The objective of this review is to critically assess the contributing role of the gut microbiota in human obesity and type 2 diabetes (T2D).Experiments in animal and human studies have produced growing evidence for the causality of the gut microbiome in developing obesity and T2D. The introduction of high-throughput sequencing technologies has provided novel insight into the interpersonal differences in microbiome composition and function.The intestinal microbiota is known to be associated with metabolic syndrome and related comorbidities. Associated diseases including obesity, T2D, and fatty liver disease (NAFLD/NASH) all seem to be linked to altered microbial composition; however, causality has not been proven yet. Elucidating the potential causal and personalized role of the human gut microbiota in obesity and T2D is highly prioritized. The current obesity pandemic is increasing worldwide, thus contributing to the rising incidence and prevalence of type 2 diabetes (T2D) mellitus. Particularly alarming is the equal trend of increase in obesity in children , 2. T2D,Increasing evidence indicates a fundamental role of excess central adiposity in developing the metabolic syndrome . HoweverThe gut microbiota is a collective term for the microbial community in the gut , whereasFirmicutes) and proteobacteria (Enterobacteriaceae), whereas the more distal large intestine shows higher concentrations of Firmicutes  and anaerobes such as Bacteroidetes . The representation of Verrucomicrobia, Akkermansia muciniphila, is also mainly located distally [Diversity of the gut microbiota is the result of strong host selection and co-evolution, influenced by many factors. It includes thousands of species with a collective genome, the metagenome, which is close to 5 million genes. Our insight into the composition of the gut microbiota is based on culture-based studies and became more precisely characterized with high-throughput sequencing technology . Severaldistally –36.Metagenomic-wide association studies combine metagenomic data with clinical features. Such studies presented significant differences on a metagenomic level between metabolically healthy versus metabolically unhealthy subjects , 38. TheSymbionts and commensals are not the only microorganisms the host encounters. The host immune system is continuously challenged by distinguishing beneficial microbes from pathogens. The interactive role of phages  and fungThere are different factors influencing the development of the microbiome in the early years of life, starting with the mode of birth , 45, breDiet is a modulator of the composition and the function of the gut microbiota . Human iIn line with their finding that diet is an important driver of gut microbiota composition, Yatsunenko et al. found that gut microbiomes differ over human populations and that Malawian and Amerindian infants have more shared features than when compared to those of American infants .Gammaproteobacteria and Erysipelotrichi and this is directly associated with fatty liver development [. experimented in atherosclerosis-prone mice, supplementing a diet with either choline or TMAO, which resulted in increased levels of TMAO in plasma; the latter enhanced atherosclerotic plaques. The previous link was negated in atherosclerosis-prone mice with antibiotic-suppressed microbiota [It is thought that (aromatic) metabolites derived from gut microbiota processing of dietary compounds are important factors influencing host physiology; conversely, the responses to nutrients are influenced by the gut microbiota. Choline and trimethylamine-N-oxide (TMAO) are two of those impacting micronutrients. For example, Spencer et al. showed that a choline-deficient diet in humans modulates the gut bacteria with altered levels of elopment . Wang etcrobiota , underscBacteroidetes and an increase of Firmicutes in comparison to their lean siblings [Several studies, mostly animal models, demonstrated the microbiota as a major factor in the development of obesity , 63, 64.siblings . These dLactobacillus gasseri demonstrated positive effects on body weight in overweight and obese subjects treated in a double-blind randomized controlled intervention trial [A similar composition of the microbiota is seen in obese human subjects, and the microbial ecology in humans changes with weight loss . The traon trial . This alCurrently, the most effective treatment for obesity in the long term is bariatric surgery . IntriguLactobacilli and Enterobacteriaceae was observed in addition to a significantly decreased abundance of the butyrate-producing Roseburia spp./E. rectale group in iron-deficient rats. Repletion of iron significantly increased cecal butyrate concentrations [Bacteroidetes-to-Firmicutes as well as an increased proportion of Bacteroidetes [Diet is a major source of substrates for the production of small molecules by the gut microbiota. After absorption, these molecules may have a direct effect on hepatocytes via uptake and transportation in the portal vein. After escaping first-pass metabolism in the liver, these small molecules circulate systemically, and in this way, may contribute to diverse effects on host physiology . Dependetrations . SCFA actrations . In thistrations , 78. On trations . Indeed,roidetes . Conversroidetes , 81 Moreroidetes .Finally, Gpr43 activation by SCFA in  intestinal enteroendocrine L cells induces GLP-1 release that can also affect insulin sensitivity via incretin response . In concAs described above, the intestinal microbiota should not be viewed as an isolated community. Instead, it functions as a large metacommunity, in which microorganisms reciprocally affect each other. In this way, they can disperse and colonize new gut habitats . The impEscherichia coli in the T2D MLG, a bacterial strain that is thought to be one of the major sources of LPS in humans [As described above, low-grade inflammation of visceral adipose tissue may provide a link between obesity and insulin resistance . Adiposen humans . LPS andn humans  and flagn humans , activatn humans .Roseburia and Faecalibacterium prausnitzii), known for their anti-inflammatory properties [Akkermansia muciniphila was associated with improved metabolic control and less obesity in mouse models of T2D [Akkermansia muciniphila in the T2D MLG in humans [Akkermansia muciniphila. Moreover, ethnic differences between human populations may also affect microbiota composition. Karlsson et al. compared data of T2D-associated metagenomes between Chinese and Swedish subjects with T2D, which indicated that different intestinal bacterial species were involved in similar metabolic functions [. performed a meta-analysis, pooling results from four studies, including publicly available data from HMP and MetaHIT. This meta-analysis confirmed the differences in phylum-level taxonomic composition between lean and obese subjects. However, it could not reproduce the association between BMI and taxonomic composition [. performed metabolomics and microbiome profiling in subjects with T2D and without diabetes. They reported increased plasma levels of branched-chain amino acids (BCAA) as characteristic of individuals with insulin resistance, which in turn correlates with specific bacterial species (P. copri and B. vulgatus) in their gut microbiota [. [Microbiome sequencing also revealed that butyrate-producing Clostridialis  lipids . The nutG-coupled protein receptors (Gpr) are also activated by both diet-derived metabolites and lipids. For example, the activation of Gpr43 by the dietary-derived metabolite acetate directly reduces lipolysis in adipocytes leading to decreased plasma-free fatty acid levels. This suggests a potential therapeutic role for Gpr43 in the regulation of lipid metabolism .Lipid accumulation in conjunction with low-grade inflammation is a pathophysiological hallmark of atherosclerosis . There iOther key intestinal regulators of lipid and cholesterol metabolism are bile acids, which are involved in facilitating intestinal absorption and transport of diet-derived nutrients, vitamins, and lipids. Whereas bile production takes place in the liver (and is facilitated by products derived from lipid catabolism), 95% of all bile acids will be reabsorbed in the terminal ileum and subsequently re-absorbed by the liver, constituting the so-called enterohepatic circulation. The intestinal microbiota is responsible for converting primary bile salt to secondary bile salts via bile acid de-hydroxylation . AlthougObesity is defined as an imbalance between energy intake  and energy expenditure . The braSCFA has also been implicated in intestinal gluconeogenesis (IGN), which is thought to influence food intake and glucose metabolism in mice . Recent As seen in animal models, IGN induces release of glucose into the portal vein where glucose sensors are located and these sensors transmit signals to the brain by the peripheral nervous system, which curbs hunger and improves insulin resistance .Animal studies describe IGN as an important factor in metabolic control both after protein-enriched diet –125, andChanging the gut microbiome composition with prebiotics has also been shown to affect portal vein levels of other hormones including GLP-1, which in turn affected food intake, followed by a decrease in body weight and fat mass . This suThe studies discussed in this review suggest that individuals who are obese are likely to have an imbalance in gut microbiota composition and a ‘gut signature’ varying with metabolic control . This poMoreover, conclusions on the magnitude and causal role of the intestinal microbiota in human metabolism are blurred by other confounding factors, such as (ethnic) differences in study populations, differences in sequencing and analytical techniques, and variability of diets used for the studies. Therefore, guidelines for standardized techniques are essential.Shifting towards large prospective cohort studies will provide us with more evidence and information on clinical relevance. Similarly, mapping patients with pre-diabetes and diabetes, and observing their diet, type of obesity, metabolic parameters, medication, and inflammatory state is essential for clarifying whether microbiome composition and the presence of metabolites is a result of disease phenotype or if it is causally interlocked with the underlying pathophysiology. Identifying the most important microbiota-related metabolic pathways will enable us to develop new therapeutic agents by influencing these microbiota-related pathways.Treatment with prebiotics or with newly identified beneficial bacterial strains are potential interventions that will be used in the near future, and it will be important to evaluate their efficacy. Likewise, interventional studies with metabolites of microbiota will be performed (including SCFA butyrate supplementation) to evaluate if this compound has similar effects on food intake, energy expenditure, and improved metabolic features in humans .Newly identified bacterial strains with probiotic potential will probably be found using FMT. For example, our recent study illustrated how donor FMT can (transiently) improve insulin sensitivity in male recipients with metabolic syndrome, bringing new insight in pathophysiology ••. NeverIn conclusion, the modifiable effects of the human gut microbiota on the development of metabolic syndrome make its manipulation a promising therapeutic approach. Analyzing and mapping individual microbial composition on a metagenomic level provides insight into specific targets for treatment and contributes to personalized therapeutic interventions.


