Meat and milk production needs to increase ~ 70–80% relative to its current levels for satisfying the human needs in 2050. However, it is impossible to achieve such genetic gain by conventional animal breeding systems. Based on recent advances with regard to in vitro induction of germ cell from pluripotent stem cells, herein we propose a novel embryo-stem cell breeding system. Distinct from the conventional breeding system in farm animals that involves selecting and mating individuals, the novel breeding system completes breeding cycles from parental to offspring embryos directly by selecting and mating embryos in a dish. In comparison to the conventional dairy breeding scheme, this system can rapidly achieve 30–40 times more genetic gain by significantly shortening generation interval and enhancing selection intensity. However, several major obstacles must be overcome before we can fully use this system in livestock breeding, which include derivation and mantaince of pluripotent stem cells in domestic animals, as well as in vitro induction of primordial germ cells, and subsequent haploid gametes. Thus, we also discuss the potential efforts needed in solving the obstacles for application this novel system, and elaborate on their groundbreaking potential in livestock breeding. This novel system would provide a revolutionary animal breeding system by offering an unprecedented opportunity for meeting the fast-growing meat and milk demand of humans. As the main dietary protein sources, meat and milk production requires approximately 70–80% increase relative to current levels , 2 in orDuring the past decades, researchers have made encouraging progresses in improving animal breeding efficiency and have realized many proposed concepts. From 1990’s, several landmark studies , 13 concRecent advances in stem cell biology offer an unprecedented opportunity for revolutionizing the animal breeding system. Using pluripotent stem cells (PSCs), including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), germ cells can be induced in vitro to complete the entire gametogenesis processes and form functional spermatids or oocytes , 18. In This renewed in vitro life cycle perpetuates the direct cross-generational transmission of genetic information from parental embryos to offspring embryos (E-to-E), distinct from the natural cross-generational transmission from parental individuals to offspring individuals (I-to-I) Fig. . In vitrThe animal embryo-stem cell breeding system completes a livestock breeding scheme in a dish by integrating in vitro germ cell induction, IVF, genome sequencing, and genomic selection. Based on the in vitro reconstituted life cycles, an animal breeding cycle can be renewed by directly selecting and mating embryos rather than adult individuals, thereby achieving rapid genetic improvement of important economic traits.Similar to the conventional breeding system, the stem cell-embryo breeding system also needs to first create a breeding scheme based on market demand and genetic resources of the breeding herd and then establish a platform for genomic selection or use an established platform. The breeding value of each individual should be evaluated and elite candidates will be selected to establish a base breeding population Fig. , Part A.Using the sperms and oocytes from individuals with the best breeding values in the breeding population, IVF will be performed to generate male and female base embryos according to the breeding scheme. Genomic estimated breeding values (GEBV) will be evaluated for all base embryos. Embryos with the top GEBV will be used as parental embryos to establish a nuclear breeding population of elite embryos Fig. , Part B.Selective breeding of parental embryos: Single cells (blastomeres or trophectoderm cells) will be isolated from parental preimplantation embryos and they will be genotyped by whole-genome sequencing. The genotyping results will be used to predict the embryo genomic estimated breeding value (eGEBV) by which elite parental embryos will be selected as candidates for controlled mating.Controlled mating of parental embryos: Selected parental embryos will be used to derive ESCs, which will be subsequently induced in vitro to form sperm or oocytes. Based on the eGEBV-based breeding scheme, in vitro derived gametes will undergo IVF to generate offspring embryos to complete the controlled mating of parental embryos.Transgenerational breeding of embryos: Following selective breeding and controlled mating of the parental embryos, the offspring embryos will undergo a new round of selective breeding and controlled mating, which in turn starts a new selection cycle Fig. , Part C.This step includes three essential breeding components similar to the conventional breeding system: selective breeding of parental embryos; controlled mating of parental embryos; and multigenerational breeding of embryos.The generation interval of the embryo-stem breeding system spans from parental embryos to offspring embryos, involving ESC derivation, in vitro germ cell induction, and IVF. It should be mentioned that ESCs are more preferred for constructing transgenerational breeding cycles. In contrast, the use of iPSCs, or germline-potential stem cells, will prolong the breeding cycle because differentiated fetal or adult somatic cells are needed. The entire breeding cycle is independent of the lengthy processes of pregnancy and postnatal growth. Aside from the classic major factors of a conventional breeding system, this system is characterized by direct selection and mating of candidate embryos, followed by an E-to-E breeding cycle, entirely distinct from individual-based conventional breeding selection. A detailed comparison between the embryo-stem cell breeding system and conventional breeding system is summarized in Table During the transgenerational breeding of embryos, the reference population can be updated as frequently as needed. In general, individual production performance and genome-wide single-nucleotide polymorphisms (SNPs) are required to construct original reference population under the breeding plan guidelines as discussed in detail previously . The pro=σg), selection intensity (i), selection accuracy (r), and the generation interval (L) [.Key factors affecting genetic gain  . Genomicrval (L) . CompareThe generation interval is about 5–7 years for sire(s) or dam(s) of bulls in the conventional dairy breeding scheme. This can be drastically reduced to approximately 2.5 years by