Vaccines represent a remarkable success in the history of medicine since they have prevented and, in some instances, eradicated a range of infectious diseases. However, for many existing vaccines, immunogenicity is limited, requiring multiple booster doses, and we are still unable to target many pathogens due to intrinsic features of the microorganism, such as genetic/antigenic variability between strains, and our limited understanding of the variables that regulate vaccine responsiveness, including age- and sex-specific differences. in vitro models, and in novel adjuvant and formulation strategies offer unprecedented opportunities to dissect the human immune response to vaccines and inform dramatic improvements in vaccine efficacy. These approaches are poised to have a major scientific and translational impact in vaccinology.Vaccines represent a remarkable success in the history of medicine since they have prevented and, in some instances, eradicated a range of infectious diseases. However, for many existing vaccines, immunogenicity is limited, requiring multiple booster doses, and we are still unable to target many pathogens due to intrinsic features of the microorganism, such as genetic/antigenic variability between strains, and our limited understanding of the variables that regulate vaccine responsiveness, including age- and sex-specific differences. Moreover, the traditional approach to vaccine development is often empirical, relying on inactivation of microorganisms or purification of their components, which are usually less immunogenic than the whole microorganism from which they derive. This approach has yielded multiple important vaccines but has failed to consistently generate vaccines that are sufficiently immunogenic in populations with limited immune responsiveness such as newborns and elderly individuals. In an effort to trigger impactful collaborations, a community of scientists gathered in Boston in the United States for the first biennial International Precision Vaccines Conference, sponsored by the Boston Children’s Hospital Precision Vaccines Program, to discuss innovation in vaccinology. Recent advancements in the field of systems biology that can identify vaccine immunogenicity biomarkers for target populations, in human  Vaccines are among the most effective interventions available to prevent infectious diseases and represent a prominent success in the history of medicine . Neverth3–http://www.childrenshospital.org/research-and-innovation/research/departments/medicine/precision-vaccines-program), whose aim is to promote collaboration in development of vaccines tailored to distinct vulnerable populations.To develop strategies and synergies in vaccine development, a community of experts in a range of fields, including immunology, pediatrics, vaccinology, systems biology, biostatistics, and bioinformatics as well as vaccine adjuvantation and formulation, gathered at the Joseph B. Martin Conference Center at Harvard Medical School  on 23 and 24 October 2017 for the first biennial International Precision Vaccines Conference (IPVC) . The folin vitro culture systems as well as in adjuvantation and formulation science, is paving the way for the development of precision vaccines, defined as vaccines that (i) take into account the target population; (ii) are formulated to selectively activate the immune system by targeting anatomic sites, cells, and pathways that generate a protective response; and (iii) may, as needed, contain adjuvantation systems known to optimally enhance immunogenicity in a target population. In order to accomplish this complex and crucial task, systems biology methodologies such as transcriptomics, proteomics, and metabolomics as well as preclinical human in vitro models that take into account age- and sex-specific differences can be leveraged to generate hypotheses to be tested in appropriate animal models and eventually in targeted clinical trials , Ofer Levy  introduced the Precision Vaccines Program that sponsored the conference. Based in the Division of Infectious Diseases at Boston Children’s Hospital, and comprised of >200 members from academia, government, private-public foundations, and industry, the Precision Vaccines Program is a platform for international collaboration to foster development of vaccines targeted at vulnerable populations such as the young and elderly. Recent advances in molecular and systems biology and genetics as well as in translational medicine have informed a precision medicine strategy of defining subpopulations of patients sharing similar characteristics and tailoring medical interventions according to a patient’s responsiveness . The usel trials . Key to Bordetella pertussis, human immunodeficiency virus (HIV), rotavirus, and respiratory syncytial virus (RSV). The efficacy of influenza vaccines is hindered by influenza virus variability, and ways to improve it include the addition of adjuvants and/or of a conserved antigen such as stalk hemagglutinin  outlined major obstacles in defining effective vaccine approaches for specific microorganisms and possible strategies to overcome them. Examples of these are influenza viruses, glutinin , 17. A memployed –20. Thesmployed ––26. Rotaployed ––, as wellployed ––, 29. Morployed ––. Additioployed –––34. In aloyed –––, 36.The session on ontogeny, the change in a given individual across time, considered how the developing early life immune system impacts infection risk and interventions aimed to enhance host defense as well as systems biology tools to study the immune system in early life. James Wynn  highlighted the high incidence and mortality rates of sepsis early in life, especially in preterm newborns . In partMycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine to characterize the molecular ontogeny of the peripheral blood compartment in the first week of human life and then assess vaccine-induced perturbations of that ontogeny to define molecular signatures predicting immunogenicity.Tobias Kollmann  described how finding biomarkers for successful vaccination can provide useful information for the optimization of vaccines in which low or partial responsiveness is observed in early life. Challenges in biomarker identification include (i) uncertainty regarding which biomarkers to pursue  due to our limited knowledge of the underlying diseases and (ii) the fact that biomarkers that correlate with protection might not necessarily give insight into mechanisms of protection—i.e., they may not be mechanistic biomarkers. An unbiased approach using systems biology tools holds promise to define biomarkers of newborn vaccine immunogenicity , 36. Howin vitro Toll-like receptor (TLR) responses in Gambian infants across the first year of life, significant differences were noted between different TLR agonists, with TLR8 agonists demonstrating robust activation of Th1-polarizing cytokine induction in early life and between birth and 1 month of age  described examples of how combining clinical trials and immunological studies can provide insights into the ontogeny of the immune response as well as into maternal factors shaping newborn risk of infection and response to vaccines. For example, in a cross-sectional study of h of age . Analysinization . LookingPeter Richmond  also highlighted the importance of carefully designed clinical trials to define vaccine efficacy, especially in high-risk populations. For example, in a study based in Papua New Guinea (PNG), a pneumococcal polysaccharide vaccine given to children between the ages of 6 months and 5 years reduced pneumonia mortality . Howeverin vitro and in vivo immune responses elicited by the concurrent administration of HBV vaccine and BCG. Stimulation of whole blood isolated from preterms, newborns, and adults with a combination of BCG and HBV vaccine enhances the production of several cytokines/chemokines, in particular, interleukin-1β (IL-1β), some of them in an age-specific manner. Interestingly, in vivo administration of BCG and HBV vaccine to mice enhanced anti-HBV IgG titers in mice immunized on day of life 0 or 7 but not in adult mice  reported her recently published results on ult mice . These rInnateDB (http://www.innatedb.com/), a publicly available database of the genes, proteins, experimentally verified interactions, and signaling pathways involved in the innate immune response, was used for pathway overrepresentation analysis. Hanno Steen  and Tue Bennike  highlighted the challenges encountered in applying mass spectrometry to the study of the plasma proteome, especially in newborns and infants. The plasma proteome is highly complex  and exhibits an extreme dynamic range (11 to 12 orders of magnitude) and significant interpersonal variability. A