All cells release a multitude of nanoscale extracellular vesicles (nEVs) into circulation, offering immense potential for new diagnostic strategies. Yet, clinical translation for nEVs remains a challenge due to their vast heterogeneity, our insufficient ability to isolate subpopulations, and the low frequency of disease-associated nEVs in biofluids. The growing field of nanoplasmonics is poised to address many of these challenges. Innovative materials engineering approaches based on exploiting nanoplasmonic phenomena, i.e., the unique interaction of light with nanoscale metallic materials, can achieve unrivaled sensitivity, offering real-time analysis and new modes of medical and biological imaging. We begin with an introduction into the basic structure and function of nEVs before critically reviewing recent studies utilizing nanoplasmonic platforms to detect and characterize nEVs. For the major techniques considered, surface plasmon resonance (SPR), localized SPR, and surface enhanced Raman spectroscopy (SERS), we introduce and summarize the background theory before reviewing the studies applied to nEVs. Along the way, we consider notable aspects, limitations, and considerations needed to apply plasmonic technologies to nEV detection and analysis. Nanoscale extracellular vesicles (nEVs) encompass a heterogeneous grouping of naturally occurring nanoparticles that are endogenously secreted by all cells tested to date  analyses of maturing reticulocytes, cells that specialize in recycling a large portion of their contents, and nEVs spent much of the last 30 years associated primarily with their function in secreting cellular waste  and apoptotic bodies (both of which bleb directly from the cell membrane) that are often co-enriched during standard isolation preps . Furthermore, detection based on light scattering is typically incapable of absolute sizing of nEVs smaller than ~70 nm, despite TEM evidence that a major fraction of EVs are smaller . These are referred to as localized surface plasmon resonances (LSPRs) when the SP is confined to a nanoparticle surface with dimensions far below the wavelength of incident light. Particularly strong electromagnetic fields can be formed at the interface where two SPs meet, known as a gap-mode plasmon  and metal substrates with nanoscale surface roughness  as an experimentally observed phenomenon dates back to the late 1960s, based on the understanding that polarized light is capable of exciting electrons residing at the interface of a dielectric and conductive metal surface  a prism is tightly connected via a refractive index matching material to a glass sensor chip coated with a thin (~50 nm) film of gold, though many other configurations exist, comprising diffraction gratings, optical fibers, and optical waveguides  is measured as the incident light angle θ0 is varied. As the incident light angle reaches the surface plasmon resonance condition, the energy transfers from photons to surface plasmons, and the resulting resonance can be observed as a reflectance minimum  of interest using appropriate mathematical fitting.In a  minimum . AnotherThe physical principles of LSPR are fundamentally similar to SPR-based sensing. Instead, while SPR is typically carried out using planar metal substrates, LSPR relies heavily on the utilization of metal nanoparticles suspended in solution or micro- and nano-fabricated metallic structures such as gratings, nanopillars, and nanoarrays  to create a non-propagating free electron oscillation in the conduction band of the metal. As discussed in the context of SERS below, a remarkable electric field enhancement at the close proximity of the metal nanoparticle/structure can be observed. At the key plasmon resonance frequency, the absorption and scattering of light by the metallic nanostructures takes place  it is relatively inert, (ii) thiol chemistry enables straightforward functionalization, and (iii) its plasmon frequency allows for detection using inexpensive UV-Vis spectrophotometers (sometimes even an unaided eye)  to capture the analyte of interest. The non-functionalized surfaces and sites in the nanostructures are blocked by inactive components to avoid non-specific adsorption of co-analytes. Given that the target analyte  typically has higher RI than the blank background, the local dielectric environment near the plasmonic nanostructure experiences a change, which can be seen as the LSPR peak shift toward higher wavelength -nEVs with surface-immobilized antibody  nEVs was developed for predicting the existence and stage of coronary heart disease (CHD)  platform using multiplexed antibody microarrays was introduced  in samples exacerbate reaching adequate specificity and sensitivity as all these components may bind to biosensor surface and change the local RI at its vicinity. Advancements in optics  could be harnessed to address the instrument-related shortcomings. On the other hand, rapidly developing understanding of chemical modification of plasmonically-active materials may provide new insights for high-precision biosensor surface preparation  metallic nanostructure arrays on a solid support generated using micro- and nano-fabrication lithographic techniques or (ii) nanoparticles, freely suspended in solution or deposited onto a solid support.Nanohole and nanopillar arrays. The nano-plasmonic exosome (nPLEX) is an LSPR-based assay utilizing optical transmission through an array of periodic nanoholes patterned to a 200 nm thick gold film on a glass substrate  was observed when the nanoholes were functionalized with anti-CD63, and subsequent capturing of CaOV3-derived nEVs was performed. In comparison to WB and ELISA, the nPLEX protein quantification performed better . Notably, secondary labeling significantly amplified signaling of the nPLEX platform: spherical Au nanoparticles showed a 20% increase and larger, star-shaped Au NPs showed 300% amplification. Another OvCa cell line, OV90, was used in parallel with CaOV3 to compare the capabilities and correlation of nPLEX against ELISA . Correlation of R2 > 98% was found. Concurrently, the captured nEVs were eluted from the nanostructures, and a quantitative real-time PCR (qRT)-PCR was implemented to investigate the mRNA contents of the nEVs. Next, the researchers did a molecular screening of nEVs encompassing various non-cancerous and cancerous cell lines. Subsequent observations were applied to a cohort of OvCa (n = 20) and cirrhosis  patients to determine the nPLEX performance. nEVs were enriched only using 0.2 μm membrane filtration. The amounts of EpCAM and CD24 were significantly higher in the OvCa group. Lastly, a small cohort (n = 8) of OvCa patients undergoing chemotherapy were profiled to evaluate patients' response to treatment. Levels of EpCAM and CD24 showed a decreasing trend in the corresponding patients. In future, the use of larger patient cohorts and additional disease models will strengthen the power and applicability of the platform.ubstrate   and further validated with a cohort of 43 human samples, with an accuracy of 84%, sensitivity of 86%, and specificity of 81%. A correlation study was performed between the PDACEV signature and gold standard markers (CA19-19 and CEA), finding none. Sixty-one percent of PDAC patients showed elevated CA19-19 levels and 17% of PDAC patients showed elevated CEA levels, while PDACEV values revealed 89% of PDAC patients. High specificity and sensitivity, scalability, throughput, automated operation, molecular printing capabilities, cost effectiveness, and low sample consumption were the advantages of the developed method. Meticulous statistical analyses complemented the different phases of the study and underlined the high translation potential of the platform.In a similar study, nanohole-based surface plasmon resonance platform named intravesicular nanoplasmonic system (iNPS) was developed to screen both surface as well as intravesicular proteins trafficked by nEVs released from ovarian cells , nor assess purity of the preps. A concern can be raised, whether the speculated intravesicular filaments pictured in the final image of the study are due to non-nEV impurities, thus resulting in the larger plasmon sensing area, which was considered to contribute to the higher sensitivity of the 3D PCC nanostructures. A second suggestion would be to measure several additional sources/types of nEVs for validation.Two-dimensional (2D), quasi-three-dimensional (quasi-3D), and 3D plasmonic photonic crystal (3D PPC) nanostructures have been developed and evaluated for nEV detection  were introduced to the LSPRi sensors using a microfluidic system. The authors state that single-nEV binding events are resolvable from free protein as a result of the subsequent discrete step function response in comparison with a smoother integrated response that smaller analytes would produce. For this work, it is noted that precipitation kits may induce artifacts to the observed signals in the form of aggregated nEVs or larger non-EV contaminants due to the isolation reagent.Single nEV detection was pursued by a LSPR imaging (LSPRi) system  was recently introduced  bound the anionic nEVs. When integrated with fluorescent microscopy and the nEV lipid membrane was labeled with DiIC18, fluorescence signals correlated well spatially with the iPM image analyses. Through the iPM analysis, nEVs demonstrated a continuous distribution between 30 and 150 nm with a peak value of ~62 nm. Notably, the NTA analyses showed higher values at ~120 nm. Although rigorous biophysical work was represented, the known EV/liposome shape reformation upon binding onto the Au surface was not addressed—which may contribute to the observed results.plasmonic interferometer array (PIA) was invented for real-time detection of circulating nEV proteins with high sensitivity and portability . Without surface functionalization, reported LOD of 0.194 μg/mL and linear dynamic range of 0.194–100 μg/mL were obtained. However, a concern is raised regarding isolation methods, which varied for each type of sample; cell culture supernatants employed UC and filtration, while serum and urine EVs were isolated using different commercial isolation reagents. The vaguely described MV isolation method stands out since the study essentially focuses on investigating nEVs vs. MVs, and the outcomes likely depend heavily on the chosen MV enrichment procedure. Also, given that each method likely yielded varying levels of purity, which were not directly assessed, non-EV