applying genomic selection . HoweverIVF makes it possible to produce 100,000 or 1,000,000 embryos at the same time, which is equivalent to that of 100,000 or 1,000,000 of selected individuals. We can design the best sequencing strategy for genomic selection to achieve the best selection progresses in considering the breeding cost and genetic improvement.Monotocous animals, such as cows and ewes, naturally produce only a few offspring in its lifetime. The elite females cannot produce enough offspring as needed, even if some IVF technologies can assist females to have more offspring. If we overcome the obstacles in stem cell biology of farm animals and apply them in this system, monotocous females will make much more genetic contribution than conventional breeding program. Thus, the breeding system will introduce more genetic variations to the breeding population, especially for monotocous animals.The breeding system provides easy access to the latest technologies for further improvement of the in vitro breeding system because it relies on manipulation of embryos and ESCs that can be performed in a dish. For example, more sophisticated genome editing can be integrated into the system. Harmful mutations within the population can be eliminated via whole-genome sequencing and genome editing. Promotion of alleles by genome editing (PAGE) combined with genomic selection can be 1.08–4.8 times more efficient than genomic selection itself .The proposed novel embryo-stem cell breeding system is mainly based on the recently developed technologies for in vitro germ cell induction and the established routines including IVF, genome sequencing, and genomic selection. Recently, functional haploid male and female gametes have been successfully induced in vitro in mice. These works provide a robust paradigm for achieving in vitro germ line induction in farm animals, and could make the proposed breeding system technically feasible, although a series of obstacles need be overcome. A recent study reported that stable bovine ESCs can be efficiently derived from bovine blastocysts, which offers a technical basis for further establishment of in vitro germ cell induction in farm animals . Here, wPrdm1, also known as Blimp1) and developmental pluripotency–associated 3 , Hayashi et al reported that the combination of bone morphogenetic protein 4, leukemia inhibitory factor  and stem cell factor are highly competent for inducing PGC marker expression in epiblast-like cells (EpiLCs); these cells in turn become PGC-like cells (PGCLCs) to facilitate in vitro induction of PGCs  is more frequent than that from in vivo germline differentiation . Although de novo mutations induced by the manipulation of pluripotent cells have minimal contributions to the reference sites of genome selection, the biological significance and potential application as well as the risk of de novo mutations should be re-evaluated based on offspring phenotypes.From the feasibility perspective, a relative low-frequent but noticeable de novo generation of single-nucleotide variants (SNVs) can be elicited in the proposed breeding system, along with the derivation culture and passage of ESCs, especially by the induced reprogramming of iPSCs , 51. ForIVF is the process of creating embryos from oocytes by fertilizing them with sperm cells in a dish. A broader definition of IVF in cattle industry often involves oocytes retrieval from the ovaries, including recovery and in vitro maturation of oocytes, and in vitro fertilization and culture of embryos. The high-efficient IVF methodology is an important component of embryo-stem cell breeding system to support large-scale production of highly competent embryos for ESCs derivation. According to data from International Embryo Transfer Society (IETS), global production and transfer of IVF bovine embryos increased over 10-fold during the past decade. In 2015, over 60,000 embryos were produced in vitro and approximately 40,000 were transferred globally, contributing to ~ 50% of total transferred embryos . Large iCompared with in vivo conceived embryos, the IVF embryos often have compromised developmental potential, particularly in certain domestic species. By using standard or chemically defined culture conditions in combination with different growth factors during oocyte maturation or embryo culture, e.g., colony-stimulating factor, bone morphogenetic protein 15, LIF, natriuretic peptide type C, and/or biologically active small molecules, e.g., 3-isobutylmethylxanthine, 5-aza-2′-deoxycytidine, the efficiency in producing IVF embryos and their developmental potential have been substantially improved –59. ThusSingle-cell genomic DNA amplification technology has been fully established and can sequence the genome of various species –62. ThusGenomic selection is a milestone in animal breeding. Compare to conventional animal genetic selection programs that use individual GEBV, an important feature of our proposed system is to use embryonic GEBV instead. Numerous theoretical breeding studies and applications have confirmed that the GEBV can replace the conventional pedigree-based estimated breeding value entirely as long as the reference population, number of markers, and prediction equation meet the basic requirements , 65, 66.However, the accuracy of genomic selection might decrease as multiple generation selection using the same referene population. At first several generations, our proposed breeding system can still achieve high accuracy of selection as the large reference population and whole genome variations will be used. For later generations, we can add production population phenotypes in the reference population to maintain the accuracy of GS Fig. . During As the significant shorter generation interval for this E-to-E breeding system, it is possible that some detrimental mutations would accumulate in the embryo breeding populations before more phenotypes show up. This should be carefully considered when executing the breeding program. Efforts should be taken to reduce the potential damages of harmful mutations by considering all known mutations, and also develop more powerful prediction for these new mutations. Ineed, several algorithms such as SIFT, PolyPhen-2, and CADD, and EVmutation are available to estimate the mutation effects . More bioinformatic analysis may need to be included in the GS pipeline for novel mutations.Compared to whole-genome sequencing, chip-based genotyping techniques are limited in detecting