artery disease, and associated with unfavorable clinical outcomes ,137. StuExperimental and our pilot clinical studies reveal that chronic RIC is capable of robustly improving cerebral blood flow in chronic cerebral ischemia –145. ThiAlthough RIC strategies in different organs/tissues may recruit similar signaling transduction molecules, variations are still recognized, . ClarifyA growing number of clinical trials have implied that RIC is a promising approach to enable multi-organ protection, even though a few studies displayed inconsistent results. Details are shown in Fig. 5 and 6Supplemental Table 1 to 4.Evidence collected so far with regard to the scenarios of target organs are listed below.(1) Acute ischemic stroke ,147, RIC(1) Selective coronary artery bypass surgery (CABG) ,162–175,(1) Renal dysfunction after elective coronary revascularization –211, in (1) Pulmonary dysfunction following cardiac surgeries ,220,221,(1) Liver injury after cardiac valve replacement surgery , the lev(1) Intestinal injury after open infrarenal abdominal aortic aneurysm repair surgery , one sma(1) Cutaneous microcirculation in healthy volunteers –231, RICAs the average human lifespan has increased markedly over the past decades, the mounting incidence of ageing-related disorders in the elderly, and the related economic and social burden, impel scientists to develop novel strategies to slow down or prevent these disorders, alleviating the suffering at the end of life.Decreased physiological reserve and tissue resilience are characteristics of biological ageing, which render the human system more susceptible to pathological threats. Given the fact that elderly patients usually have at least two afflicted organs or tissues, therapeutic approaches with systemic actions (inducing protective responses in a wide range of organs and tissues) are warranted. In addition to the efficacy, safety and compliance issues are of great importance in an ageing population. The emerging area of RIC builds upon this foundation. The capability of this non-pharmaceutical and non-surgical intervention to protect vital organs simultaneously by enhancing the body’s powers to adapt to pathological threats could provide a safe, less burdensome, minimally-invasive way for ageing-related disorders. Currently, RIC is being evaluated in a variety of clinical settings such as cerebrovascular disease, coronary artery disease and renal injury that predominantly influence the older population.To date, regarding the safety and tolerability of the methodology, no RIC-associated adverse events have been reported in the published clinical studies. Although the prospect of clinical transformation of RIC on multi-organ protection is promising, challenges still exist. The controversial findings raise the question “Can RIC have impact on clinical outcomes in patients under different clinical conditions?” To the best of our knowledge, a number of factors should be reconsidered thoroughly in the future work.For instance, although previous experimental work has implied that the number and duration of IR cycles might affect the efficacy of RIC, there is a paucity of clinical data comparing the effectiveness of different RIC protocols, and no convincing evidence of the most favorable conditioning strategy has been established . NotablyEstablishment of biomarkers has been considered as a paramount step for translating RIC from preliminary data into real clinical practice. Many hurdles still lie ahead to screen and discover the appropriate set of biomarkers in order to accurately evaluate the clinical efficacy of RIC. The investigations of a wider panel and combination use of genomics, proteomics and imaging markers are warranted. For instance, it was reported that more than 150 genes including apoptosis, cell survival, and immunity-related genes were differentially expressed in healthy subjects who were preconditioned with repetitive arm ischemia . FollowiEssentially, the potential applications of RIC are numerous and may be extended far more than aforementioned clinical settings. Fox example, RIC has been found to reduce the severity of acute mountain sickness symptoms, enhance the exercise performance and benefit patients with diabetic foot ulcer –237. On There are conceivably a number of existing and potential stakeholders when it comes to the extrapolation of RIC from test bench to bedside. One of the major explanations for these discrepancies lies in the impact of comorbidities on the ischemic tissues or organs in response to the benefits from IC. Animals used in experimental studies are mainly juvenile, genetically homogenous, housed in similar living environments with the same diets, and carried with limited comorbidities. Nevertheless, a significant challenge in clinical trials is that enrolled individuals are more aged with large comorbidities and polypharmacy. In fact, sets of comorbidities including ageing, hypercholesterolemia, hypertension, diabetes, and chronic kidney disease have been found to inhibit or abolish the potency of IC according to previous experimental findings. It seems that concomitant medications including volatile anesthetics such as