originating contaminant proteins cannot be excluded and concentration estimates based on protein content may not be accurate. A critical suggestion would be to use a uniform isolation panel for all the different preparations and a consequent endeavor to qualitatively or quantitatively determine the purity of the preparations [further suggestions can be found e.g., in references:  have been recently introduced as sensitive and inexpensive optochemical biosensors, produced by a two-step deposition-annealing procedure  sensitivity was reported. Pancreatic cancer cells (PPCs) are characteristic to pancreatic ductal adenocarcinoma (PDAC), and PCCs are known to overexpress miR-10b in PDAC. The aim was to study whether PCCs release miR-10b into the cell culture medium or circulation, and to explore whether the miR-10b levels in nEVs could differentiate between chronic pancreatitis (CP), and individuals with and without PDAC pathology. Gold nanoprisms were synthesized, attached onto a glass substrate, and functionalized with complementary oligonucleotides. The developed LSPR sensor was demonstrated to work with PCCs -derived conditioned media and nEVs. Successful differentiation was also performed between PDAC or CP patients and normal controls in plasma, nEVs, and post-UC supernatants. The authors pinpoint various structural attributes in the LSPR sensors that enable high sensitivity, in particular the charge transport of the gold nanoprisms through the DNA backbone upon forming duplexes with the ssDNA. The regeneration capability and stability of the LSPR biosensors was also tested, and the authors concluded that the sensitivity remains unchanged at least over 5 days.Another more recent LSPR sensor using AuNIs was reported, decorated with the peptide Venceremin (Vn96), which exhibits strong affinity to nEV surface heat-shock proteins (HSPs) overexpressed on many tumor cells  assay was developed and tested for pancreatic cancer as a model approach , chronic pancreatitis (n = 48), and pancreatic cancer patients (n = 49) at different stages of the disease. The non-diagnostic biomarker CA19-9  was used for benchmarking the clinical performance of the EphA2-EV nPES levels. As 23 pancreatic cancer patients were evaluated pre and post neoadjuvant therapy, the EphA2-EV levels in comparison to CA19-9 more accurately reflected the treatment status for patients with good or partial therapy responses. The study was well outlined, and the results supported by additional experiments  as well as carefully designed statistical analyses. As noted by the authors, the specificity and generalizability of the nPES platform could be improved further by replacing the secondary anti-CD9-AuNR probe with additional cancer-specific probes.The To summarize, LSPR techniques complement SPR methods well, and they can be used as stand-alone applications for nEVs. The needed instrumentation is relatively cost-effective and operator-friendly, simultaneously showing potential for miniaturization, automation and integration with e.g., microfluidics. A good example is the work by Maiolo et al. , where t−1) on the abscissa and scattering intensities on the ordinate. Raman spectroscopy affords high chemical specificity, minimal to no sample processing, is inherently non-destructive, and relatively inert to aqueous background. The main disadvantages for spontaneous Raman spectroscopy are weak signal and high fluorescent/photoluminescent backgrounds, particularly for biological specimens. However, these shortcomings can be efficiently overcome using SERS. Notably, even single molecule sensitivity can be achieved  of the conductive electrons in metals. Thus, in SERS, the high chemical specificity of Raman spectroscopy is combined with unprecedented sensitivity accomplished by plasmon-assisted scattering of molecules on or near  metal nanostructures. The formation of “hot spots”—where the highest signal enhancement is observed—is an important physical phenomenon, occurring where two or more SERS-active regions come into spatial contact, i.e., gap-mode plasmons  has been applied to characterize nEVs and MVs in several cases, enabling quantitative measurement of global chemical composition analysis . Spontaneous Raman of both bulk EVs  to capture and analyze nEVs binding α3ß1 integrin overexpressed on OvCa cell-derived nEVs, though should be extended to clinical samples for increased relevance  NP with a silica shell. Both Au@Ag NRs and magnetic nanobeads were further decorated with antibodies, enabling formation of a sandwich complex by capturing vesicles between the nanobeads and NRs. The resulting magnetic properties of the immunocomplexes permitted separation by magnet for SERS signal detection in collected precipitates. Using anti-CD63 and anti-HER2 antibody, nEVs secreted by SKBR3 could be readily distinguished from control MRC5 cell-derived nEVs. Decreasing concentrations of vesicles were immunoprecipitated to find an LOD of ~1,200 vesicles, with a total assay time of just 2 h. Another study incorporated three specific SERS probes decorated with aptamers as alternative biorecognition components to supplement antibodies  to be used as capturing substrates. CD63 surface protein aptamer was decorated onto the MB@SiO2@Au probes. In conjunction, AuNPs with complementary specific aptamers (AuNP@aptamer) were developed for use as a readout signal. Target nEVs were added to a mixture of the two probes to form a sandwich apta-immunocomplex. Similar to the previous study, the complexes could be precipitated by magnet for SERS readout. In this case, the signal became weaker with the addition of EVs that bind to the SERS probes while the signal from control non-specific probes remain unchanged. nEVs isolated using commercial kits from breast cancer cells (SKBR3), PCa cells (LNCaP), and colorectal cancer cells (T84) were each probed by various specific AuNP@aptamer complexes, using aptamers for H2, PSMA, and CEA, respectively. As the enhancements in various regions of SERS spectra varied widely between the samples, each presented a unique LOD: 32 nEVs per microliter for SKBR3, 203 nEVs per microliter for LNCaP, and 73 nEVs per microliter for T84. A similar study fabricated gold nanostars (AuNSs) “over-coated” with an outer gold film to trap Raman reporter molecules (4-MBA) between the layers  in the form of patterned silicon micro-pillars decorated with silver nano-aggregates on the pillar top  of liquids are being manipulated in channels with dimensions of tens to a few hundred microns. Furthermore, nEV analyses can be carried out in a short time, inexpensively, and isolation in addition to characterization is possible with high resolution and sensitivity. Microfluidics in combination with optics, nanoplasmonics and advanced data analysis tools offer an immense palette of opportunities for robust, automated, high-throughput and statistically relevant nEV research as well as moving from bench to bedside and commercial applications. We guide the reader to two excellent reviews that elucidate how modern microfluidic lab-on-chip techniques can be applied for nEV isolation, detection, and characterization (Gholizadeh et al., A nanomechanical resonator was recently demonstrated to weight the mass of single NPs down to 10 nm with attogram precision (Olcum et al., Still other emerging tools may be boosted by nanoplasmonic additions, such as the recent introduction of ZnO nanowire lateral displacement arrays for nEV fractionation and enrichment (Wunsch et al., Diagnostic platforms for disease-associated nEVs have so far failed to achieve meaningful clinical translation, partly due to confusion and difficulties regarding their isolation and characterization, but also due to their low frequency in circulation compared to healthy background, particularly for early-stage patients. Nanoplasmonics offers the potential to realize the promise of exosomes and related nEVs to non-invasively report in real-time on the condition of cells and tissues. It is clear that nanoplasmonics-based approaches to detect and molecularly characterize nEVs offer spectacular signal enhancement compared to standard field-adopted methods. Yet, the majority of reported nanoplasmonic approaches are firmly ground at the level of proof-of-concept, displaying only incremental effects with limited scope. Although rational synthesis of plasmonic materials has been advancing tremendously in the past several years (Xi et al., Researchers should focus on improving statistical power in clinically relevant sample sets, especially increasing number of patient samples, with the goal of high-power clinical trials.Adherence to the ISEV suggested minimal information to characterize nEVs, along with careful reporting using the EV-TRACK database is highly advisable. Specifically, care should be taken to provide stringent chemical and physical characterization of EV preps using complementary field-validated methodology and protocols.Development of materials engineering and chemical methods to improve the homogeneity of plasmonic probes and substrates is needed, the lack of which severely limits reproducibility and system stability or predictability. The effect of heterogeneity of nanoplasmonic probes and substrates is rarely considered and should be measured, reported, and the results contextualized accordingly.Label-free approaches should be chosen when possible, in order to avoid known artifacts with respect to heterogeneous nEV labeling.Plasmonic NP and substrate geometries should be tailored toward accounting for heterogeneity in size, a hallmark of nEVs. For example, nanohole arrays may be varied in hole diameter to accommodate a range of EV sizes, rather than a one-size-fits-all approach.The vast majority of assays are currently limited to detecting transmembrane or lipid-bound biomolecules, based on immunoaffinity of whole nEVs to device surfaces. New assays should be developed to investigate and correlate intravesicular content, for example, plasmonic analysis before and after EV lysing.in vivo context. Diffraction-limited nanoplasmonic approaches may take advantage of patterning/spacing to address single or small groups of vesicles in their appropriate dimension range.Approaches capable of quantitative analysis, particularly toward single vesicle measurements, are critical to interpret and understand detailed structural and chemical information of nEVs in TR, BP, HK, and RC wrote and revised the manuscript 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.