further challenge for plasma proteome analysis is that represented by the relatively small volumes obtainable from newborns and infants. Through optimization of sample preparation protocol and mass spectrometry methodology  described a novel analytic approach named DIABLO  to integrate large-scale molecular OMICS data sets of various characteristics, including transcriptomics, proteomics, and metabolomics. By integrating multiple data sets, this software offers the opportunity to uncover the biological pathways involved in different disease subgroups, thereby identifying biomarkers of disease or of vaccine immunogenicity. These powerful approaches also generate some challenges, including data collection and management, analysis, and integration. Al Ozonoff  described the important role of data management in multidisciplinary systems vaccinology studies. He gave as an example the data management core (DMC) that serves the EPIC-HIPC project. Focused on systems vaccinology of newborns, this project will generate thousands of biological samples to track, process, and analyze. In this context, the DMC is responsible for accurate and reliable data capture, secure data management, project and analytical computing, and quality assurance. To accomplish these important tasks, a cloud computing platform and a decentralized governance structure were chosen, such that EPIC-HIPC cores and projects generate and process data and are also responsible for quality control. Providing cloud-based access to data and integrative analysis tools, the DMC will ultimately be responsible for quality assurance, data access, and public archiving to NIH websites such as ImmPort (http://www.immport.org).At the core of precision vaccinology studies is the use of systems biology tools that can shed new light on molecular requirements for successful vaccination. In the systems biology session, several members of the EPIC-HIPC consortium detailed the various OMICS approaches used and which strategies are used for data integration, storage, and dissemination. As part of EPIC’s goal of defining the molecular ontogeny of early life, Erin Gill  described preliminary transcriptomic results from a pilot cohort of Gambian infants who were receiving immunization at birth or at day of life 7 and who were sampled across the first week of life. hodology , 199 proJames Crowe  outlined the efforts to characterize the human immunome, that is, the whole set of recombined genes encoding human T cell and B cell receptors. This project will leverage Illumina’s genetic sequencing technologies and is poised to uncover novel diagnostic biomarkers as well as enable the development of highly targeted vaccines and immunotherapies for a wide range of diseases.William Mohn  reported a study of the impact of early life gut microbiome on asthma severity. Mice treated with vancomycin had an altered gut microbiome and experienced more inflammation in a papain-induced asthma model. Interestingly, administration of a cocktail of short-chain fatty acids (fermentation products of gut bacteria) along with vancomycin had no effect on gut microbiome but reduced inflammation in the same asthma model.Vaccines provide pathogen-specific protection, but growing evidence suggests that some vaccines can also exert heterologous (“nonspecific”) effects, potentially protecting against unrelated pathogens. Live attenuated vaccines, including measles vaccine (MV), BCG, and oral polio vaccine (OPV), have positive effects on survival that are substantially greater than those accounted for by a specific pathogen . AlthougChristine Benn  further described the principles of heterologous (“nonspecific”) effects of vaccines. She also reported the results of the Danish Calmette Study, which evaluated the effect of BCG immunization at birth on all-cause and infectious disease hospitalization . Interesin vitro and induce distinct levels of cytokines in neonatal cord or adult peripheral blood.Asimenia Angelidou  discussed differences between the BCG formulations produced and given in different locations of the world. She showed that different BCG formulations have different growth properties Nigel Curtis  presented the Melbourne infant study, a randomized controlled trial that aims to define whether BCG immunization at birth protects infants from infections while reducing the risk of developing allergies. The scientific basis for linking BCG immunization to allergy development relates to the hygiene hypothesis, in accordance with the idea that exposure to microbes in early life promotes a balanced development of the immune system, thereby reducing risks of allergies and autoimmune diseases . That stAn effective vaccine design usually requires proper adjuvantation and formulation systems. While adjuvants may modulate the magnitude and polarization of the immune response, formulation systems may ensure cell- or organ-specific vaccine targeting. Of note, formulation systems can dictate adjuvant properties ; therefore, adjuvantation and formulation systems need to be considered in a holistic and intertwined manner. Peter Andersen  described the adjuvantation system cationic adjuvant formulation 01 (CAF01), which consists of the Mincle agonist TDB , formulated with the cationic liposome-forming surfactant dimethyl dioctadecyl ammonium bromide (DDA), which is internalized by and activates dendritic cells (DCs). CAF01 was developed for generating a BCG booster vaccine and has since demonstrated enhancement of antibody production, germinal center reaction, T cell memory, and Th1/Th17 polarization in several immunization models, including newborn mice. Indeed, CAF01 is now being tested for multiple vaccine indications, including tuberculosis, HIV, malaria, and chlamydia, in phase I clinical trials. In addition, the CAF01 system is amenable to combination with other TLR agonists, such as TLR3 and TLR7/8 agonists , 53.in vivo, therefore requiring appropriate formulation. The first formulation strategy consists of encapsulating TLR8 agonists in polymersome nanoparticles. Encapsulated TLR8 agonists induce robust activation of human and murine neonatal DCs. Moreover, their administration in vivo to humanized TLR8 mice with the BCG-specific peptide Ag85B induced an increase in antigen-specific CD4 T cell counts comparable to that seen with BCG immunization  reported two formulation strategies that enable use of small-molecule TLR7/8 agonists for early life immunization. TLR7/8 agonists robustly activate neonatal DCs but lead to high levels of reactogenicity when delivered in soluble form nization . The secnization . These rnization , 56.Jeffrey Hubbell  presented a new system to target antigen and adjuvant to myeloid cells by coupling them to mannose residues. This complex binds to mannose receptors and enters the endocytic compartment and is therefore particularly well suited to molecules that activate endosomal receptors such as TLR7 agonists [p(Man-TLR7)]. When p(Man-TLR7) is administered in a complex with the experimental antigen ovalbumin [OVAp(Man-TLR7)], it enhances both humoral cell immunity and T cell immunity as well as Th1 polarization. Interestingly, OVAp(Man-TLR7) is more active than OVApMan plus TLR7  or OVA plus p(Man-TLR7) . These observations highlight the importance of targeting both the antigen and the TLR7 agonist to the endosomal compartment. Of note, comparable results were obtained using the circumsporozoite protein (CSP) malaria antigen, supporting the idea of the translational relevance of this formulation system.in vitro models that enable their characterization and evaluation of translational relevance is key to identify novel adjuvant targets. Jonathan Kagan  has proposed the provocative concept that not only pathogen-associated molecular patterns (PAMPs) such as that represented by CpG but also specific danger-associated molecular patterns (DAMPs) may exert adjuvant effects. Although the stimulation of DCs with a combination of a PAMP and a DAMP  leads to pyroptosis (release of proinflammatory cytokines associated with cell death such as IL-1β), use of oxidized phospholipids  as a DAMP enhances production of cytokines (including IL-1β) without inducing cell death. Accordingly, concurrent in vivo administration of LPS and oxPAPC with OVA leads to enhanced T cell activation and polarization compared to administration of LPS or oxPAPC alone . Accordingly, study of innate immune signaling pathways along with the development of proper PC alone .