insertions/deletions (indels) and CNVs. Therefore, whole-genome sequencing data can yield more informative genetic variations and can further improve the accuracy of genomic selection . Except As the number of traits, SNPs, and candidate breeding animals and size of reference population will continue to increase and simultaneous whole-genome sequencing of thousands or more individuals produces massive data, it is possible that computation time will be an important limitation in commercial breeding programs. Continuous optimization will be required for reducing computation expenditure for genomic selection analysis–related processes, such as analysis of massive sequencing data, haplotyping, imputation, and model selection. A robust and scalable data handling and analysis pipeline will be desired for these sequencing data and phenotypic data.The embryo-stem cell breeding system has significant advantages compared to the conventional breeding system, especially in shortening generation interval, increasing the number of female monotocous offspring, and selection intensity. Taking breeding dairy cows as an example, ideally, our envisioned system is expected to be 30–40 times more efficient in comparison to the conventional system, meaning that 1-year genetic gain of in vitro breeding can be the same as that of 30–40 years of conventional breeding.The establishment of in vitro germ cell induction, as well as the generation of subsquent embryos and offspring in farm animals, remain the most fundamental challenges for creating an embryo-stem cell breeding system. High-quality and stable ESC lines are prerequisite for achieving in vitro germ cell induction. During the preparation of our manuscript, a recent study reported the efficient derivation and stable propagation of bovine ESCs, and this provides us an unshakeable confidence for constituting the proposed breeding system. However, huge effort remains to be required since high-quality ESCs have not been proven in pigs or other domestic species. As well, even using the recently-reported pluripotent ESCs, the in vitro germ line induction in bovines will be a great challenge. In addition, the improvement and optimization of IVF and genomic selection technologies, highlighting their integration in the embryo-stem cell breeding system, are also needed.Farm animal populations harbor numerous genetic variations with phenotypic effects and thus serve as a unique model for understanding the genetic basis of phenotypic diversity. The breeding practice of our system will extend the understanding of genetic basis, e.g. genetic transmission, recombination, and variance under in vitro-reconstituted E-to-E life cycle. Based on our breeding system, one can create an embryo-stem cell conservation system of endangered animals. Many of the endangered or rare species encounter a notable difficulty in propagation due to poor fertility, especially for those with a long generational interval and a small litter size. In our proposed system, the population of these species can be quickly expanded and live offspring can be obtained through embryo transfer as needed Fig. . Lastly,


OBJECTIVES/SPECIFIC AIMS: Biostatistics and Epidemiology courses within clinical research or public health training programs are typically developed and taught separately. As a result, students may have trouble in their research outside the classroom, where biostatistical and epidemiological concepts must be well integrated. Case method teaching is a participant- and discussion-centered pedagogical approach that has been used in business and law schools for more than 50 years to improve student learning, yet has taken longer to be adopted in health professional schools. The case method is distinguished by presenting learners with a real-world problem without a single unique solution. Designed to mimic the constraints and incomplete information found in real life, it is an ideal approach for integrating multiple related disciplines. A team of Clinical and Translational Science (CTS) faculty from the Tufts CTSI collaborated to develop a new course that integrates epidemiology and biostatistics disciplines using the case method. METHODS/STUDY POPULATION: We developed an intermediate-level, case-based course integrating epidemiology and biostatistics topics using modern, real-world clinical examples. Recognizing the importance of technical skill building, this intermediate-level Tufts CTS course adopted a hybrid approach, incorporating lecture and in-class laboratory exercises, alongside cases. We surveyed CTS faculty to identify a set of core methodological competencies. These included randomized trials, case-control and cohort studies, confounding, effect modification, propensity scores, linear and logistic regression, and survival analysis. Faculty provided us with clinical questions and deidentified data sets corresponding to these competencies; we also reviewed publicly available data sets. RESULTS/ANTICIPATED RESULTS: CTS faculty collaborated to develop 10 cases (with accompanying data sets) from modern clinical research examples that illustrate the connections between epidemiology and biostatistical concepts. Each case contains a background section, a statement of the core problem, a data set with data dictionary, articles from the primary literature (often the publication of the data set) with discussion questions and in-class lab exercises (R programming). One case presents students with the challenge of whether acupuncture may be an effective therapy for pain associated with chronic headache. Through case activities, students gain experience weighing observational Versus experimental evidence, apply directed acyclic graph theory, and analyze clinical trial data. Qualitative evaluations in 2015 (pilot year) and 2016 indicate students preferred the integrated approach to separate courses, and found the integration facilitated application of methods to their independent research projects. Significant rewards for faculty include cross-disciplinary collaboration, sharpened teaching skills, and engaging with learners in a dynamic classroom environment. DISCUSSION/SIGNIFICANCE OF IMPACT: Despite administrative and pedagogical challenges, a case-based, integrated curriculum offers rewards for faculty and students. The case method may be a useful pedagogical strategy to integrate other closely related topics or courses in translational science to better prepare scholars for the challenges of independent research.