Genome-wide association studies (GWAS) are widely used to identify regions of the genome that harbor genetic determinants of quantitative traits. However, the multiple-testing burden from scanning tens of millions of whole-genome sequence variants reduces the power to identify associated variants, especially if sample size is limited. In addition, factors such as inaccuracy of imputation, complex linkage disequilibrium structures, and multiple closely-located causal variants may result in an identified causative mutation not being the most significant single nucleotide polymorphism in a particular genomic region. Therefore, the use of information from different sources, particularly variant annotations, was proposed to enhance the fine-mapping of causal variants. Here, we tested whether applying significance thresholds based on variant annotation categories increases the power of GWAS compared with a flat Bonferroni multiple-testing correction.Whole-genome sequence variants in dairy cattle were categorized according to type and predicted impact. Then, GWAS between markers and 17 quantitative traits were analyzed for enrichment for association of each annotation category. By using annotation categories that were determined with the variants effect predictor software and datasets indicating regions of open chromatin, “low impact” variants were found to be highly enriched. Moreover, when the variants annotated as “modifier” and not located at open chromatin regions were further classified into different types of potential regulatory elements, the high impact variants, moderate impact variants, variants located in the 3′ and 5′ untranslated regions, and variants located in potential non-coding RNA regions exhibited relatively more enrichment. In contrast, a similar study on human GWAS data reported that enrichment of association signals was highest with high impact variants. We observed an increase in power when these variant category-based significance thresholds were applied for GWAS results on stature in Nordic Holstein cattle, as more candidate genes from previous large GWAS meta-analysis for cattle stature were confirmed.Use of variant category-based genome-wide significance thresholds can marginally increase the power to detect the candidate genes in cattle. With the continued improvements in annotation of the bovine genome, we anticipate that the growing usefulness of variant category-based significance thresholds will be demonstrated.The online version of this article (10.1186/s12711-019-0463-9) contains supplementary material, which is available to authorized users. Cattle is one of the most important domestic animals in human history. Both breeding programs and genetic studies in cattle depend largely on the availability of a reliable cattle reference genome  and refeTo address this problem, additional information from independent sources are needed. For example, gene expression data have facilitated the identification of candidate genes from GWAS data , and expDue to many reasons, such as LD, inaccuracy of imputation, random sampling errors, etc., the lead single nucleotide polymorphism (SNP) may not be the causative one . Using a2 > 0.2) with the lead SNPs, as potential causal variants to estimate the enrichment of each of the annotated variants’ categories. We make the hypothesis that the category-based significance threshold will increase the power of a GWAS study. We tested this hypothesis by performing an association study on stature in cattle and comparing the results with those of a previously reported large meta-analysis in cattle stature and genes reported for human height.In this study, we implemented a previously proposed, category-based Bonferroni adjustment based on the enrichment  of variant annotations observed for association signals  that werSince, no animal experiments were performed in this study, approval from an ethics committee was not required.http://www.nordicebv.info/). For details on the genetic evaluations performed for these 17 traits/indices in Nordic countries, see http://www.nordicebv.info/production. The phenotypic values used in the association analysis included de-regressed proofs that were derived for animals based on the effective daughter contributions of sires and maternal grandsires .


Using this system, we found that the blood-enriched lipid mediator, sphingosine-1-phosphate, controlled the migratory behavior of osteoclast precursors. We also developed pH-sensing chemical fluorescent probes to detect localized acidification by bone-resorbing osteoclasts on the bone surface in vivo, and identified two distinct functional states of differentiated osteoclasts, “bone-resorptive” and “non-resorptive.” Here, we summarize our studies on the dynamics and functions of bone and immune cells within the bone marrow. We further discuss how our intravital imaging techniques can be applied to evaluate the mechanisms of action of biological agents in inflammatory bone destruction. Our intravital imaging techniques would be beneficial for studying the cellular dynamics in arthritic inflammation and bone destruction in vivo and would also be useful for evaluating novel therapies in animal models of bone-destroying diseases.Bone is a highly dynamic organ that is continuously being remodeled by the reciprocal interactions between bone and immune cells. We have originally established an advanced imaging system for visualizing the  In this review, we discuss how the advances of imaging methods in living mice have contributed to our understanding of the bone–immune cell interaction in bone destruction. Furthermore, we introduce our recent studies, including evaluation of the interaction between osteoclasts and osteoblasts, and our novel approach for evaluating the mechanisms of action of different biological agents used for the treatment of bone-destructive diseases.The interdisciplinary research field focusing on the crosstalk between the bone and immune systems, termed “osteoimmunology,” has revealed extensive reciprocal interplay between the two systems –3. Over in vivo information on dynamic cell movements and cellular interactions is not available  compared to confocal microscopy, which yields data to only a depth of <100 μm. Thus, two-photon excitation microscopy affords efficient light detection, reduces phototoxicity, and penetrates deeper into tissues, which makes it an important imaging tool for intravital visualization of the dynamic cellular behavior of deep tissues , 6.in vivo visualization of the real-time behavior of bone and immune cells in bone marrow cavities, such as osteoclasts, osteoblasts, macrophages, and lymphocytes. Moreover, such imaging may be useful when it is desirable to evaluate the effects of novel drugs targeting skeletal disease.Bone marrow is surrounded by calcium phosphate crystals of the bone matrix, which can readily scatter light of near-infrared wavelengths. However, in parietal bones of mice, the distance from the bone surface to the bone marrow cavity is only 80–120 μM, which is sufficiently thin to permit controlled fluorophore excitation within the cavity. Intravital two-photon imaging of skull bone tissue allows in vivo behavior of osteoclast precursor macrophages in the bone marrow  are specifically expressed along the ruffled border membrane to maintain highly acidic conditions in the resorption pit  via interactions between integrin αvβ3 on the osteoclast membrane and bone matrix components . A numbetion pit . V-ATPastion pit , 20. To in vivo  cells that are firmly adherent to bones and dissolve the bone matrix by secreting acids, and non-resorbing (N) cells that are relatively loosely attached to bones and moved laterally along bone surfaces  cells, but not Th1, preferentially adhere to mature osteoclasts, although both T cell types migrate into bone marrow cavities to the same extent  in the cytosol of osteoclasts (TRAP-tdTomato mice)  9). To . To 9). entified . Time-la+ for bone resorption when they are not in contact with osteoblasts, whereas osteoclasts in contact with osteoblasts are non-resorptive, suggesting that osteoblasts inhibit the bone resorption capacity of osteoclasts in a contact-dependent manner. Intermittent administration of parathyroid hormone led to a mixed distribution of osteoblasts and osteoclasts, thus increasing cell–cell contact to induce bone anabolic effects. The precise molecular mechanisms involved in the direct cell–cell contact should be explored in detail.In addition, a pH-sensing fluorescence probe revealed that osteoclasts secrete HAn earlier study used another mouse line featuring an osteoblast reporter, the Col2.3–GFP reporter line, to explore the interactions between T-cell acute leukemia and bone marrow microenvironment via two-photon microscopy . FurtherArthritic bone erosion in RA has been a major research topic in osteoimmunology. Works on the interplay between the immune and bone systems have suggested many useful drug development strategies. For example, proinflammatory cytokines, such as interleukin (IL) 6 and tumor necrosis factor α (TNFα), promote osteoclast differentiation by inducing RANKL in mesenchymal cells, and may directly stimulate both osteoclastogenesis and the bone-resorbing capacity of mature osteoclasts –37. Biolin vivo behavior of mature osteoclasts and their precursors during inflammatory bone destruction, and explored how different biological agents affect the dynamics of these cells in vivo (Using the LPS injection model, we directly visualized the  in vivo . We foun in vivo . Taken tIn addition, macrophages of osteal tissues are reported to be involved in the regulation of osteoblast function, and subsequently bone dynamics . The addin vivo and determine the precise interplay between bone and immune systems in future studies.Considerable progress has been made in clarifying the interplay between bone and immune cells under both physiological and inflammatory conditions. However, their dynamic crosstalk within living animals is still largely obscure. Intravital two-photon imaging provides unbiased spatiotemporal information on the biological phenomena in living organisms, which are often much more complex than we may have hypothesized. Therefore, it is important to incorporate technical developments in imaging, such as two-photon microscopy, to directly observe the biological phenomena TH, JK, and MI contributed to the discussion and wrote and reviewed the manuscript.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.