+ T cells, it was possible to model the age-specific prime and boost responses to licensed vaccines such as HBV and BCG. Interestingly, the CD4+ T cell responses changed depending on whether newborn plasma or adult plasma was added into the system.Guzman Sanchez-Schmitz  presented an innovative three-dimensional tissue culture system to model neonatal and adult innate and adaptive immune responses to vaccine formulations. This system consists of a human extracellular matrix covered by endothelial cells. Monocyte migration into the extracellular matrix and back into the medium across the endothelium (reverse transmigration) can differentiate monocytes into dendritic cells. By adding adjuvants or vaccines to this system and allowing monocyte reverse transmigration in their presence and then coculturing vaccine-pulsed monocyte-derived DCs with autologous CD4Simon van Haren  presented novel insight into the mechanism of action of an age-specific combined adjuvantation system comprised of an agonist of TLR7/8 together with an agonist of the C-type lectin receptor Mincle. This combination is particularly effective in overcoming impairments in the ability to induce a Th1 response in early life . The molRepresentatives from institutions that support vaccine research also presented at the conference. Mercy PrabhuDas  discussed NIAID programs that support the study of immunity in distinct populations such as the infant and elderly immunity programs as well as new initiatives in immune mechanisms at the mother-fetus interface.http://www.humanvaccinesproject.org), a public-private partnership whose goal is to decode the human immune system in order to inform and accelerate vaccine development.Theodore Schenkelberg  described the recently established Human Vaccine Project  effects of live vaccines in rigorously designed studies. Accordingly, the panel highlighted the need for better definitions of how vaccines protect and the relevant differences between distinct but related vaccine formulations that differ in protective efficacy, such as BCG. Opportunities for collaboration include using biomarkers of protection to guide development of novel adjuvantation and formulation strategies poised to usher in a new era in vaccine design, for example, by targeting less-responsive populations at the extremes of life or achieving effective single-shot immunizations that induce durable protection.A panel discussion moderated by Ofer Levy included Peter Andersen, Beate Kampmann, Tobias Kollmann, Stanley Plotkin, Mercy PrabhuDas, Alexander Rumyantsev (Sanofi Pasteur), Ted Schenkelberg, and Geert van den Bossche  and considered conclusions from the conference and the path ahead. The Precision Vaccines Conference highlighted the importance of a multidisciplinary approach in translational vaccinology. Novel and powerful systems biology tools enable an unprecedented depth of analysis and may be particularly useful for vaccinology, providing the opportunity to obtain large amounts of molecular and cellular information across the human life span from relatively small biological samples, leading to the following conclusions: (i) vaccine studies should be designed to include appropriate sample collection and storage for downstream systems biology; (ii) computational tools will be critical to convert big data to knowledge; and (iii) once potentially protective signatures are identified, these represent hypotheses that require testing Overall, the First International Precision Vaccines Conference highlighted important advancements that are creating new paths for vaccine discovery and design. Important work lies ahead that will benefit from synergistic interaction between academia, funding agencies, government, and industry. In this context, the Second International Precision Vaccines Conference, to be held in 2019 at the Harvard Medical School , will be at the forefront of these changes by gathering a multidisciplinary community of scientists to review progress and encourage partnerships focused on the most current challenges and opportunities in vaccine research.


These analyses show that, while factors intrinsic to cancer cells such as oncogenic mutations, alter cellular metabolism, cell-extrinsic microenvironmental factors also substantially contribute to the metabolic phenotype of cancer cells. These findings highlight that microenvironmental factors within the tumor, such as nutrient availability, physical properties of the extracellular matrix, and interactions with stromal cells, can influence the metabolic phenotype of cancer cells and might ultimately dictate the response to metabolically targeted therapies. In an effort to better understand and target cancer metabolism, this Review focuses on the experimental evidence that microenvironmental factors regulate tumor metabolism, and on the implications of these findings for choosing appropriate model systems and experimental approaches.Cancers have an altered metabolism, and there is interest in understanding precisely how oncogenic transformation alters cellular metabolism and how these metabolic alterations can translate into therapeutic opportunities. Researchers are developing increasingly powerful experimental techniques to study cellular metabolism, and these techniques have allowed for the analysis of cancer cell metabolism, both in tumors and in  Summary: Here, we discuss the model systems for cancer metabolism studies and highlight their key strengths and pitfalls in addressing different questions, such as how the microenvironment affects cancer metabolism and response to therapy. RAS)  (MYC) (TP53) (VHL) (KEAP1)  , phospho PIK3CA)  and MYC A) (MYC)  have bee) (TP53) , von Hip3) (VHL)  and Kelc (KEAP1)  are also (KEAP1) . These m (KEAP1) , metasta (KEAP1)  and resi (KEAP1) ; thus, tAuxotrophic: in the context of metabolism, auxotrophy refers to the inability of cells to synthesize a particular compound or metabolite and thus auxotrophic cells require access to that nutrient from extracellular sources.Entosis: a cellular phenomenon that involves the engulfment and degradation of neighboring cells; a type of cell death.Exchange flux: a phenomenon that can complicate analysis of stable isotope tracing experiments (see definition below) in which substrate-product interconversion leads to metabolite labeling regardless of the net direction of the reaction.Glutaminase (GLS): an enzyme that catalyzes the first step in glutamine catabolism by converting glutamine to glutamate and ammonia. Can localize to the cytosol or mitochondria.Macropinocytosis: a nonselective endocytic process by which bulk extracellular materials are internalized into cells.Mass spectrometry (MS): a method usually coupled to gas chromatography or liquid chromatography and used to assess the chemical composition of metabolites in a given sample.Matrix effect: a phenomenon that can complicate analysis of stable metabolites by MS (see definition below) in which compounds in the sample interfere with the ionization process necessary to detect a particular metabolite by MS. This can result in ionization suppression or enhancement and thus affect the accuracy of quantitative metabolite measurements. Compounds with high mass or polarity can cause matrix effects.Microtome: a tool used to thinly slice tissues. Although typically used for producing thin tissue sections for histology, a microtome can be used to cut tumor slices that can be then cultured in vitro for several days.Nuclear magnetic resonance spectroscopy (NMR): a method that can be used to assess metabolites in a given sample.Organoids: a type of cell culturing method by which cancer cells [or other type(s) of cells] are embedded in a 3D matrix, such as collagen or basement membrane, with or without particular factors to promote growth as a 3D structure. These types of culturing methods better recapitulate the spatial organization and diversity of cells in tissues and tumors.Pooled genetic screens: a tool to identify genes that contribute to a particular phenotype. Pooled genetic screens involve using multiple short hairpin RNAs (shRNAs) or single guide RNAs (sgRNAs) to silence or inhibit the expression of various genes in a target cell population, which may cover most of the genome, or a subset of genes such as metabolic enzymes. In this method, the genes for shRNAs or sgRNAs are integrated in the cell's genome. The depletion or enrichment for specific shRNAs or sgRNAs is measured with next generation sequencing techniques. A depleted or enriched shRNA or sgRNA that targets a particular gene provides information on selection for or against loss of that gene in a particular context.Spheroids: a type of cell culturing method by which cells are grown in clusters or aggregates, typically without the addition of ECM or special factors to the culture medium. Spheroid culture can be promoted via a variety of manipulations including culturing cell clusters in low-attachment plates. This culturing method maintains some