Collectively, our findings provide a novel molecular framework for how ceramides execute their widely acclaimed anti-neoplastic activities.Ceramides are central intermediates of sphingolipid metabolism that can activate a variety of tumor suppressive cellular programs, including cell cycle arrest, senescence and apoptosis. Indeed, perturbations in ceramide generation and turnover are frequently linked to cancer cell survival and resistance to chemotherapy. Consequently, the potential of ceramide-based therapeutics in the treatment of cancer has become a major focus of interest. A growing body of evidence indicates that ceramides can act directly on mitochondria to trigger apoptotic cell death. However, molecular details of the underlying mechanism are scarce. In our recent study (Dadsena S  The release of cytochrome c into the cytosol is a point of no return in the suicide program, initiating activation of caspases that execute an ordered self-destruction of the cell. Consequently, how a rise in mitochondrial ceramides triggers the release of cytochrome c is a hotly debated issue. One model postulates that ceramides can self-assemble into channels in the OMM that are large enough to enable passage of cytochrome c. An alternative view is that ceramides form lipid microdomains into which the pro-apoptotic Bcl2 protein Bax inserts and oligomerizes into a cytochrome c-conducting pore. In addition, some experiments with purified mitochondria indicate that ceramides may require metabolic conversion to gain apoptogenic activity.Sphingolipids are unusually versatile membrane components that participate in mechanical stability, signaling, and molecular sorting. As the path to their production is paved with toxic intermediates, cells must monitor sphingolipid biosynthesis closely to avoid jeopardizing their viability. Notably, ceramides, the direct precursors of all complex sphingolipids, are potent mediators of cell cycle arrest, stress signaling and apoptosis. Ceramide engagement in apoptosis is clinically relevant. Many anticancer regimens, including ionizing radiation and chemotherapeutic agents, have been shown to trigger ceramide-mediated cell death. While these findings raised considerable interest in targeting ceramide-metabolic enzymes for cancer therapy, mechanistic insights into how ceramides exert their tumor suppressor activities are limited. Converging lines of evidence indicate that ceramides can act directly on mitochondria to promote permeabilization of the outer mitochondrial membrane (OMM) for cytochrome de novo on the cytosolic surface of the endoplasmic reticulum (ER). In mammals, the bulk of newly synthesized ceramides is transported to the Golgi by a ceramide transfer protein, CERT, for metabolic conversion into sphingomyelin (SM) by a Golgi-resident SM synthase. Thus, newly synthesized ceramides largely bypass vesicular trafficking to reach the Golgi, where they run in a metabolic trap set by SM synthase. An obvious advantage of this arrangement is that it allows cells to readily clear the ER of a potentially toxic class of metabolic intermediates that are synthesized there. To study the consequences of diverting the biosynthetic flow of ceramides to mitochondria, we designed a CERT variant equipped with an OMM anchor, mitoCERT . We found that cells expressing mitoCERT import newly-synthesized ceramides into mitochondria and undergo Bax-dependent apoptosis. Apoptosis induction by mitoCERT was blocked by mitochondrial targeting of a bacterial ceramidase, indicating that apoptogenic activity relies on intact ceramides rather than any downstream metabolic intermediate of ceramide turnover. These results confirmed that a rise in mitochondrial ceramides suffices to activate Bax and trigger apoptosis.Most previous studies on ceramide-activated cell death pathways relied on the application of truncated ceramide analogues, isolated mitochondria and/or the treatment of cells with apoptotic stimuli that affect ceramide pools in multiple organelles. To circumvent some of the drawbacks of these approaches, we have begun to exploit lipid transfer proteins as experimental tools. By mediating lipid transport at contact sites between two organelles, these proteins enable lipids to reach their destination independent of vesicular trafficking. Ceramides are synthesized Ka valuethat is closely tuned to the physiological pH of the cytosol. In its deprotonated fully charged state, this residue could be seen to mediate direct contact with the ceramide head group. Protonation, substitution or chemical modification of the Glu residue in each case abolished photolabeling of both channels with the ceramide probe. Moreover, photolabeling was progressively reduced when carried out in excess of native ceramides, indicating that the charged