propofol and isoflurane, opioids, statins, anti-platelets, ACEI, beta-blockers, nitrates and hypoglycemic agents such as pioglitazone and glimepiride might either abrogate or restore the beneficial effects afforded by IC ,241–246.Intriguingly, pharmacological manipulation targeting the signaling cascades and related molecular participants that can modulate the endogenous mechanisms of RIC is an emerging notion with substantial clinical value. Figuring out common mechanisms of protection afforded by IC and some pharmacological agents in a given clinical set-up may facilitate the development of novel approaches against deleterious effects such as IRI. In addition, the presence of time windows during which the signaling cascades in responsive to IRI are engaged might affect not only the effectiveness of IC but medical interventions that target perpetrators of reperfusion injury. Clearly, research should be continue for the sake of identifying pharmacological mimetic candidates that are safe and efficacious, verifying time window for conditioning, and further delineating the mechanism behind as well as human genetic and proteomic responses to conditioning.RIC is an innovative therapeutic strategy, whereby transient, repetitive, sublethal ischemic exposure to a particular tissue or organ is able to confer systemic protection to distant susceptible organs or tissues against subsequent IRI.The application of RIC is simple and non-invasive, and has been extended from the arena of initial cardiac protection to a number of other organs such as the brain, kidney, lung and liver. The feature of multi-organ protection is quite significant since it allows RIC to a variety of clinical settings in which organs or tissues are vulnerable to acute IRI. It is hopeful that future well-designed clinical trials with adequate size and power will turn RIC into a routine clinical practice for either prevention or attenuation of disorders in more extensive scenarios than currently expected.In this study, we provided an overview of the basic concept and proposed protective mechanisms of remote ischemic conditioning (RIC). In addition, we comprehensively reviewed and summarized the clinical studies of RIC in multi-organ protection published so far. Potential obstacles, which may retard the clinical translation of RIC, were discussed as well. We found that:Proof-of-concept clinical studies demonstrated benefits with RIC as an adjuvant to thrombolysis, mechanical thrombectomy or primary angioplasty in patients with acute cerebro- or cardiovascular attack, such as ischemic stroke and acute myocardial infarction; in light of advantages including simplicity, low expenditure and safety, RIC may be an ideal intervention to be employed during the transportation to qualified stroke or chest pain centers, and before and/or after intrahospital treatment.Long-term, repeated use of RIC has the potential to improve vascular endothelial function and enhance tissue perfusion, whereby decreasing the incidence or recurrence of unfavorable clinical endpoints in diverse clinical settings, such as chronic cerebral ischemia and chronic heart failure.Although two large, multicenter, randomized clinical trials of RIC in coronary artery bypass graft surgery yielded negative outcomes, factors including patient selection, RIC protocol and potential confounders should be further explored before reaching a final conclusion.Regarding the multi-organ protection feature, RIC seems to be a promising non-pharmaceutical and non-surgical approach for preventing and treating systemic vascular diseases, which can inflict multiple systems simultaneously. As multi-organ ischemia-reperfusion injury, particularly amongst elderly patients with clinical scenarios such as atherosclerotic vascular disease, heart surgery and organ transplantation, is one of the most common real-world situations that besets clinicians, the authors believe that this paper will certainly arouse a wide interest from medical workers in both tertiary and community-based medical facilities.Future experimental or clinical work should focus on addressing the issues that may influence the translation of RIC from test bench to bedside, such as identifying the protective mechanism underlying ischemic conditioning, optimizing the conditioning regimen, establishing biomarkers to accurately evaluate the efficacy of RIC, and figuring out the impact of potential comorbidities, medications and other factors on RIC.We searched PubMed and EMBASE to identify randomized controlled trials (RCTs), meta-analyses, observational studies and systematic reviews that published between 1986 and 2018, via key words of ‘remote ischemic conditioning’, ‘remote ischemic preconditioning’, ‘remote ischemic perconditioning’, ‘remote ischemic postconditioning’ etc. We subsequently reviewed references of the retrieved articles for additional reports. Meta-analyses and systematic reviews were prioritized; case series and reports were included only for interventions for which RCTs were not available. Some data from prospective clinical trials were processed using RevMan5.0 software provided by Cochrane collaboration and transformed via relevant formulas.Supplemental Figure 1Supplemental Table 1-4