Maternal and child health (MCH) and chronic disease programs at state health agencies may not routinely collaborate. The objective of this study was to describe a project that enhanced relationships between MCH and chronic disease epidemiologists at the Florida Department of Health, increased epidemiologic capacity, and informed both programs.We collaborated to assess hypertension-related severe maternal morbidity (H-SMM) and hypertensive disorders  among women at delivery of their live birth to help determine the burden on health care systems in Florida. We identified ways to improve the health of women before they conceive and to help them manage any chronic diseases during the perinatal period.We found differences by maternal characteristics in H-SMM rates among 979,660 women who delivered live births. We proposed strategies to support collaboration between state MCH and chronic disease staff. First, increase the screening, monitoring, and management of hypertension before, during, and after pregnancy. Second, examine H-SMM concurrently with maternal mortality to help find prevention strategies. Third, include reproductive-aged women in ongoing hypertension prevention and intervention efforts. Fourth, expand team-based care to include obstetricians, midwives, and doulas who can work together with primary care providers for hypertension management. And fifth, create and share data products that guide various groups about hypertension and related risk factors among reproductive-aged women.The collaboration between the Florida Department of Health MCH and chronic disease epidemiologists produced 1) a program-relevant indicator, H-SMM and 2) strategies for enhancing program and clinical activities, communication, and surveillance to reduce H-SMM rates. Maternal and child health (MCH) outcomes and chronic diseases can be interconnected. At the Florida Department of Health, MCH programs examine how hypertensive disorders affect maternal morbidity and mortality, and chronic disease programs focus on how hypertension affects the general population of men and women.We described an example of MCH and chronic disease staff members collaborating to develop a meaningful program indicator — hypertension-related severe maternal morbidity — and potential data-to-action strategies to integrate efforts.The relationship between MCH and chronic disease program staff members can be enhanced by intertwining efforts, unique perspectives, and resources on projects with shared visions and objectives.Maternal and child health (MCH) and chronic disease epidemiologists may not routinely work together at state health agencies. Possible reasons include organizational structures that preclude effective communication and a limited understanding of how MCH and chronic disease morbidity intersect . For exaSMM refers to unexpected outcomes of labor and delivery that result in major consequences to women’s health . SMM incThe objective of this study was to describe our collaborative process to further analyze SMM data, develop a program-relevant measure — hypertension-related SMM (H-SMM) — and identify opportunities for translating H-SMM results into potential public health practices.During 2014–2016, our team of MCH and chronic disease epidemiologists participated in a capacity-building program sponsored by the Centers for Disease Control and Prevention (CDC) and taught by staff at the University of Illinois at Chicago School of Public Health . Our parFirst, we analyzed SMM data and observed that Florida and national SMM rates  were similar: 162.4 in Florida during 2010–2013  and 162.We then shared those results with staff members who administered or supported various MCH or chronic disease programs to receive guidance on further analyses. To best meet Florida’s program needs, the staff advised the following:Hypertension is an important risk factor for other adverse MCH outcomes, including maternal mortality ,14.Further analyses of SMM, specifically related to hypertension, could inform current MCH and chronic disease programs because the prevalence of hypertension is increasing among women of reproductive age .Measures of frequency or disease burden  are more understandable and useful than measures of association . Measures of frequency describe the effect of health outcomes on populations, making these data more useful and more likely than other types of data to resonate with public health practitioners, government leadership, and other nongovernmental organizations such as health care systems. In contrast, measures of association are useful for conducting more in-depth analyses, such as estimating causal associations with minimal confounding or other bias.International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes in the hospital discharge data  preeclampsia, 2) preexisting hypertension, and 3) gestational hypertension. We examined each subtype separately. We used a hierarchical approach to classify women into only 1 subtype when codes for multiple conditions were present. First, women with preeclampsia were identified, regardless of other hypertension-related codes, then those with preexisting hypertension, followed by those with gestational hypertension. These subtypes differ clinically and can have varying associations with adverse birth outcomes . The subWe analyzed hospital deliveries during 2010–2014  by selected maternal characteristics using Florida hospital discharge data linked to birth certificate data. We investigated median hospital length of stay, median hospital charges, and H-SMM rates, and whether these differed by type of hypertensive disorder, to help identify the burden each disorder places on health care systems in Florida. We tabulated prevalence ratios, but we did not interpret these data because they resulted from ad hoc analyses, not discussions with programmatic staff. The FDOH Institutional Review Board approved this analytic project.Of 979,660 delivery hospitalizations during 2010–2014 in Florida, 4,021 recorded at least 1 H-SMM disorder — a rate of 41.0 cases per 10,000 delivery hospitalizations . Among cWomen with H-SMM used more health services — resulting in higher median length of stay and hospital charges — than women with no H-SMM. Women with preexisting hypertension or preeclampsia used more health services than those with gestational hypertension or those without hypertension. Lastly, we observed a greater burden on the health care system when H-SMM and hypertensive disorders were experienced simultaneously. Women with H-SMM and either preexisting hypertension or preeclampsia had a significantly longer length of stay (2 days longer) and higher hospital charges  than those without H-SMM with the same hypertensive disorders.Given these findings, our team proposed the following strategies that our state health agency and its partners could use to support MCH and chronic disease efforts.In our study, women with preexisting hypertension and preeclampsia incurred higher charges and longer hospital stays. This finding suggests that standardized, evidence-based interventions to prevent and manage hypertensive disorders during the preconception and prenatal periods could be critical for reducing morbidity and mortality .The American Heart Association added preeclampsia to its list of risk factors for later-life cardiovascular disease. They recommended adding preeclampsia to the method for calculating the risk score for cardiovascular disease and posited that cardiovascular disease should be tested before the age of 40 years among women with a history of preeclampsia .Our data indicate H-SMM is more prevalent among women with hypertension than among women without hypertension. Given that women with hypertension also have increased risk of pregnancy-related mortality, statewide programs throughout the nation, including perinatal quality collaboratives   and mateCDC has offered 5-year funding support for maternal mortality review committees to identify and characterize maternal mortality to better understand pregnancy-related deaths, develop recommendations for prevention, improve quality of care, and address social determinants of health to reduce health inequities. Grantees who seek to expand their work may find Strategy 2 useful for identifying prevention factors. Additionally, federal partners might consider funding opportunities for programs looking to integrate SMM and maternal mortality surveillance efforts.FDOH’s Bureau of Chronic Disease Prevention (BCDP) works to optimize the use of electronic health records in health systems, including county health departments and federally qualified health centers. BCDP also alerts health professionals about which patients may have undiagnosed hypertension and may require monitoring. These interventions include algorithms specific to certain blood pressure readings and exclude pregnant women. In our study, women with preexisting hypertension had higher H-SMM rates, longer lengths of stay, and greater hospital charges than those without hypertension. Our findings support enhancing the next phase of the electronic health record intervention to include women of reproductive age by pregnancy status into the algorithm, which will ensure the timely diagnosis of hypertension and help prevent pregnancy-related disease and death.BCDP also works with the FDOH-funded Medication Therapy Management Support project, which trains community health workers (CHWs) to work in health care systems to help patients better manage their hypertension. CHWs primarily work in low-income communities with limited access to health care and in populations that face multiple barriers to optimal health outcomes, including non-Hispanic black women. We found that H-SMM was more prevalent among non-Hispanic black women than among members of other racial/ethnic groups, a finding that was similar to findings in another report . We suggBCDP plans to work with health care systems in Florida to help them become recognized as Hypertension Control Champions through the Million Hearts Initiative . This inBCDP works with partners to implement team-based care. This evidence-based approach uses nonphysician and physician provider care teams to help patients better manage their chronic conditions. Women in our sample with preexisting hypertension had an H-SMM rate 3 times higher than the overall rate and used health care services at a higher rate. We suggest expanding primary care teams to include obstetricians, midwives, and doulas for bidirectional consultation for women of reproductive age. Primary care providers can encourage their chronic disease patients seeking pregnancy to obtain a preconception consultation with an obstetric care provider and conversely obstetric providers can assist postpartum patients in following up with their primary care physicians to appropriately manage hypertension after pregnancy.An example of such an expansion is North Carolina’s Pregnancy Care Management Program, which pairs low-income women who have medical needs with a social worker or nurse care manager to help address factors that affect low birth weight and preterm birth . The carBCDP is creating a communications plan to identify, produce, and share data products, such as infographics and social media posts, to inform the public, health care providers, and partners about hypertension and related risk factors. MCH and chronic disease prevention programs could collaborate to adapt these products for women of reproductive age, especially women with preexisting hypertension who are at increased risk of H-SMM during pregnancy. Communication products for the general public should adhere to key components of effective health communication and patient education   has been identified as a priority in Florida’s State Health Improvement Plan . Strong Our analysis has several limitations. It was subject to limitations of the ICD-9-CM codes described elsewhere , includiThis collaborative experience enhanced our understanding of claims-based analysis and enriched our professional relationships. Although external skill-building activities can help agencies initiate multidisciplinary partnerships, MCH and chronic disease epidemiologists can also benefit from examining their existing infrastructure to find ways to develop and sustain collaborations on program-relevant projects. No matter the size, state health agencies by design have multidisciplinary capacity and thus are in unique positions to serve as best practice examples for fostering collaboration and aligning activities for maximum effect. Actively strengthening these relationships by intertwining efforts, unique perspectives, and resources on projects with shared objectives may ultimately benefit and guide the enhancement of MCH and chronic disease programs.


Therefore, the quantity and purity of the substances in pharmaceutical compounds are continuously monitored during drug manufacturing with various chemical or instrumental analysis techniques. The possibility of impurities development and chemical or quantity changes of active drug species at various stages  makes them redundant and risky for human health. So, sensitive and accurate analysis methods for qualitative and quantitative analysis are highly demanded by pharmaceutical companies and medical centers. The present mini review emphasizes on application of carbon based modified electrodes in health care and pharmaceutical analysis. Electrochemical determination of drugs employing carbon nanostructured modified electrodes will be reviewed and their advantages and disadvantages will be mentioned.Pharmaceutical drugs play an important role in human life since they caused a revolution in human health. Notably, their administration to a living organism helps body to stay healthy. Commonly, they are employed to diagnose, prevent, or treat and cure a disease via a biological effect on a human body. Administration of impurity Pharmaceutical analysis should be performed during all steps of pharmaceutical development -3, from Development of modified electrodesElectrochemical techniques have been widely employed for both qualitative and quantitative analysis of organic -14 and iIn voltammetry measurements, potential and current of resulted peak (s) in recorded voltammograms show two important characteristic of the target species. Peak potential specify the electro-active spices , while pCarbon nanostructured based electrode modificationVarious ways have been applied for electrode modification by carbon nanostructures. Generally, electrode modification includes bulk or surface modification. In simplest method, suspension of various types of carbon nanostructures is prepared in a suitable organic or inorganic solvent such as water, dimethyl form amide (DMF), or mixture of them , 51. TheVarious types of carbon based nanostructures were employed in electrode modification such as single and multi-walled carbon nanotubes -79, grapThe combination of CNSs with other nano-structured materials such as metals, metal oxides, and polymers has provided multifunctional hybrid or composites that can significantly improve the sensing properties of the electrochemical sensors. Nano-materials with various type of morphology such as nano-particles, nano-wiers, nano-ribbons, nano-spheres are integrated with CNSs. The prepared heterogeneous nanostructures show outstanding promising potential in sensing applications.et al.  employed for dopamine (DA) analysis  analysis -120, TabAdvantages and limitations in clinical complex samples bioanalysisEven though, carbon nanostructures based modifiers significantly have improved the analysis sensitivity  and 77, Electrochemical methods employing carbon-based nanostructures can be efficiently employed for pharmaceutical and clinical analysis. However, to improve the selectivity and to access to more sensitivity, it is recommended to apply mixture, hybrid, or composite of these nano-materials with other organic or inorganic compounds. Therefore, it is possible to benefit synergetic or enhancement effect of them together. So, we can access to selective determination of active ingredients of drugs in both bulk pharmaceutical formulation or after administration in biological fluid of the patients’ bodies.