membrane-buried Glu is part of an authentic ceramide binding site. To address whether VDACs play a role in ceramide-induced apoptosis, we next determined the impact of VDAC removal on mitoCERT-mediated cell death in human HCT116 colon carcinoma cells. Unlike VDAC1 removal, loss of VDAC2 rendered the cells resistant to mitoCERT-mediated cell death. Importantly, substitution of the membrane-buried Glu in VDAC2 greatly reduced its ability to restore mitoCERT-mediated cell death in VDAC1/2 double knockout cells. Collectively, these findings qualified VDAC2 as a direct and critical effector of ceramide-induced apoptosis.The foregoing raised the question whether ceramide-mediated activation of Bax relies on direct and specific interactions between Bax and ceramides or involves other mitochondria-resident proteins. This led us to use a photoactivatable and clickable ceramide probe in a screen for mitochondrial ceramide binding proteins. Photoaffinity labeling experiments with purified mitochondria consistently revealed a 33 kDa protein that was efficiently crosslinked by the ceramide probe. Affinity purification and mass spectrometric analysis revealed that this 33 kDa protein actually corresponds to two proteins, namely the voltage-dependent anion channels VDAC1 and VDAC2. Besides mediating metabolic crosstalk between mitochondria and the cytosol, VDAC2 serves as a mitochondrial platform for Bax translocation whereas VDAC1 oligomerization has been implicated as a critical step in mitochondrial apoptosis. Molecular dynamics simulations revealed that both channels harbor a ceramide binding site buried in the membrane interior on one side of the barrel wall. This site includes a uniquely positioned glutamate (Glu) that faces the bilayer's hydrophobic core and that has a pc. Overexpression of hexokinases and their association with VDACs are typical features of hyperglycolytic cancer cells. When bound to VDACs, hexokinases enable an effective coupling between oxidative phosphorylation and glycolysis by capturing ATP released from mitochondria to phosphorylate glucose, the rate-limiting step in the glycolytic cascade. In the clinic, there is great interest in drugs that dissociate hexokinases from VDACs, as such drugs would have a dual anti-neoplastic effect. First, they would facilitate induction of apoptosis. Second, they would revert the hyperglycolytic state that allows tumors to grow under conditions where oxygen is in short supply. Our finding that ceramides bind VDACs at a site that overlaps with the binding site for hexokinases implies that ceramides may exert their tumor suppressor activities in part by influencing the interaction between VDACs and hexokinases . How could this work? Our simulations and photoaffinity labeling studies indicate that ceramide binding to VDACs is pH sensitive and controlled by the protonation state of the membrane-buried Glu. Interestingly, acidification has been shown to promote association of two VDAC1 monomers into a dimer, presumably by enabling formation of H-bonds between the Glu and a serine residue at the dimer interface. Under normal stress-free conditions, the membrane-buried Glu is in its deprotonated fully charged state at least a significant amount of time, thus keeping VDACs at the brink of oligomerization. We speculate that binding of ceramides to the negatively charged Glu may lower the threshold for VDAC oligomerization at neutral pH. Such ceramide-driven oligomerization of VDACs may then trigger apoptosis by: i) blocking binding of anti-apoptotic hexokinases; ii) creating a suitable platform for mitochondrial translocation of Bax . In sum, our recent study provides a novel molecular framework that will serve as a valuable guide to further unravel how ceramides execute their tumor suppressor activities.Previous work revealed that the charged membrane-buried Glu in VDACs is essential for binding of hexokinase I, which antagonizes cell death through inhibition of Bax-mediated release of cytochrome


The mitochondrial pyruvate carriers mediate pyruvate import into the mitochondria, which is key to the sustenance of the tricarboxylic cycle and oxidative phosphorylation. However, inhibition of mitochondria pyruvate carrier-mediated pyruvate transport was recently shown to be beneficial in experimental models of neurotoxicity pertaining to the context of Parkinson’s disease, and is also protective against excitotoxic neuronal death. These findings attested to the metabolic adaptability of neurons resulting from MPC inhibition, a phenomenon that has also been shown in other tissue types. In this short review, I discuss the mechanism and potential feasibility of mitochondrial pyruvate carrier inhibition as a neuroprotective strategy in neuronal injury and neurodegenerative diseases. Regulatory nodes of