aspects of spatial architecture and cell-to-cell contact observed in vivo.Stable isotope tracing: a method by which cells, tissues or animals are exposed to stable isotope-labeled nutrients, such as 13C-glucose, and the incorporation of the isotope-labeled atoms into various metabolites is measured using NMR or MS to infer metabolic pathway utilization.The development of more sophisticated cellular metabolism assays has increased our understanding of the functional effects of oncogenic alterations in metabolic enzyme regulation, activity and pathway use. Analytically, advances in nuclear magnetic resonance (NMR) , mass spin situ, such as in genetically engineered mouse models of cancer, can recapitulate human cancer progression in some cases . To quantitatively assess total metabolite levels across experimental conditions, researchers can use a variety of chromatography–MS- or NMR-based analytical platforms. Depending on the approach, metabolite levels can be measured in a targeted (for a pre-determined set of metabolites) or untargeted manner, with a trade-off between the scope of detected metabolites and assay sensitivity . FurtherInterpreting the biological meaning of metabolite level differences across conditions can be challenging, because measurements of metabolite pool sizes are static by nature. The observed differences in metabolite pool size between conditions could be caused by (1) changes in metabolite transport, (2) changes in metabolite production or (3) changes in metabolite consumption, and researchers need to conduct further experiments to understand why metabolite levels have altered . CombiniAnother method to assay cellular metabolism is to measure the consumption and release of nutrients, also referred to as the extracellular flux of nutrients. Extracellular flux analysis was used in the pioneering studies that determined the glycolytic nature of cancer, by measuring changes in glucose and lactate levels between the afferent and efferent circulation of a tumor . Current14C or 3H) or stable isotope tracers (such as 13C or 15N) have long been used to study metabolism in microorganisms and the metabolic alterations associated with diabetes. These techniques allow for dynamic assessment of cellular metabolism outside of the static snapshot provided by the analysis of metabolite pool sizes. The various uses of isotopic tracers have been reviewed extensively elsewhere  as an essential gene for cancer cell proliferation under limiting mitochondrial electron transport chain function  or CRISPR-based pooled genetic screens  interrogfunction . CRISPR-function .In addition to genetic-based screening approaches, small molecule inhibitors, when available, can also be used to assess the functional requirements for a metabolic enzyme. A renewed interest in metabolism has sparked the development of metabolic enzyme inhibitors, and these inhibitors have been used to identify the molecular signatures of cancer cells that rely on specific metabolic pathways for proliferation. For example, pancreatic ductal adenocarcinoma cell lines were stratified based on their intracellular metabolite levels and subsequently treated with inhibitors based on this metabolic categorization. This process allowed for identification of cell lines that differentially respond to various metabolic inhibitors . FurtherFunctional metabolic studies using genomic or pharmacological interventions can be highly informative; however, extrapolating these results to understand how metabolism functions in a cell can still be challenging. For example, GOT1 essentiality induced by mitochondrial dysfunction does not indicate the directionality of flux through GOT1, or why the cell requires this enzyme, which is involved in multiple cell processes. Nevertheless, functional screening approaches hold promise for defining the metabolic requirements of cancer cells.Assays of cellular metabolism are only one facet of what researchers need to study cancer metabolism. These assays must be deployed in appropriate disease model systems to gain insight into how metabolism is altered in cancer. Cancer models range from traditional monocultures to mouse cancer models to humans, and this section discusses how metabolic assays can be utilized in these systems. We highlight a key trade-off: with increasing complexity and physiological relevance of the model system, deriving insight from metabolism assays becomes more challenging .Fig. 1.in situ .Cultured microtome  slices oin vivo models might better represent the physiological complexity and heterogeneity of cells observed in human cancers, and also allow interactions between the tumor and normal host tissues. However, when using mouse models to study metabolism, factors such as diet, strain, sex, age, husbandry and environmental stressors can affect metabolism and should thus be considered  cycle substrates can be utilized differently between tumors and 2D cultured cells. For example, in 2D culture models of lung cancer, glutamine is a primary carbon substrate for the TCA cycle, rendering glutaminase-mediated glutamine catabolism essential in most 2D culture settings . In contCA cycle . InsteadCA cycle , and lunCA cycle . SimilarCA cycle ; howeverCA cycle . These rin vitro versus in vivo. Interestingly, genetic perturbations of some metabolic pathways can cause profound phenotypes in vivo, yet cause relatively minor phenotypes in culture and vice versa. Recycling metabolism such as autophagy or acetate recapture are striking examples of this phenomenon. Specifically, depletion of autophagy related 5 (ATG5) or autophagy related 7 (ATG7) (loss of autophagy), or acyl-CoA synthetase short chain family member 2 (ACSS2) (acetate recapture loss) is well tolerated by most cells in culture, but can dramatically decrease tumor growth in vivo  are required to have antiproliferative activity in culture models . High glutamate export causes an increased reliance on glutamine catabolism in cultured cells. Thus, cystine indirectly affects glutamine catabolism via transport of glutamate, an intermediate in glutamine catabolism. Therefore, glutaminase inhibition has a greater antiproliferative effect in vitro, where cystine is abundant. Importantly, these examples illustrate that nutrient levels within the microenvironment alter metabolism in ways that might be distinct from the metabolic pathways in which these nutrients directly participate.The amino acid cystine is another example of how differences in nutrient levels between culture models and tumors alter cancer cell metabolism . High le12 , again potentially altering cancer cell metabolism  and ammonia (an amino acid catabolism by-product). Lactate and ammonia have long been considered to be metabolic waste products of tumors, as pioneering tumor metabolism studies found that nearly all tumors net secrete lactate  and ammoin vivo can consume and utilize these macromolecules as metabolic substrates. For example, pancreatic tumors acquire amino acids from circulating proteins via macropinocytosis  depletion increased the autophagic flux required to process albumin . This reThe cellular heterogeneity observed in tumors suggests that cancer cells exist in a metabolic community within the tumor microenvironment  that is Different cell populations within tumors can also engage in communal cell-cell nutrient sharing. Proper assessment of intercell nutrient sharing with current biochemical and metabolic tracing approaches is challenging, as these methods are unable to resolve net nutrient transfer versus exchange flux. Future studies that address nutrient sharing in co-cultures using chemical engineering-based analytical methods, such as performing metabolic flux analysis on co-cultures with appropriate tracers to infer metabolic phenotypes, or modeling techniques, such as using cell-type specific reporter proteins to assess cell-type specific labeling data, could help to unravel intercell nutrient sharing . Thus, aGlutamine is another nutrient that is reported to be shared between stroma and cancer cells. Both glioblastoma and ovarian cancer cells in culture behave as glutamine auxotrophs, relying on an external supply of this nonessential amino acid . HoweverBeyond nutrient sharing, stromal cells in the microenvironment can themselves serve as alternative sources of nutrients. For example, exosomes shed by fibroblasts contain metabolites that can sustain nutrient-starved cancer cells . StromalThe tumor microenvironment also displays chemical and physical properties that are distinct from normal tissues and the circulation. Outside of animal models, cancer models commonly miss these aspects, which can play an important role in regulating cellular metabolism. For example, although