PLOS Neglected Tropical Diseases to continue to reevaluate the scope of the journal and to define which syndromes and diseases continue to disproportionately impact neglected populations in low-resource settings.The past decade has seen a dramatic increase in global attention to neglected tropical diseases, with remarkable momentum in policy, programs, funding, and research. As we look to the future, it is critical for PLOS Neglected Tropical Diseases have profound impacts on the gastrointestinal system. There is increasing recognition of the role of the intestine as the critical site where the human host encounters pathogens, initiates the immune response, protects from pathogen invasion, controls nutrient and calorie absorption, and modulates hormonal response. Enteric dysfunction—as a result of diarrheal disease, parasitic infections, alterations in the gut microbial environment, and other causes—has been linked to malnutrition, delayed cognitive development, increased susceptibility to other infectious diseases, poor oral vaccine response, and increased mortality risk. The collective long-term impacts of these conditions on the most marginalized communities are tremendous. Given the importance of these diseases and syndromes, PLOS Neglected Tropical Diseases has decided to include a new section focused on enteric infections and enteric dysfunction. Of course, many diseases and syndromes—including the soil-transmitted helminths, schistosomiasis, giardiasis, cholera, and other enteric pathogens that impact the physiology of the gut—are already included within the scope of PLOS Neglected Tropical Diseases and have been highlighted previously in this journal [Many pathogens highlighted in  journal . It is oShigella spp., rotavirus, adenovirus, heat-stable toxin enterotoxigenic Escherichia coli (ST-ETEC), Cryptosporidium spp., and Campylobacter spp. as the most common pathogens associated with moderate-to-severe diarrhea in infants and young children in low-resource settings [Campylobacter pathogen burden was associated with a significant decrease in height for age z-score among children enrolled in a multisite birth cohort study (MAL-ED), independent of diarrhea symptoms [Diarrhea remains a leading cause of child mortality globally. The Global Enteric Multicenter Study (GEMS) reported settings , 3. Howesettings . For exasymptoms . In addisymptoms –8. AlthoIn many low-resource environments, environmental enteric dysfunction (EED)—a small bowel intestinal disorder characterized by mucosal inflammation, reduced barrier integrity, and nutrient malabsorption —is widespread among infants and children, including children with no other identified comorbidity –11. NoniPLOS Neglected Tropical Diseases is uniquely positioned to highlight research addressing these gaps from basic research to the implementation of interventions at scale.However, despite the highly prevalent nature of EED and the associated morbidity and mortality risk, there are major gaps in our understanding of EED and enteric dysfunction across the entire spectrum of translational research. Because the etiology and physiology of enteric dysfunction is incompletely defined, it is essential that model systems  be developed to elucidate some of the underlying features of the condition. Few published studies have evaluated the histopathologic features of EED. One such study produced a mouse model of EED using a combination of protein-deficient diet and exposure to a specific microbial environment . OngoingThere remain major challenges in establishing formal case definition(s) for enteric dysfunction that can be used in clinical trials and surveillance studies. The lack of well-validated biomarkers suitable for use in low-resource settings and predictive of morbid sequelae limits the ability of clinicians to diagnose and manage EED. The optimal method to assess gut abnormalities is endoscopy with biopsy to determine villus blunting and other histopathologic features. However, these invasive procedures may not be practical or ethical to utilize among children in low-resource settings. Because several forms of enteric dysfunction are often coendemic in low-resource settings, it is critical to harmonize case definitions to ensure proper classification of similar clinical entities from differing etiologies, because the etiology is likely relevant for elicitation of response to a given treatment approach. A combination of anthropometric, intestinal biomarker, and systemic biomarker information could potentially be used as the foundation for a case definition for EED ; howeverMany candidate biomarkers that can be analyzed noninvasively from stool, blood, and urine samples have been evaluated, and several have been associated with linear growth and other health outcomes . NoninvaLactobacillus rhamnosus GG) nor antibiotic (rifaximin) reduced lactulose excretion in children living in Malawi [A number of interventional approaches for enteric dysfunction have been tested in children over the past 10 to 15 years, including water, sanitation, and hygiene (WASH) interventions; zinc; antibiotics; probiotics; and the targeted administration of locally active anti-inflammatory drugs. Most of these studies have used the dual sugar permeability test as an outcome measure, with many focusing on lactulose excretion alone (rather than the ratio with mannitol or rhamnose) due to its reduced complexity. Neither a probiotic (n Malawi , 34. Altn Malawi , 36. A sn Malawi . A smalln Malawi . An upcon Malawi . Resultsn Malawi , 41, andn Malawi , 43. AddPLOS Neglected Tropical Diseases looks forward to publishing the results of studies and trials targeting the definition, classification, mechanisms, treatment, and/or prevention of enteric infection and dysfunction. PLOS Neglected Tropical Diseases is prepared to serve as a platform to bring together and disseminate important discoveries and innovations that will drive solutions to these enteric conditions that disproportionately impact populations in low-resource settings.


Mythimna  separata armyworm; the cytokine was named growth-blocking peptide (GBP), reflecting its actions in combating parasitic invasion. The peptide’s name has been retained, though the list of its regulatory activities has greatly expanded. All members of this family are small peptides, 19–25 amino acid residues, whose major source is fat body. They are now known to regulate embryonic morphogenesis, larval growth rates, feeding activities, immune responses, nutrition, and aging. In this review, we will describe recent developments in our understanding of the mechanisms of action of the GBP family, but we will also highlight remaining gaps in our knowledge.The widespread distribution of insects over many ecological niches owes much to evolution of multiple mechanisms to defend against environmental stress, especially because their ectothermic nature and small body size render them particularly susceptible to extremes in temperature and water availability. In this review, we will summarize the latest information describing a single, multifunctional cytokine family that is deployed by six orders of insect species to combat a diverse variety of environmental stresses. The originating member of this peptide family was identified in  Mythimna  separata armyworm upon infection by the parasitic wasp Cotesia kariyai (M. separata (Ms) GBP suppresses hemolymph JH esterase activation is still unknown, this initial observation led us to focus on its hormone-like function , release of inositol triphosphate (IP3), and the elevation of cytoplasmic Ca2+ concentrations (Growth-blocking peptide (GBP) was initially found as a peptidergic factor which blocks JH esterase activation in the hemolymph of early last instar larvae of host  kariyai . GBP-ind kariyai . Althougfunction . Furtherhe brain , 2003. Ttrations . Althougtrations .MsGBP, over 10 GBP orthologous peptides have been found in several lepidopteran species (Drosophila-Mthl10 (see below). MsGBP itself was demonstrated to have this multifunctionality  GBP exhibits some sequence similarity with human defensin BD2, a member of the immunomodulatory ß-defensin family, that can also regulate cell proliferation; BD2 is small, cationic peptides produced by specific proteolytic processing just like DmGBP  expression independently of the canonical receptors that at that time were known to be associated with the inflammatory pathways mediated by Toll- and IMD-dependent pathways , while inhibiting humoral pathways (production and release of AMPs), through an IP/Ca2+ signaling-mediated activation of a receptor-regulated kinase cascade (the PVR/ERK pathway).Second, it was demonstrated that egulated . DmGBP pDmGBP receptor has been determined to be the G-protein-coupled receptor Methuselah-like 10 (Mthl10)  . KnockdoDmGBP regulates the release of insulin-like peptides (ILPs) from the brain depending on nutrient levels in the hemolymph through target of rapamycin (TOR) in Drosophila . MsGBP regulates not only immune active plasmatocytes and granulocytes in Lepidoptera  that functions as a scavenger of armyworm . As mentidoptera , but alsatocytes . Therefoca sexta , Bombyx byx mori , Spodopta exigua , Spodoptugiperda , and Helarmigera . Bacteriarmigera . For exaarmigera . It is pMsGBP acts as a bipolar growth regulator: high concentrations  suppress larval growth but low concentrations  enhance larval growth and cell proliferation  was first identified, four other Drosophila genes encoding the proGBP-like peptide were also found: CG11395, CG12517, CG14069, and CG17244. Koyama and Mirth recently found that the CG11397 gene product regulated the release of ILPs from the brain in the similar manner of GBP (CG15917) and they named CG15917 and CG11397 for GBP1 and GBP2, respectively  will turn out to expand the functionality of the DmGBP family.When ectively . The rolMsGBP and DmGBP have been clearly demonstrated, it remains unclear to what extent the GBP-signaling pathways and functionalities are conserved in other insects. For example, it is not yet known if Mthl10 orthologous gene occurs in the armyworm. Moreover, SRP and GBP-BP have been examined only in the armyworm. It will be important to identify all these essential components for GBP-signaling function and regulation in broad insect species, so as to identify species-, development-, and stage-specific expression of such components. Elucidating commonality and difference of such GBP-associated factors in insects may hint at the conservation of some of these important homeostatic mechanisms in mammals.Although the multiple functionalities of 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.