Glioblastoma multiforme is the most common and aggressive primary brain tumor. Even with aggressive treatment including surgical resection, radiation, and chemotherapy, patient outcomes remain poor, with five-year survival rates at only 10%. Barriers to treatment include inefficient drug delivery across the blood brain barrier and development of drug resistance. Because gliomas occur due to sequential acquisition of genetic alterations, gene therapy represents a promising alternative to overcome limitations of conventional therapy. Gene or nucleic acid carriers must be used to deliver these therapies successfully into tumor tissue and have been extensively studied. Viral vectors have been evaluated in clinical trials for glioblastoma gene therapy but have not achieved FDA approval due to issues with viral delivery, inefficient tumor penetration, and limited efficacy. Non-viral vectors have been explored for delivery of glioma gene therapy and have shown promise as gene vectors for glioma treatment in preclinical studies and a few non-polymeric vectors have entered clinical trials. In this review, delivery systems including viral, non-polymeric, and polymeric vectors that have been used in glioblastoma multiforme (GBM) gene therapy are discussed. Additionally, advances in glioblastoma gene therapy using viral and non-polymeric vectors in clinical trials and emerging polymeric vectors for glioma gene therapy are discussed. Glioblastoma multiforme (GBM) is a type of glioma that arises from astrocytes, defined by the World Health Organization (WHO) as a grade IV glioma ,2. GBM iHigh-grade gliomas are typically located in undesirable locations in the cerebral hemisphere and are classified as diffuse gliomas due to their high rate of infiltration into surrounding brain tissue. These factors allow for persistent tumor growth and lessen the chance of remission, with progression to grade III or IV gliomas likely even in most low-grade diffuse gliomas ,5. The cThis review discusses gene expression in GBM, the limitations to conventional therapy, and current approaches to circumvent these barriers using gene therapy. Advances in gene delivery systems will be reviewed, highlighting viral and non-viral vectors used for GBM gene therapy. Trials bringing these therapies closer to clinical approval to date will be discussed, as well as preclinical studies, particularly using polymeric nanoparticles, which have shown promise as future vectors for delivery of gene therapy in GBM patients.de novo, and occurs in about 95% of cases, only requiring about 3–6 months to develop . ,23. 5,23Drug resistance is another major barrier in the treatment of GBM due to overexpression of drug resistance genes. Additional protection of the BBB exists in the form of various efflux transport systems which remove unwanted substances that are able to traverse the BBB. A largely studied efflux pump, P-glycoprotein or multidrug resistance protein 1 (MDR1), encoded by the ATP-binding cassette sub-family B member 1 (ABCB1) gene, has been a persistent challenge in therapeutic delivery due to its efficacy in removing small molecules from the brain . A wide Gene therapy for cancer treatment conventionally includes the introduction of growth regulating or tumor suppressing genes. More recently, RNA interference (RNAi) has been introduced to inhibit the activity of oncogenes causing tumorigenesis or proliferation. Suicide gene therapy is another approach that is commonly used in viral gene therapy to convert non-toxic prodrugs into lethal active compounds. Other approaches include oncolytic and immunomodulatory gene therapy . Gene orViral vectors were the first delivery vehicles used for gene therapy in glioma clinical trials and have been studied for glioma gene therapy over the past 25 years. Viral vectors commonly used for GBM gene therapy in clinical trials include neurotropic retroviruses  and adenRetroviral vectors were the first delivery systems evaluated in clinical trials for glioma gene therapy. The initial trial evaluated the combination of modified murine cells containing retroviral herpes simplex virus-thymidine kinase (HSV-tk) with ganciclovir (Cytovene) and began in 1992 (NCT00001328). HSV-tk functions as a suicide gene and converts the prodrug, ganciclovir, into its active form, ganciclovir-triphosphate, which inhibits DNA replication and cell division in HSV-tk-transfected cells . The res+ T-cells were found in tumors analyzed post-treatment [Adenoviral vectors have also been widely evaluated in clinical trials. A phase 1 trial of an adenoviral vector carrying the wild-type p53 gene (Ad-p53) demonstrated that Ad-p53 successfully transfected astrocytic tumor cells with minimal toxicity when intratumorally injected pre- and post-resection of the glioma tumor; however, transfected cells were only detected on average within 5 mm of the injection site . Additioreatment , indicatreatment . Additioreatment . Half ofreatment .Although viral vectors have been studied extensively, they have only resulted in marginal increases in overall survival and have yet to achieve