it has long been appreciated that tumors are more acidic than the circulation , recent It is also increasingly apparent that the physical properties of the ECM within the tumor alter cancer cell metabolism . Althougin vivo, and by better characterizing the tumor microenvironment to design new ex vivo models of cancer that better represent the tumor microenvironment and remain experimentally tractable.As discussed in this article, various components of the tumor microenvironment affect the metabolic behavior of cancer cells, and thus noncell-autonomous factors play a major role in dictating cellular metabolism. Importantly, this affects cancer cell response to some therapies, including those that target metabolism . Thus, iTowards the first goal, emerging technologies such as imaging MS will allow spatial examination of metabolite levels in tumors , and couex vivo cancer models possible. We anticipate that this will provide new insight into the metabolism of cancer and stromal cells in tumors, and the microenvironmental variables that constrain metabolism. This information will ultimately provide insight into the metabolic processes that should be targeted for cancer therapy and the contexts in which the therapies will be most efficacious.Toward the second goal, a better understanding of how microenvironmental variables influence tumor metabolism is needed. There is increasing knowledge of which cell populations are present in a tumor  and the http://dmm.biologists.org/collection/cancermetabolism.This article is part of a special subject collection ‘Cancer metabolism: models, mechanisms and targets’, which was launched in a dedicated issue guest edited by Almut Schulze and Mariia Yuneva. See related articles in this collection at


Zika virus is an emergent pathogen that gained significant importance during the epidemic in South and Central America as unusual and alarming complications of infection were recognized. Although initially considered a self-limited benign infection, a panoply of neurologic complications were recognized including a Guillain–Barré-like syndrome and in-utero fetal infection causing microcephaly, blindness, and other congenital neurologic complications. Numerous Zika virus vaccines were developed, with nine different vaccines representing five different platforms entered into clinical trials, one progressing to Phase II. Here we review the current landscape and challenges confronting Zika virus vaccine development. The Zika virus, discovered in Uganda in 1947 . IsolateIn this paper, we provide a brief update of current progress in Zika virus vaccine development and explore the challenges to vaccine assessment and eventual licensure.Zika virus infection presents with a symptom complex consisting of a diffuse maculo-papular rash, fever, asthenia, myalgias, arthralgias, headache, and retroorbital pain. The frequency and degree of symptomatology has, however, varied between studies. A retrospective study immediately following the outbreak on Yap Island found that only 19% of survey participants reported symptoms consistent with Zika virus infection , a figurIn adults, the most common reported complication of Zika virus infection is a Guillain–Barré-like illness that had a prevalence of 0.24 cases per 1000 cases of infection in the French Polynesian epidemic . Of inteIn contrast, infection during pregnancy has been associated with fetal microcephaly and a number of other congenital illnesses including visual deficits, hearing disorders, neural calcifications, learning disabilities, and arthrogryposis that may affect as many as 30% children born to mothers infected during pregnancy ,19,20,21Zika virus can also be transmitted through sexual contact, first reported for a researcher returning from Senegal in 2008 . NumerouIn mice, Zika virus infection causes testicular atrophy and significantly decreases spermatic function and fertility rates ,29,30. IFemales may also excrete Zika virus RNA for extended times. A prospective study of five women showed that Zika RNA was detected in vaginal fluid for as long as 6 months and a month or more in three of the five . Murine Thus, while Zika virus infection is mildly symptomatic and self-limited for the vast majority of individuals, with infrequent neurologic complications in adults, vaccination of the general population may not be warranted. However, vaccination of females at or entering reproductive age and their male partners is prudent. As discussed previously, vaccination during pregnancy is non-ideal due to the time to generate protective immunity and unknown vaccine safety . The onehttps://www.paho.org/). The almost complete disappearance of Zika has created significant barriers to ongoing vaccine studies as attack rates have fallen below those need to meet reasonable sample sizes [In late 2015 and through mid-2016, global concern for this spreading epidemic was increasing, however, the experience in the two prior epidemics was predictive of the subsequent epidemiology in the Americas. The Zika virus outbreaks in Yap Island and French Polynesia were characterized by rapid onset and rapid resolution over a span of 3–4 months . The ZikThe WHO removed the designation of Zika a virus of global concern in 2018 as case rates have fallen. In March 2018, the WHO and the National Institutes of Allergy and Infectious Diseases (NIAID) convened a meeting of academics, representatives from industry, and regulators to discuss the approach to Zika virus vaccine development in an era of waning incidence . AlthougVaccine development against the Zika virus began in earnest in late 2015 following the reports of microcephaly in fetuses and infants in Brazil. Of note, the first demonstration of immunoprotection was as part of a 1953 study to define the ultrastructural characteristics of Zika virus, that found intramuscular vaccination of mice with infectious viral filtrates protected against cerebral infection .Poland et al. provide a comprehensive listing of almost 40 Zika virus vaccines in development as of 2017 . DiamondA total of three DNA vaccines have entered into human testing ,42, inclGLS-5700, a DNA vaccine encoding for the Zika virus prME genes designed as a consensus based on available Zika virus sequences through December 2015 , was theTwo additional DNA vaccines, based on the French Polynesian H/PF/2013 strain were developed as chimeras that included the JEV prM signal sequence followed by the Zika envelope (E) gene (VRC5283) or a similar construct with the terminal 98 amino acids of E, representing the stem and transmembrane regions, exchanged for the analogous JEV sequence (VRC5288) . Both vaThree inactivated vaccines have entered into clinical studies, of which clinical data for only ZPIV vaccine has been published . ZPIV isClinical trial data for the other vaccines has not yet been reported as of the date of this monograph, however, pre-clinical data has been reported for three candidate vaccines. An mRNA vaccine that incorporates the prM-E genes of a Micronesian strain of Zika virus was created incorporating with or without four-point mutations in the fusion-loop segment of the DII region of the envelope gene that abolished binding of antibodies directed against the fusion-loop region to reduce the potential risk for antibody-dependent enhancement of infection . ImmunizA number of animal models have been assessed for vaccine development and have been reviewed elsewhere . ImmunocThe logistical difficulties in pursuing standard vaccine evaluation have created significant interest in the possibility of controlled human infection models (CHIM). The conduct and planning for human challenge trials raises unique medical and ethical considerations. For Zika virus trials, one must balance the relative benign and self-limited infection experienced by the vast majority against the risk for less benign complications and transmission risks.As reviewed above, published studies provide estimates that Zika virus infection is relatively asymptomatic and self-limited for the majority of individuals, however, methodology in these studies varies widely. In adults, two sets of complications warrant consideration for a proposed CHIM study. As reviewed above, a Guillain–Barre-like syndrome occurs in approximately 1 of 5000 Zika virus infections , whereasTo address safety concerns, there has been work to develop attenuated viral strains deleted for potential neurotropic regions  with a gIn summary, Zika vaccine development continues with multiple candidate vaccines in clinical trials. Because of the significant decline in incidence, evaluation of vaccine efficacy is increasingly difficult. There has been renewed interest in animal model and human infection models of infection.