Drug addiction is a complex disorder driven by dysregulation in molecular signaling across several different brain regions. Limited therapeutic options currently exist for treating drug addiction and related psychiatric disorders in clinical populations, largely due to our incomplete understanding of the molecular pathways that influence addiction pathology. Recent work provides strong evidence that addiction-related behaviors emerge from the convergence of many subtle changes in molecular signaling networks that include neuropeptides (neuropeptidome), protein-protein interactions (interactome) and post-translational modifications such as protein phosphorylation (phosphoproteome). Advancements in mass spectrometry methodology are well positioned to identify these novel molecular underpinnings of addiction and further translate these findings into druggable targets for therapeutic development. In this review, we provide a general perspective of the utility of novel mass spectrometry-based approaches for addressing critical questions in addiction neuroscience, highlighting recent innovative studies that exemplify how functional assessments of the neuroproteome can provide insight into the mechanisms of drug addiction. Drug addiction is a chronic relapsing disorder characterized by compulsive drug-seeking, the loss of control in limiting drug intake and the emergence of negative emotional states during drug abstinence . In the Drugs of abuse are widely recognized to facilitate dopamine transmission in brain reward systems, contributing to a powerful hedonic and euphoric response that reinforces drug-taking behavior. As drug use continues, increased dosage and frequency of use induces pivotal changes in brain reward pathways  such that the same circuits recruited initially respond differently upon re-exposure. Pharmacological manipulation of the dopamine system has long been recognized to reduce drug intake in preclinical models of addiction ,4; howevAlthough the deregulation of brain reward remains a critical symptom of addiction, the maladaptive behaviors exhibited by addicts during drug abstinence are indicative of a more complex pathology driven by changes in additional brain structures. While a diverse set of hypotheses have been developed to explain the transition from casual use to dependence ,5,6,7,8,While the population of drug-addicted individuals continues to grow in the United States and worldwide, the small number of available treatment options has failed to address this growing burden. Drugs that are FDA-approved for other diseases have been evaluated off-label to treat addiction, yet have been met with limited success, highlighting a critical need to identify addiction-specific changes in the central nervous system (CNS) that can be harnessed for clinical therapeutics.in vivo monitoring of signaling peptides (neuropeptidomics). In each of these cases, proteomics offers a valuable means to identify targets in a more unbiased manner than conventional protein assays, and together they establish a foundation for the “hit-to-lead” optimization of novel druggable targets for addiction and related psychiatric disorders. Here, we demonstrate the value of applying a proteomics approach to the CNS by highlighting recent studies that utilize novel methods for elucidating the mechanisms of addictive disorders.From a pre-clinical perspective, a better understanding of the molecular alterations in the CNS would offer a wealth of information regarding the mechanistic underpinnings of disease, products of disease pathology (biomarkers) and the identification of high-value targets for the development of precision medicine. In the simplest case, a small molecule therapeutic would selectively bind a protein target in order to intercept a molecular change that underlies an important behavioral construct, such as drug reinforcement. Here, we depict the brain reward pathway  consistiTraditional proteomic approaches have evaluated broad-scale changes in protein abundance in the brain following chronic drug exposure. The results have yielded a plethora of information on candidate targets, summarized elegantly in review articles for alcohol , morphinObtaining a viable sample that is likely to contain relevant targets of addiction poses a critical challenge for neuroscientists. While anatomical structures provide some level of specificity, there is substantial heterogeneity at the molecular and behavioral level. An interesting example of this involves the dorsal striatum known to contain afferent dopaminergic terminals that are activated by drugs of abuse. While this structure is often dissected and analyzed as a singular region, site-specific inactivation has informed the rationale for distinguishing critical areas. In this regard, the dorsal medial region is important during acute drug exposure given its role in influencing goal-directed behavior that establishes early drug-cue associations; however, the dorsal lateral region becomes increasingly important as addictive behaviors become more automated, thus reflecting one of the core symptoms of addiction that influence compulsive intake ,20,21. RIdentifying a druggable target from the many changes observed in the proteome remains a persistent challenge in neuroproteomics research. One strategy is to employ pathway analyses that can distill large amounts of information into known signaling networks, biological functions and associated disease states. A notable example from Salling et al. identified 29 proteins that were dysregulated by moderate alcohol drinking in the mouse amygdala using two-dimensional difference gel electrophoresis (2-DIGE) . IngenuiThe emergence of sample-labeling and multiplexing procedures provides an added dimension of research analysis of the brain proteome. Several strategies currently exist and are well documented in the literature , each displaying their own unique advantage in terms of reducing the technical variation between mass spectrometric analysis and other aspects of labeling efficiency and sample preparation ,27. The A common strategy in proteomics research is to seek confirmatory evidence/validation of the targets derived from large-scale analyses with antibody-based molecular approaches. For instance, Nimitvilai et al. confirmed changes in a subtype of glutamate AMPA receptors (GluA1) in alcohol-dependent macaques using traditional western blot techniques , as valiTo realize the ultimate goal of identifying druggable targets, the proteomics field would benefit from employing multiple strategies in elucidating proteome-derived targets. Exploring functional relevance with respect to whole organism behavior would enhance the translational value and application to the clinical population in question. For example, Salling et al. expanded on the initial proteome work in alcohol-drinking mice to elucidate a functional role for increased CaMKII subunit α expression in the amygdala, providing molecular and electrophysiological evidence of the strengthening of long-term potentiation (LTP) signaling ostensibly driven by CaMKII binding sites on GluA1 receptors . These fRecent advancements in the isolation of neuronal circuits using chemogenetic or optogenetic tools clearly demonstrate the heterogeneity of brain structures involved in addiction ,38. EvenN-methyl-d-aspartate (NMDA) that is abundantly expressed in the hippocampus. To functionally evaluate this complex, they developed and validated small peptides that interfered with the formation of this complex without altering receptor-mediated signaling by either nicotinic α7 and NMDA. The intracerebroventricular delivery of this interfering peptide to the brain prevented the reinstatement of nicotine self-administration in rodents, providing evidence that this complex represents a viable druggable target for nicotine addiction.While substantial work has demonstrated an important role for nicotinic receptors in the development of nicotine dependence by targeting the ligand binding site, the proteins they interact with  also participate in many important synaptic and immunological processes. Thus, drug-selective signaling complexes may represent unique opportunities for pharmacological intervention to disrupt addiction-related behaviors while minimizing therapeutic side-effects inherent in directly targeting nicotinic receptors. One such receptor complex in the hippocampus facilitates nicotine relapse-like behavior in rats . In thisin vitro and in vivo preparations [Affinity-based enrichment can also provide insight into the network of protein interactors that may alter cellular signaling. For example, Wills et al. immunoprecipitated the NMDA receptor NR2B in hippocampal preparations to reveal a network of interacting proteins including scaffolding and PDZ-domain binding proteins that were dysregulated by chronic alcohol exposure . Of the arations . These sarations .The detection of phosphoproteins has provided addiction scientists with a useful tool for measuring changes in activated states that may be devoid of changes in respective protein levels. Protein phosphorylation constitutes one of the most common post-translational modifications in protein biology, whereby the enzymatic addition or subtraction of a phosphate group onto nucleophilic residues  can alter the structural conformation of a protein, rendering it active, inactive or otherwise modifying its function. Though not exclusively linked to conformational changes, protein phosphorylation has been shown to serve as a molecular switch influencing a wide range of biological activity including signal transduction, cell differentiation/proliferation, protein-protein/-gene interactions and subcellular localization. Indeed, a large number of hypotheses recognize the importance of protein phosphorylation in directing the flow of molecular signaling, converging on key regulators of gene transcription , membrane receptors  and other important binding partners  that modulate neuroplasticity ,46,47. ISeveral aspects of the phosphoproteome are conducive to the identification of novel addiction-related targets. First, global assessments of the phosphoproteome often reveal an abundance of phosphoproteins involved in the regulation of phosphorylation states . Indeed,Presently, there are only a few studies that have applied a discovery-based phosphoproteomics approach in addiction models. Our laboratory recently utilized metabolic labeling procedures to quantify phosphopeptides enriched from the prefrontal cortex of rats receiving the psychedelic compound phencyclidine (PCP) . We applAnother exceptional study by Rich et al.  utilizedNeuropeptides play a central role in the development and persistence of addictive behaviors . For exaNeuropeptides are short sequences of amino acids that can act like neurotransmitters, but with some critical distinctions that underlie their importance as potential druggable targets. While both neurotransmitters and neuropeptides are packaged in vesicles and released during neuronal activity, neuropeptides are expressed discretely throughout the CNS to facilitate specific behavioral responses. Neurons and glia have robust, rapid reuptake systems for most neurotransmitters, allowing the recycling of these molecular signals to reduce energy consumption in the CNS. In contrast, substantially more time and energy is required to produce a neuropeptide ; first, ex vivo bioassays including the induction of uterine contractions, intestinal contractions, and secretion of other hormones. Likewise, their localization within the CNS was evaluated using antibody-based immunohistochemical techniques. The technical limitations of these approaches have biased studies toward high abundance neuropeptides, and as a result low abundance signals potentially dysregulated by abused drugs and other psychiatric disorders remain understudied.Most established neuropeptides currently implicated in addiction were discovered prior to the development of modern proteomic approaches . The ideModern genetic and proteomic technologies have facilitated more comprehensive studies of neuropeptides. The human genome project (and corollary projects in related species) has identified over 70 genes that contain sequences capable of producing more than 1000 potential neuropeptides . After cNeuropeptide expression in discrete regions allows them to exert their effects on specific