energy metabolism have emerged as potential therapeutic targets in multiple human diseases such as cancer, aging and neurodegeneration ,4,5,6,7.In view of the above, it may, therefore, be somewhat non-intuitive but interesting that the inhibition of pyruvate transport by MPC was recently shown to be beneficial in experimental models of neurotoxicity, particularly in the context of Parkinson’s disease (PD) models , and is SLC25A gene family  glucose tracer found in TCA cycle intermediates were significantly diminished by UK-5099. The reciprocal regulation of pyruvate and glutamate oxidation could also be discerned to some degree in UK-5099 treated organotypic hippocampal slice cultures with mixed neural cell types. A profiling of metabolites and TCA cycle intermediates showed, among expected changes in citrate and alanine, UK5099 treatment reduced total glutamate levels by half, whereas there was a twofold increase in aspartate. This suggests an increased in oxidation of glutamate (or glutamine), which could occur via aspartate aminotransferase or oxidative deamination by glutamate dehydrogenase ,88, and +/K+-ATPase) and an N-methyl aspartate (NMDA) pulse. Furthermore, cortical neurons treated with UK-5099 were protected from excitotoxic cell death by glutamate added in culture medium in a non-additive manner to the protective effects of the excitotoxicity-mediating NMDA receptor antagonist MK801. Post-synaptic currents in neurons treated with UK-5099 also produced a smaller current density peak in excitatory postsynaptic current elicited by a hypertonic sucrose shock. Together, these findings therefore suggest that enhancing glutamate oxidation by MPC inhibition could indeed attenuate the glutamate pool released upon depolarization. Perhaps the most intriguing finding of Divakaruni and colleagues is that this glutamate oxidation promoted by MPC inhibition could ultimately be neuroprotective. Glutamate is an excitatory neurotransmitter, and its uncontrolled release during massive glutamatergic neuron depolarization, resulting from acute oxygen-glucose depletion accompanying ischemic or traumatic injury, mediates excitotoxic neuronal death. Use of neuronal glutamate for oxidation may conceivably decrease the glutamate released upon depolarization, and could thus limit excitotoxic neuronal injury. Indeed, neurons treated with UK-5099 released less glutamate extracellularly upon depolarization induced by veratridine (which prevents sodium channel closure), ouabain , thus feeding into the TCA cycle   between CA cycle . A switcThe data of Divakaruni and colleagues appear to suggest that the synaptic glutamate pool of glutamatergic neurons may plausibly equilibrate with that of the mitochondria, thus increasing mitochondrial glutamate. How this might occur is not particularly clear. The mitochondrial glutamate carrier 1 (SLC25A22)  is mainlGiven the critical role of MPCs in mammalian development and the problems of patients harboring MPC mutations, MPC inhibition might be perceived as being overly detrimental for cellular and organismal energetics and survival to be of benefit. However, as the findings above in liver, muscle and neurons have demonstrated, genetic ablation or pharmacological inhibition of MPC activities could be contextually beneficial in diseased conditions. This is very much down to the previously underappreciated metabolic adaptability exhibited under the condition of mitochondrial pyruvate transport inhibition. As far as neuropathological conditions are concerned, a metabolic shift to the usage of glutamine/glutamate that would reduce glutamate release and excitotoxicity, as well as the possibility of mTOR pathway inhibition that could lead to elevated autophagy and suppression of neuroinflammation, would constitute neuroprotective benefits. The reprogramming of mitochondrial metabolism resulting from MPC inhibition could therefore provide context-dependent benefits under neuronal disease conditions, which could potentially also be extended to diseases in other organs or tissues.There are also indications that MPC inhibition could be beneficial in other neuropathological conditions. TZDs have been shown to be beneficial for a range of neurodegenerative diseases, including Alzheimer’s disease (AD) ,104,105,What then, are the clinical prospects of MPC inhibition in neuroprotection? TZDs which are standard diabetic drugs with ample clinical testing already done, have been and should be further investigated in trials . The aerAs an epilogue, therefore, findings in neuroprotection resulting from MPC inhibition are intriguing. Although there is much more to be learned before the underlying mechanisms are fully explained and the strategy could be implemented effectively and safely, the translational potential is fairly obvious.