Deep venous thrombosis of the lower extremities following orthopaedic surgery is well-documented. Though less common than its lower extremity counterpart, upper extremity deep venous thrombosis (UEDVT) has been documented in the literature as well, largely in the context of arthroscopic shoulder surgery. However, there is a paucity of literature documenting UEDVT following surgical fixation of upper extremity fractures, specifically fractures involving the proximal humerus. We present a case of UEDVT following a fracture to the proximal humerus and subsequent surgery. Though UEDVT is considered a rare complication following this type of surgery based on a lack of documentation within the literature, we believe a high-index of suspicion is required to prevent potentially life-threatening sequelae, such as pulmonary embolism (PE) and post-thrombotic syndrome. Deep venous thrombosis (DVT) is the formation of a thrombus within the deep veins of the upper or lower extremity. Though more common within the lower extremities, DVT can occur in any of the veins of the upper extremity or thoracic inlet, including the jugular, brachiocephalic, subclavian, axillary, and brachial veins . The occA 66-year old right hand dominate female, with no significant past medical history, presented to the emergency department at our institution with left shoulder pain after an accidental trip and fall onto an outstretched hand that occurred the same day. She had noticeable deformity of the upper arm and was neurovascularly intact distally. Radiographs were obtained which demonstrated a four-part proximal humerus fracture and dislocation of the left glenohumeral joint . The patDVT following orthopaedic surgery is well documented within the literature. In particular, much is known about DVT after reconstructive hip, knee, and shoulder arthroplasty , 5 as weThe pathogenesis of UEDVT can be divided into either primary or secondary causes. Primary causes are less common and include venous thoracic outlet syndrome, Paget-Schroetter syndrome, and idiopathic causes. Paget-Schroetter related thrombosis occurs in individuals who develop muscle hypertrophy and vessel wall damage following strenuous exercise in the context of narrowed thoracic inlet . SecondaWe believe that the formation of an UEDVT in this patient was directly related to her proximal humerus fracture and subsequent surgery with immobilization. Blunt trauma increases the circulation of proinflammatory cytokines and procoagulant microparticles, creating a hypercoagulable state . In addiThe signs and symptoms of UEDVT are relatively nonspecific such as swelling, pain, edema, and erythema, regardless of the structure involved . As sympAnticoagulation is the mainstay of a multimodal approach to treatment for DVT. Goals of treatment include reducing further clot propagation, preventing embolization and promoting resolution of the existing thrombus. As such, patients should be started on a parenteral anticoagulant upon diagnosis. Such parenteral anticoagulants include unfractionated heparin or LMWH . FollowiAdequate treatment of UEDVT is absolutely critical to prevent possible life-threatening sequelae, including PE. Symptomatic pulmonary emboli occur in approximately 12% of all UEDVT cases while asymptomatic pulmonary emboli occur in an additional 36% of such cases . AdditioAlthough uncommon, UEDVT can occur following surgical fixation of an upper extremity fracture. Sequelae, such as PE and post-thrombotic syndrome, necessitate the need for a high index of suspicion. Persistent or worsening symptoms of swelling and pain should trigger a diagnostic workup consisting of doppler ultrasound imaging. Because pain and swelling are almost universal, to some degree, after all cases of surgical treatment of a proximal humerus fracture, we raise this issue so that clinicians continue to keep UEDVT in the differential diagnosis when swelling seems more excessive than normal, or when the patient presents with significant concern.If a diagnosis is confirmed then prompt treatment with systemic anticoagulation should be initiated and continued for three months. The risk of recurrence and possible life-threatening sequelae warrant consideration for a multimodal approach to treatment if symptoms do not promptly resolve.This case report highlights the risk of UEDVT as a complication following surgical fixation of a proximal humerus fracture. Knowledge of this complication along with close vigilance of post-operative symptoms and clinical course should trigger a diagnostic work up of UEDVT followed by appropriate therapy.


The short-chain fatty acid butyrate, either in unprotected or protectedform, is widely applied as a growth-promoting feed additive in poultrynutrition; however, its possible effects on the carcass composition of broilers have not been fully elucidated. Further, lowering dietary crude protein (CP) levelsis an important issue in poultry farming, contributing to ecologicallybeneficial lower nitrogen excretion. The main aims of this study were to testhow unprotected and protected forms of butyrate and decreased dietary CPcontent with essential amino acid  supplementation (“LP-EAA” diet) affect carcass parameters and the chemical composition of muscles in broilers. Ross 308 chickens wererandomized to seven groups ( As broilermeat is one of the most common, valuable and easily available proteinsources in human nutrition, the significance of efficient poultry husbandryhas greatly increased in the last decades. Animal welfare and economicaspects should also be addressed when improving productivity in poultryfarming. Nutritional changes, such as altering nutrient composition orapplying feed additives may provide great tools for enhancing meat qualityand production efficiency. As the application of antibiotics as growthpromoters has been prohibited in the EU since 2006 ,using alternative feed additives is of particularly high interest in poultrynutrition.The short-chain fatty acid . However, butyric acid glycerides had no significanteffects on the carcass composition of chickens .In another study, carcass yield was increased and abdominal fat content wasdecreased by butyric acid as a feed additive in broilers . However, it is not known if butyrate as a feed additive, either as anunprotected free salt or in a protected form, may influence musclecomposition and thus the meat quality of broilers.Based on its widespread biological actions, butyrate can increase thegrowth performance of broilers, indicated by a significantly higher bodyweight gain and decreased feed conversion ratio (FCR) values . It was also reported that butyric acid glycerides increased the carcassweight and breast meat yield of broilers, which effects became even morepronounced under suboptimal health conditions, such as Altering dietary crude protein (CP) content and free amino acidsupplementation can also affect growth and meat production of broilers.Decreased dietary CP levels could cause impaired body weight gain and FCR ifthe digestible amino acid profile is unbalanced or an amino acid is limiting. However, this effect could be ameliorated by thesupplementation of the diet with limiting free amino acids, maintainingphysiological productivity . The application of low-CPdiets increased the fat content of the carcass  but decreased the nitrogen excretion, the latter being highly beneficial forthe environment . Similarly, the abdominal fat mass andthe lipid content of the carcass were both significantly increased inchickens fed a low-CP diet. This effect was overcome when arginine andlysine were supplemented, indicating that arginine and lysine concentrationswere deficient before the supplementation . Theincreasing action of low-CP diets on the fat mass of the carcass is alsomainly associated with the unbalanced dietary amino acid profile becauseexcess amino acids are broken down and may be utilized for triacylglycerolsynthesis . However, abdominal fat mass was reduced whenlow-CP and low metabolizable energy (ME) diets were fed to broiler chickens. Lowering dietary CP and ME content also decreasedthe final body weight but increased the relative weight of thighs and wingsin broilers .The main goal of this study was to assess if unprotected sodium butyrate orvarious protected (film-coated and fat-embedded) sodium butyrate types mayinfluence carcass characteristics and muscle composition of broilerchickens. Further, the suggested effects of reduced dietary CP levels withessential amino acid supplementation were also to be analyzed tooptimize the dietary conditions required for sustainable and economic broilermeat production. By combining these two factors, we wanted to monitor ifunprotected sodium butyrate as a pure substance has different effects oncarcass traits under different dietary conditions, such as normal or loweredCP levels, possibly due to the suggested interplay of dietary amino acidsupply, composition of the gut flora, and microbial butyrate utilization orproduction.22.1The housing and treatment of the animals were carried out in accordance with thenational and international laws as well as with the institutionalguidelines. Experimental procedures were approved by the Government Officeof Pest County, Food Chain Safety, Plant Protection and Soil ConservationDirectorate, Budapest, Hungary (number of permission: PEI/001/1430-4/2015).2.2Day-of-hatch male Ross 308 broiler chicks, obtained from a commercialhatchery , were randomly allocated toseven dietary groups . Two groups of chickens received a low-CP, amino-acid-supplemented  diet. The amino acid levels in all diets werecalculated, and the four first-limiting commercially available amino acids were supplemented to meet the recommendations of thebreeder . Starter diets were switched to growers on day 10,while growers were switched to finishers on day 25. All diets