circuits to produce distinct behavioral outcomes. In the striatum, dopaminergic neurons regulate excitatory and inhibitory signaling to facilitate drug reward. Neuropeptides produce maladaptive effects by acting on specific neurons in distinct locations in the striatum and other limbic regions, ultimately contributing to drug craving and compulsive behaviors that influence relapse. Thus, the spatial location of precise neuropeptides plays a critical role in the development of addiction.in situ. Hishimoto used MALDI-IMS with sufficient resolution (200 μm) to identify these structural features using substance P and enkephalin as molecular markers, and thereby demonstrated that nicotine administration decreased nigral substance P levels while increasing enkephalin levels. In total, 768 features were identified from mouse striatal spectra, and more than half of these were significantly regulated by nicotine. More importantly, they found that nicotine produces a negative correlation between substance P and other m/z species identified in those samples, whereas a positive correlation emerged between m/z identified in regions with enkephalin. These results suggest a more broad-scale remodeling of the direct (substance P) and indirect  pathways in the striatum, working in concert to facilitate behavioral changes produced by nicotine exposure.Hishimoto et al. investigated the localization of neuropeptides in the striatum using MALDI imaging mass spectrometry (MALDI-IMS) . Mice exRecent work has also identified the habenula as an important regulator of nicotine dependence. The habenular nuclei are morphologically and biochemically distinct structures that innervate reward circuitry: the medial habenula is thought to play a critical role in the maintenance of nicotine consumption, while the lateral habenula drives the aversive effects of the drug . Immunohm/z that correspond to potential neuropeptides and discovered that many of these peptides were regulated by behavioral manipulation [In addition to exhibiting differences between brain structures, neuropeptide levels vary dramatically within brain structures at the subcellular level. Genetic sequence analyses predict that over 1000 potential neuropeptides may exist, with many having already been identified in CNS tissue using peptidomic approaches. In the striatum, Ye et al. identified 419 pulation . SpecifiFor most studies, the analysis of neuropeptide content has typically been performed on samples from bulk tissue. As illustrated by analogous work studying neurotransmitters and endocannabinoid lipids in the CNS, there are some potential limitations to using this approach ,89. Briein vivo microdialysis addresses many of these concerns and provides a useful approach for identifying signaling-competent peptides in the CNS during drug exposure [m/z features released in the rat striatum. From these data, they identified 29 potential neuropeptides from 6 different genes, including two peptides from enkephalin (PENK 198–207 and BAM 8–22). A similar study by Bernay et al. used multiple microdialysis and mass spectrometry approaches to more comprehensively investigate the striatal neuropeptidome and identified 97 peptides from these samples, including additional pro-enkephalin peptides PENK 114–133 and PENK 239–260 [Sampling using exposure ,92  for subsequent distribution in the brain to their biological targets [in vivo using peptidomic strategies, and these peptides should be explored further as potential druggable targets.The reverse-dialysis of neuropeptides, a complementary technique to microdialysis, provides a rapid and effective readout for establishing  targets . Thus, r targets ,94, demoThe transition from peptide identification to target discovery remains a critical bottleneck in neuropeptidomics. As illustrated in the studies above, traditional behavioral and molecular biology approaches can establish a functional role for neuropeptides identified using mass spectrometry. As reported by Ye et al., peptides can be site-specifically infused into brain structures to evaluate behavioral outcomes and establish a functional link with the underlying biology . Likewisi/o) and gene expression information , they limited the possibilities to four likely targets, ultimately revealing GPR171 as the endogenous big LEN receptor. Cells overexpressing GPR171 showed characteristic Gi/o-mediated decreases in cAMP levels following exposure to big LEN, and GPR171 siRNA delivered to the hypothalamus led to increased food intake in mice. Subsequent studies have identified a role for big LEN and GPR171 in anxiety-like behavior and fear conditioning [Given the importance of big LEN in food intake , Gomes eitioning , suggestitioning , and futThe proenkephalin-derived peptide BAM 8–22 has previously been identified as an endogenous anti-nociceptive peptide , and thuThe population of drug-addicted individuals continues to expand in the United States and worldwide, but the dearth of available treatment options has impeded progress in addressing this escalating burden. As our understanding of the neurobiology of addiction has evolved, it has become increasingly apparent that subtle changes in discrete brain circuits facilitate maladaptive behaviors associated with mood and cognition that ultimately lead to relapse and sustained drug use. Thus, the continued development of novel mass spectrometric-based methods such as cell-specific labeling  and sing


Dendritic cells (DCs) have a critical effect on the outcome of adaptive immune responses against growing tumors. Recent studies on the metabolism on DCs provide new insights on the functioning of these critical controllers of innate and adaptive immunity. DCs within the tumor microenvironment (TME) often exist in an inactive state, which is thought to limit the adaptive immune response elicited by the growing tumor. Tumor-derived factors in the TME are known to suppress DC activation and result in functional alterations in DC phenotype. We are now beginning to appreciate that many of these factors can also induce changes in immune cell metabolism. In this review, we discuss the functional alternation of DC phenotype by tumor metabolites. Upon administration, the DCs must migrate to the secondary lymphoid organs and induce an antigen-specific immune response. DC-based cancer vaccines have been tested in clinical trials of patients with melanoma, myeloma, prostate cancer, renal cancer, breast cancer, ovarian cancer, and gastrointestinal cancer. Although there is evidence that DC vaccines can induce anti-tumor immune responses in some patients, clinical results remain poor. Multiple clinical studies have indicated that DCs within tumors are scarce and functionally defective in vitro and in animal models. There may be multiple reasons for this lack of efficacy, including a tendency for early-phase clinical trials to enroll patients with advanced, metastatic disease and associated comorbidities, including immune suppression, which has only recently been recognized as a major barrier to the efficacy of tumor vaccines in general. Suboptimal maturation and immunogenicity of DCs within early vaccine formulations and the lack of definition of immunogenic neoantigens may also have contributed to the limited clinical efficacy of these strategies Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) and have a central role in maintaining immune homeostasis 2) are commonly used in the maturation of monocyte-derived DCs (mo-DCs) for clinical studies. The maturation stimulus is a major factor that determines the success or failure of DC therapy. TLR agonists , cytokines , costimulatory receptor ligands (CD40L), and PGE2 have all been used either alone or in various cocktails to mature and program DCs Although lipopolysaccharide (LPS) is a commonly used maturation factor in mouse studies, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and prostaglandin E2  alters the metabolism of tumor-associated immune cells, facilitating tumor cell escape and immune detection. Multiple TME-generated factors contribute to suppressing DC function by inhibiting DC recruitment, activation, antigen presentation, and Th1 polarization in vivo. Moreover, activation-induced metabolic shift markedly induces the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. This is mediated via phosphoinositide 3-kinase (PI(3)K) signaling through mammalian target of rapamycin complex 1 (mTORC1), which promotes a long-term commitment for aerobic glycolysis and anabolic metabolism in inflammatory DCs.It is increasingly recognized that the ability of DCs to become activated and prime adaptive immune responses is associated with profound alterations of the cellular metabolic state. Moreover, evidence is accumulating in the context of airway inflammation that various DC functions such as immune priming and immune polarization may have different metabolic requirements. This metabolic plasticity of DC function in airway remodeling could enhance our current understanding of DC metabolism in cancer therapy. Key exogenous metabolites induce the nutrient-sensitive anabolic and/or catabolic pathways to support DC maturation and function in vitro. ATX is associated with various inflammatory diseases including cancer, fibrosis, rheumatoid arthritis, and neural defects. Immature DC derived from LPA3-deficient mice do not migrate to LPA in vitro, demonstrating a necessary requirement for this receptor in LPA-directed motility There is a growing appreciation that to effectively take up antigen, migrate to the draining lymph node, and present antigen, and therefore to initiate appropriate T cell-mediated immune responses, DCs must undergo the correct metabolic reprogramming. For example, autotaxin (ATX), a secreted lysophopholipase D that is implicated in cancer cell motility, generates extracellular lysophosphatidic acid (LPA) from the precursor lysophosphatidylcholine (LPC). In 1992, ATX was identified as an autocrine factor in A2058 melanoma cells. LPA is a pleiotropic lipid molecule with strong effects on cell growth and migration. LPA induces diverse signaling pathways via six LPA G-protein-coupled receptors (GPCRs), resulting in cytokine production, inflammation, hyperplasia, tumor formation, and metastasis Recently, ecto-nuclase such as CD39 and CD73 were found to be novel immune check-points and potentially new therapeutic targets in solid tumors Expression of CD39 and CD73 on tumor cells has been proposed as a prognostic marker. CD39 was found to be highly expressed in pancreatic cancer and is mainly expressed by the vasculature and various stromal cells. A subsequent study performed in the setting of human rectal carcinoma demonstrated increased CD39 expression in tumors and metastases compared with normal tissue, although it was proposed that expression of both CD39 and CD73 leads to poor prognosis In solid tumors, adenosine triphosphate (ATP) is abundantly released into the extracellular environment  The effects of eATP on mo-DCs have been demonstrated in a number of studies. ATP has been found to stimulate human DC maturation, as indicated by high expression of costimulatory molecules such as CD80 To our knowledge, this is the first review focusing on that tumor-derived metabolites can be greatly associated with negative aspects of DC function in immune system, whereas previous reviews have mainly concentrated on influences on general and broad immune system. Although little research has concentrated on manipulating DC metabolism to induce activation and priming capacity, evidence is accumulating that addresses how metabolic perturbations are linked to the ability of DCs to facilitate immune responses. Taken together, these findings suggest that agents that restore DC metabolic health should be studied, developed, and tested in combination with immune checkpoint blockade. The composition of the culture medium, particularly the presence of metabolites, is as important as other therapeutic products such as cytokines and maturation agents in the function of monocyte-derived DCs, and therefore must be taken into account when establishing standard operating procedures for the generation of DC for experimental use or vaccination. The potential to enhance immune responses to other modalities of immunotherapy makes clinically effective "second-generation" DC vaccination strategies a priority for cancer immunologists.