In critically ill children admitted to pediatric intensive care units (PICUs), enteral nutrition (EN) is often delayed due to gastrointestinal dysfunction or interrupted. Since a macronutrient deficit in these patients has been associated with adverse outcomes in observational studies, supplemental parenteral nutrition (PN) in PICUs has long been widely advised to meeting nutritional requirements. However, uncertainty of timing of initiation, optimal dose and composition of PN has led to a wide variation in previous guidelines and current clinical practices. The PEPaNIC  randomized controlled trial recently showed that withholding PN in the first week in PICUs reduced incidence of new infections and accelerated recovery as compared with providing supplemental PN early (within 24 hours after PICU admission), irrespective of diagnosis, severity of illness, risk of malnutrition or age. The early withholding of amino acids in particular, which are powerful suppressors of intracellular quality control by autophagy, statistically explained this outcome benefit. Importantly, two years after PICU admission, not providing supplemental PN early in PICUs did not negatively affect mortality, growth or health status, and significantly improved neurocognitive development. These findings have an important impact on the recently issued guidelines for PN administration to critically ill children. In this review, we summarize the most recent literature that provides evidence on the implications for clinical practice with regard to the use of early supplemental PN in critically ill children. Optimal nutritional support is considered of paramount importance for critically ill children admitted to the pediatric intensive care unit (PICU), since malnutrition and inadequate nutrient delivery have been associated with worse clinical outcome ,2. Moreon = 4640) randomized controlled trial (RCT) showed that withholding supplemental PN until day eight of an ICU stay (late PN), and thus accepting a substantial macronutrient deficit, was associated with fewer ICU infections, a shorter duration of mechanical ventilation and renal-replacement therapy and a shorter ICU and total hospital stay as compared with initiating supplemental PN early (within 48 hours after ICU admission) [Several observational studies have shown that malnutrition is associated with worse clinical outcome ,2,11. A mission) . Data gemission) . Since tmission)  that invmission) . This lamission) , raisingmission) ,17,18. Tmission) . Analysemission) ,15. Moremission) . Undernomission) . The benefits of withholding supplemental PN during the first week in the PICU appeared not only present from a clinical point of view, but also from a health–economic perspective. A cost-effectiveness study indeed showed that the total direct medical costs were considerably lower with late PN as compared with early supplemental PN initiation . This coA possible limitation of the PEPaNIC RCT is the use of standard equations for the estimation of energy requirements instead of indirect calorimetry . HoweverApart from the PEPaNIC RCT, no other randomized controlled trial investigating the use or timing of supplemental PN in critically ill children has been published in the last eight years. A limited number of observational studies on the use of supplemental PN and over- and underfeeding in PICUs showed different results ,24,27. AAlthough extensive guidelines on the composition of PN in critically ill children are available , a recenBecause of new insights in diagnostic and therapeutic measures in the field of pediatric critical care medicine , centralSeveral mechanisms may contribute to an adverse clinical impact of administering PN in the early phase of critical illness in children. As early PN has affected both short-term and long-term outcomes of patients, carryover effects persisting in the long term must play a role. As discussed, it was the administration of more amino acids that was associated with adverse short-term outcomes evoked by early PN in the PEPaNIC study . The mecUnderlying mechanisms of the long-term harm caused by early PN in the context of critical illness remain largely unraveled. The clinical benefits of late PN observed far beyond the intervention window suggests that early PN induces carry-over “memory” effects with a negative impact on long-term outcome. Poor long-term outcomes in other conditions have been related to accelerated telomere shortening and the induction of aberrant so-called “epigenetic” changes. Importantly, inadequate nutrition may cause both adverse effects. Hence, these processes may also play a role in the developmental impairment of critically ill children and the adverse impact of early PN on neurocognitive development. Telomeres are nucleoprotein complexes at the end of human chromosomes that shorten with each cell cycle. Telomere shortening can be accelerated by environmental and lifestyle factors ,65, inclThe involvement of aberrant epigenetic changes in long-term consequences after acute events in life appears plausible. Epigenetics refers to the study of heritable changes in gene expression that do not involve changes in the underlying DNA sequence. Epigenetic changes play an important role in physical and neurocognitive development ,70,71,72Nutritional support is considered to be an important aspect of patient care in PICUs , with la


Borderline personality disorder (BPD) is a serious psychiatric disorder that affects multiple symptomatic domains and is associated with an increased risk of suicidality. Several empirically supported treatments for BPD have been developed in recent years for adults with BPD. More recent work has focused on tailoring or applying (or both) these existing treatments to specific patient populations, including patients with certain types of comorbidity  and younger patients. Other work has involved developing treatments and models of treatment delivery that address concerns related to access of care. Relatedly, new adjunctive and technology-assisted interventions have been developed, adding to the growing repertoire of treatment options for these patients. Advances in the last several years address specific treatment needs and offer cost-efficient options for this diverse patient population. A number of specialized treatments, including dialectical behavior therapy (DBT), mentalization-based therapy (MBT), and transference-focused psychotherapy (TFP), have been developed over the past few decades and have been found to be similarly efficacious in recent meta-analytic work3. Within the past few years, additional interventions for BPD have been developed, and specific foci are targeting comorbidity common to patients with BPD, improving access to care, tailoring treatments to younger patients, and developing