within a phase were set tobe isoenergetic. Ingredients and calculated and measured nutrient compositionof diets are shown in Table 2.The feed of two groups (one within the NP and one within the LP-EAAdietary groups) was supplemented with unprotected sodium butyrate ,deboned breast meat yield, femoral muscle weight, liver weight, heartweight, spleen weight and abdominal fat weight. Deboned breast meat yield,femoral muscle weight and abdominal fat weight were additionally evaluatedrelative to body weight to obtain information of the proportion of carcassparts.Representative samples of pectoral and femoral muscle  were taken for chemical analysis of meatcomposition. Muscle samples were minced, freeze-dried, ground and stored atDiet samples were ground in a hammer mill with a 1 mm screen and analyzed induplicate for dry matter (dm), ash, crude protein  on the measured parameters asresponse (dependent) variables. A post hoc Tukey's test was applied forpairwise comparisons. Data were normally distributed and within-groupvariances were homogenous. Differences between groups were regarded asstatistically significant when 3The body weight and feed intake of the animals matched the standards of the Rosstechnology in all phases of broiler fattening. Data on body weights arepresented in Table 3, showing significantly higher body weight values inLP-EAA birds than in NP groups at weeks 3 and 6  was significantly increased by the low-proteindiet with essential amino acid supplementation: significantly higher valueswere measured in the LP-EAA CTR group than in NP CTR animals was significantlygreater in the LP-EAA CTR than in the NP CTR group  was significantly greater in LP-EAA CTR than inNP CTR animals  was significantly decreased by the essential-amino-acid-supplemented low-CP diet and by all forms of butyrate used. In detail, a significantly decreased protein content was measured in the LP-EAA CTR compared to the NP CTR groups  was significantly affected by thedietary CP level and by butyrate supplementation as well. Significantlyhigher values were measured in the thighs of chickens kept on a low-CP, amino-acid-supplemented diet than in those of the normal CP groups . In our study, the applied protected butyrate products wereable to increase carcass weight, being more potent to increase meatproduction than unprotected butyrate, while the latter could not provoke asignificant elevation in carcass yield. Relative breast meat yield wasincreased by both unprotected and protected butyrate supplementation,indicating higher mass and a higher proportion of pectoral muscle among meattypes. The absolute breast meat mass, as a sum of the increased carcassweight and relative breast meat yield, was also elevated (with approximately34 %) by all types of protected butyrate application . The greater absorptivecapacity and more balanced microflora may contribute to increased growth andaltered carcass characteristics. In addition, the absorbed butyrate can actin several tissues as an epigenetically active molecule and may also elicitsome receptor-mediated effects . For instance, thebutyrate-associated modulation of insulin homeostasis can also be related tostimulated muscle development as insulin receptor The lipid content of the femoral muscle was increased and the protein content wasdecreased by all types of butyrate applied, but no changes were observed inthe chemical composition of breast meat. The observed alterations in thighsmay improve meat quality, and the increased muscular lipogenesis was notcoupled with abdominal fat deposition. A similar action of orally appliedbutyrate was also described in feedlot cattle, where the marbling of themeat was significantly increased by calcium butyrate as a feed additive.No relevant differences were found between the efficacy of differentprotected butyrate types; the tested protected products seemed to be moreeffective in some cases than unprotected free butyrate, such as instimulating carcass weight (which effect was lacking in the case of unprotectedbutyrate) or in increasing breast meat yield to a higher extent thanunprotected butyrate. Dietary supplementation of protected butyrate providesbutyrate release in more distal sections of the intestines, whileunprotected butyrate is rapidly absorbed from the proximal section of thegastrointestinal tract . These different kineticproperties of protected butyrate products should deliver better butyrateexposure for the intestinal microflora, and the prolonged absorption mayalso have differing effects on various extraintestinal tissues compared tothe action of unprotected butyrate .According to our results, the low-CP, amino-acid-supplemented diet increasedthe live body weight (at week 3 and 6), carcass weight and breast meat yieldof broilers. In general, reduced dietary CP levels with improper amino acidcomposition can diminish the growth of chickens due to the inadequate aminoacid supply . However, no decrease can be found ingrowth performance when providing amino acids in a well-balanced profile,possibly ensured by essential amino acid supplementation . Similarly to our results, significantly greater body weights weremeasured in chickens kept on a low-protein diet, supplemented with limitingamino acids by Khan et al. (2011). This effect is suggested to be connected to more free essential amino acids being available for theamino-acid-supplemented groups compared to the animals fed with normalprotein diets. It was also reported by Awad et al. (2014) that dietary CPlevels could be lowered to a limited extent together with essential aminoacid supplementation to maintain the normal growth and health of broilers. Thesupplementation of free amino acids to broiler feeds is a key factor inlowering dietary CP levels , reducing nitrogen excretion butmaintaining or even increasing growth and meat production. The elevation ofthe relative breast meat yield in broilers kept on an LP-EAA diet indicatedthat not only the absolute mass but also the proportion of breast meat asthe most valuable part of the chicken carcass was increased by thelow-CP and amino-acid-supplemented diet.In our study, the weight of the liver was significantly increased by theessential amino-acid-supplemented low-CP diet. In contrast, Awad et al. (2014) found no changes in liver mass when low-protein and amino-acid-supplemented diets were given to broilers; however, they investigatedchickens in the grower phase and applied slightly lower dietary lysinelevels compared to our study. As observed in the present study, the proteincontent of the femoral muscle was significantly decreased, while its lipidcontent was increased by the LP-EAA diet when compared to NPanimals. However, relative abdominal fat mass was not affected by dietary CPlevels. Presumably due to the lower dietary protein and higher carbohydratecontent (to gain isoenergetic diets), the lipogenesis could be stimulated inlow-CP groups. However, the subsequently increased triacylglyceroldisposition was realized only in femoral muscle improving meat quality anddid not result in greater abdominal fat reserves. In another study, variousisoenergetic low-CP diets (from 23 % to 18%) increased the lipid content ofthe whole carcass independently of the provided amino acid supplementationin 3-week-old broilers . Similarly, abdominal fatmass and the amount of extractable carcass fat were increased by lowerdietary CP levels (from 25 % to 18 %) but were reduced by arginine orlysine supplementation in the starter phase .The effects of unprotected sodium butyrate as a pure substance were comparedin chickens kept on an NP or LP-EAA diet to gain some preliminary dataabout the possible interaction of various dietary factors. According to ourresults it can be observed that butyrate similarly altered the chemicalcomposition of the femoral muscle in both cases. However, the stimulatoryaction of unprotected butyrate on breast meat yield was lacking in the case ofthe lowered CP supply with essential amino acid supplementation. The partlydifferent action of butyrate in normal and low-CP diets cannot be explainedbased on these data. However, it can be hypothesized that a different aminoacid supply may alter the composition of the intestinal microflora, possiblyinterfering with the utilization of exogenously applied butyrate and theendogenous microbial butyrate production. Further, orally applied butyratemay also influence the pH of the ingesta, possibly acting on proteindigestion and utilization as well. Based on our results, it can behighlighted that feed additives such as butyrate can elicit differenteffects under various dietary conditions; thus, combining more nutritionstrategies to optimize animal production should be considered carefully.Therefore, based on these initial results about the combination of puresodium butyrate and an altered dietary CP supply, future studies are neededwith regard to the possible interaction of different butyrate-containingproducts and dietary CP levels or cereal types.It has to be stressed that, in contrast to the thighs, nodiet-associated changes (either in the case of butyrate addition or dietary CPlevels) could be detected in the chemical composition of the pectoral muscle.This finding might be connected to different muscle fiber compositionand metabolic properties of various muscles and suggests a great stabilityof breast meat composition.Based on our results, it can be concluded that the development andproduction of breast meat can be effectively stimulated by the dietary CPcontent and butyrate supplementation, but its chemical composition remainsunchanged at the same time. The application of unprotected or protectedbutyrate as feed additives and decreased dietary CP levels with concomitantessential amino acid supplementation seem to be nutritional tools toincrease carcass yield and to alter the meat composition of broilers.