clinical translation through FDA approval to treat patients with GBM after decades of study. Efficient tumor penetration of viral vectors has proven to be a challenge limiting overall efficacy in treating gliomas. However, there has been some clinical success with viral vectors in other cancers. Talimogene laherparepvec is an oncolytic virotherapy consisting of genetically modified HSV-1 containing the human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and has been FDA approved for the treatment of melanoma . In addition to viral vectors, non-viral vectors, including both non-polymeric and polymeric delivery systems , have bePolymeric delivery of gene therapy is an emerging approach for cancer treatment to improve therapeutic outcomes. Current research has been focused on micro- and nanoparticles for the systemic or local delivery of genes and/or drugs. These NPs are advantageous for gene therapy because they are highly tailorable, allowing for conjugation of nucleic acids, homing peptides, or targeting ligands. Though they have not yet reached clinical trials specifically for glioblastoma treatment, several polymeric delivery systems have been studied for use in gene therapy for glioma treatment, and are discussed in detail as follows, including their advantages and limitations. The advantages and disadvantages of various vectors that have been studied for glioblastoma gene therapy are summarized in Dendrimers are highly branched 3D polymers that have been explored for a variety of applications in drug and gene delivery. Cationic dendrimers, such as poly(amidoamine) (PAMAM), are particularly useful for gene therapy in glioma treatment due to their ability to form complexes with negatively charged nucleic acids, penetrate cellular and endosomal membranes, and cross the BBB. Dendrimers have been used to deliver several types of nucleic acids, including antisense oligonucleotides , microRNl-lysine (DGL) was recently discovered as a newer class of dendritic polymers and has shown potential for delivery of nucleic acids. One major advantage of DGL over dendrimers is that DGL is composed entirely of naturally occurring lysine residues and is therefore completely biodegradable. Dendrigrafts are rich in external amino groups, which enables self-assembly with nucleic acids. DGL is also non-immunogenic and has been demonstrated to cross the BBB [Similar to dendrimers, dendrigrafts are also dendritic structures that can be used to deliver therapeutics. Dendrigraft poly- the BBB . To medi the BBB  or lamin the BBB  peptides the BBB ,75. Tran the BBB . In anot the BBB . DGL has the BBB . Similar the BBB ,80. To o the BBB . Though Polymeric micelles are amphiphilic copolymers that have a core/shell structure. They have been widely used for cancer drug delivery , but havPoly(β-amino esters) (PBAEs) are another class of cationic polymer that were designed to meet specific criteria for gene delivery, including DNA condensation, biodegradability, and minimal cytotoxicity , PBAEs mGlioblastoma multiforme is a common and currently incurable brain cancer that desperately needs new treatment modalities to improve patient outcomes. The current standard of care including surgical resection, adjuvant chemotherapy, and radiation does not result in remission for the majority of patients. Barriers limiting efficacy include inefficient delivery across the BBB and therapeutic resistance. Gene therapy represents an approach to specifically target and regulate oncogenes and tumor suppressor gene in gliomas. Further, gene therapy can be used to overcome barriers such as chemotherapy resistance by downregulating resistance genes or using approaches such as suicide gene therapy. Viral vectors, including retroviruses and adenoviruses, have been evaluated in clinical trials of GBM for the past few decades for delivery of therapeutic genes or nucleic acids in combination with other therapeutics. However, viral vectors have not reached clinical approval due to immunogenicity, limited tumor penetration and marginal improvement in patient outcomes. Non-viral delivery is an evolving alternative approach that may be used to overcome the barriers of gene delivery. Many non-viral vectors, including polymeric and non-polymeric vectors, are non-immunogenic and can be functionalized with targeting moieties to increase receptor-mediated uptake of vectors into tumor tissue. Multifunctional and multimodal non-polymeric vectors, such as liposomes and gold NPs, respectively, have the ability to co-deliver multiple therapies or be used for tumor imaging as well as therapy. Cationic polymeric vectors have the ability to self-assemble with nucleic acids, enhancing their ease of use for gene therapy over other vectors. Moreover, polymeric vectors such as PBAE, have demonstrated potential for improving tissue penetration, one of the largest barriers to increasing efficacy of gene therapy vectors in glioblastoma. To date, only a few non-viral vectors have been evaluated in clinical trials for GBM; however, further evaluation of non-viral vectors in clinical trials in the future may provide advanced treatment strategies for gene therapy in glioblastoma.