Prostate cancer (PCa) is the most common cancer in men, and more than 10% of men will be diagnosed with PCa during their lifetime. Patients that are not cured with surgery or radiation are largely treated with endocrine therapies that target androgens or the androgen receptor (AR), a major driver of PCa. In response to androgen deprivation, most PCas progress to castrate resistant PCa, which is treated with anti-androgens like enzalutamide, but tumors still progress and become incurable. Thus, there is a critical need to identify cellular pathways that allow tumors to escape anti-androgen therapies. Epidemiological studies suggest that high-fat diets play important roles in PCa progression. Lipid metabolism rewires the PCa metabolome to support growth and resistance to endocrine therapies, although the exact mechanisms remain obscure. Therapeutic effects have been observed inhibiting several aspects of PCa lipid metabolism: Synthesis, uptake, and oxidation. Since AR remains a driver of PCa in advanced disease, strategies targeting both lipid metabolism and AR are starting to emerge, providing new opportunities to re-sensitize tumors to endocrine therapies with lipid metabolic approaches. Prostate cancer (PCa) is the most common cancer in men and the second highest contributor to cancer deaths in the Western hemisphere . For menFrom the metabolic perspective, men treated with ADT experience adverse effects such as fatigue, sarcopenia, loss of bone density, increased adiposity, and metabolic syndrome ,7. AdditThis review will cover three emerging, conceptually related aspects of PCa research: Lipid metabolism, endocrine therapy challenges, and potential therapeutic opportunities targeting both lipid and androgen signaling.The observation of a close correlation between the average per capita fat intake and PCa mortality in several countries, including the USA, sparked a lot of interest in the potential link between dietary fat and prostate cancer risk . It has Lipid metabolism in cancer can be studied from two angles: Lipid synthesis and lipid breakdown, which is then likely used for oxidation and ATP synthesis. The former has received more attention in the past due to the identification of de novo lipogenesis as a biomarker of aggressive disease in many hormone-dependent cancers . In factAnother important aspect of lipid metabolism beyond de novo lipid synthesis is the ability of many tumors to desaturate their own lipids. This desaturation of newly synthesized fatty acids is necessary for the formation of lipid droplets and membranes, and promotes growth and survival of the cancer cells . ParticuCancer cells can also obtain fatty acids from the circulation, by breaking down triglycerides contained in circulating chylomicrons and lipoproteins. In fact, several studies have found significant associations between circulating lipoproteins and PCa growth and recurrence . HoweverThese results also bring attention to the fact that lipid metabolism in PCa is quite dynamic, switching between lipid synthesis and oxidation depending on the cellular context. For example, as tumors start to outgrow the vasculature network, accessibility to nutrients becomes compromised. Cells distant from blood vessels have diminished access to nutrients and oxygen, and may switch to alternative forms of metabolism like oxidation of branched amino acids and fatty acids to support viability . In factCPT1A (liver isoform) gene that confers some protection from PCa risk to arctic populations [CPT1A gene locus significantly correlates with very low density lipoprotein (VLDL) and low density lipoprotein (LDL) lipoprotein profiles [Regarding the other side of the coin, lipid utilization, significant findings have been uncovered in recent years. A key player in lipid breakdown is the rate-limiting enzyme palmitoyltransferase 1 (CPT1). For the long chain fatty acids like palmitate to get into the mitochondria, the fatty acids (acyl-CoA chains) must be enzymatically converted to acyl-carnitines across the outer mitochondrial membrane by CPT1 . This isulations . In factprofiles , pointinStudies focused on the role of lipid oxidation via CPT1A have brought attention to the role of lipid catabolism by cancer cells. ,62,63. RMYC influences multiple seemingly unrelated phenotypes, and it is commonly amplified or overexpressed in human cancers [CPT1B knockdown. Their results suggested that fat oxidation via CPT1 was an essential metabolic pathway in MYC-overexpressing TNBC cells. The oncogenic role of MYC has also been studied in the context of transgenic mouse models of PCa, where combined MYC activation and PTEN loss synergized to induce genomic instability and aggressive PCa [MYC and AKT1 in PCa lipid utilization.A critical link between cancer lipid metabolism and targeting it therapeutically is identifying the upstream regulators that modulate tumor metabolism. The proto-oncogene  cancers . MYC is  cancers . Camardasive PCa . The rolsive PCa . AdditioGiven the wide important role of MYC in cancer, a direct MYC inhibitor could be clinically valuable. However, direct targeting of MYC remains challenging, and no inhibitor has been identified yet. Thus, targeting metabolic pathways modulated by MYC, like fat oxidation, is an intriguing therapeutic opportunity for MYC-driven cancers. Active research in this area is currently ongoing for breast and prostate cancers .Androgen deprivation therapy (ADT) is the standard-of-care therapy for PCa patients experiencing relapse or metastatic disease. Castrate-sensitive PCa is characterized by non-castrate levels of testosterone in the blood. This hormone-sensitive state spans from patients with increases in the prostate-specific antigen (PSA) to those with cancer metastasis detected by imaging . The curA salient characteristic of CRPC is the reactivation of AR signaling. This is reflected by progressive rises in serum PSA, which is transcriptionally regulated by the AR. Ample research has shown that the majority of AR-regulated genes  are re-expressed in most CRPCs, and several mechanisms capable of maintaining AR activity have been established ,81,82. DAlternative splicing of AR transcripts is one of the main mechanisms implicated in the progression of CRPC as well as in resistance to anti-androgens. To date, more than 20 AR splice variants (AR-V) have been identified in prostate cancer cell lines and mouse xenografts, some of which have been validated in human cancer . These AOne characteristic of PCa is that it has a long natural history, which therefore opens opportunities to investigate how signaling and metabolic molecules interact in promoting PCa growth and progression. Importantly, there is plenty of epidemiological and correlative data postulating that a Western diet (which is rich in long chain fatty acids) can promote PCa progression to lethal disease . CurrentRecent studies have indicated that lipids can fuel lethal prostate cancer in mouse models . AlthougThe synthesis and use of lipids in PCa cells is regulated by androgens. Androgen receptor signaling in PCa is known to upregulate lipid biosynthetic enzymes such as FASN and ACACA  ,91. On tOther studies have found that 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), a key enzyme in the cholesterol synthesis pathway, is elevated in enzalutamide-resistant PCa cells . A combiAs mentioned above, a rational target for lipid oxidation in hormone-dependent cancer is CPT1. There are very few studies about the connection between CPT1A and AR in PCa ,99. Our CPT1A genetic knockdowns in PCa cells. Interestingly, when we genetically decreased CPT1A expression in LNCaP cells, we observed less growth, a greater dependence on AR, and an increased sensitivity to the anti-androgen enzalutamide [Since pharmacological approaches always produce off-target effects, we also performed lutamide . From a lutamide ,101, altLipid metabolism rewires the PCa metabolome to support growth and resistance to endocrine therapies. It is still unknown which is first—the metabolic change or the AR reprogramming. It is possible that both occur in an interdependent manner, according to the tumor microenvironment and the nutrient availability. Future studies examining the lipid preference of tumors, from androgen-sensitive tumors to CRPC, will shed light on lifestyle and pharmacological interventions specific to the patient, enhancing the cancer treatment response and the quality of life of the patient. Exciting new research awaits.