adjunctive or web-based interventions to further help these patients in a cost-efficient way.Borderline personality disorder (BPD) is a serious mental illness that is characterized by difficulties in affect regulation, interpersonal relationships, and impulsivity and is associated with an elevated risk of suicidal behavior5. Two other recent studies have examined the efficacy of two existing treatments for BPD—MBT and Systems Training for Emotional Predictability and Problem Solving (STEPPS)—for treating BPD and comorbid antisocial personality disorder (ASPD)7. Additionally, there is emerging evidence that using a transdiagnostic approach to treatment may be beneficial for patients with BPD and mood/anxiety disorders8.Several newly developed and evaluated psychosocial treatments for BPD include adaptations of existing treatments to target both BPD and commonly co-occurring conditions. These treatments include two adaptations of DBT for patients with BPD (or BPD features) and comorbid post-traumatic stress disorder (PTSD)et al.9 developed a treatment that combines standard DBT with prolonged exposure (PE), an evidence-based treatment for PTSD10. An initial study of this treatment, named DBT-PE4, found that patients with comorbid BPD, PTSD, and acute suicidality or self-injury who received DBT-PE had higher rates of remission from PTSD than patients who received DBT alone . Patients who completed DBT-PE, compared with standard DBT, also exhibited improvements in other symptomatic domains  of moderate to large effect sizes. Additionally, across both treatments, improvements in PTSD symptoms and post-traumatic cognitions predicted improvements on multiple functional domains9, suggesting a potential mechanism through which this treatment operates.With respect to the treatment of BPD with comorbid PTSD, two treatment approaches using DBT have been tested recently. Harnedet al.5, was designed for patients experiencing PTSD following childhood sexual abuse as well as difficulties with emotion dysregulation (including BPD). This modular, 12-week residential treatment combines elements of DBT with other trauma-focused exposure and cognitive approaches11. Patients who received DBT-PTSD, compared with treatment as usual, showed greater improvements in PTSD symptomatology, global social functioning, and depression. They also seemed to tolerate the treatment well.  Additionally, level of initial BPD symptomatology was not a predictor of treatment course.Another modification of DBT, developed by Bohus6. MBT aims to help patients better understand their own and others’ mental states and intentions. The developers expected that engaging in this kind of active thinking about others’ intentions rather than acting impulsively on the basis of initial instincts could reduce violence and other antisocial behaviors. Results from one investigation supported this idea, finding that patients with BPD plus ASPD treated with MBT showed greater improvements in ASPD-related behaviors than patients receiving similar outpatient treatment without MBT. These patients also showed reductions in anger, hostility, paranoia, self-harm, and suicide attempts as well as improvements in mood, general psychiatric symptoms, and interpersonal/social functioning when compared with patients receiving care as usual.Researchers have also adapted existing psychotherapies with empirical support for BPD to treat comorbid personality disorders, particularly ASPD. One such treatment has been an adaptation of MBT for patients with BPD and comorbid ASPD7. This treatment includes components of psychoeducation, cognitive-behavioral therapy, and skills training12. With respect to comorbid ASPD, two recently conducted studies  demonstrated that patients with BPD plus ASPD who received STEPPS  showed greater improvement on a variety of domains, including BPD symptomatology, general psychiatric symptoms, impulsiveness, positive affect, and positive/negative behaviors. These findings, though somewhat counterintuitive, suggest that patients with ASPD can benefit from evidence-based treatments and should not be excluded from these programs.Another form of treatment that has been adapted to treat BPD comorbid with ASPD is STEPPS, an evidence-based adjunctive group treatment for BPD13 for patients with BPD8. This treatment is generally 16 to 20 outpatient sessions and is focused on the core negative emotional processes that underlie BPD and other conditions . A preliminary study by Sauer-Zavalaet al.8, which involved a clinical replication series of five patients with BPD and comorbid mood and anxiety disorders, yielded promising results, finding that most patients experienced clinically significant reductions in symptoms of BPD, depression, and anxiety as well as improvements in emotion regulation skills.Another new treatment uses an established transdiagnostic approach to treat BPD. This treatment involves applying Barlow’s Unified Protocol for the Transdiagnostic Treatment of Emotional Disorderset al.14 and Paris15 proposed and evaluated a “stepped care” model for treating BPD, which rests on the idea that some patients—particularly those with disorders for which outcomes are variable—may benefit from briefer interventions but that other patients may require more intensive treatment. Laporteet al. note that there is already evidence that patients with BPD can benefit from short-term (ST) versions of evidence-based treatments17. In their study, patients were offered an initial “step” of ST treatment, which consisted of 12 weeks of integrative therapy delivered in individual and group modalities. If patients who received ST did not improve or needed additional treatment, they were referred to an extended care (EC) clinic after a waiting period. Patients received EC, which consisted of two types of group therapy (one similar to ST and one based on group process), individual therapy, and psychopharmacology; treatment lasted for up to 2 years. Patients who attended the ST clinic exhibited significant declines in major BPD symptom areas , suggesting that brief treatment may be viable for many patients with BPD. Patients who continued on in EC also showed significant declines in these symptom areas.Other recent developments have focused on how to improve access to care for patients with BPD, including addressing long wait times for treatment in the community. Laporte18, which integrates evidence-based principles for treating BPD into a treatment that includes psychotherapy, case management, and symptom-targeted pharmacotherapy. GPM was originally tested as a condition in a large, randomly controlled trial in which it was compared with DBT19. In this study, patients were randomly assigned to either 1 year of GPM or 1 year of DBT. GPM performed as well as DBT, and the two groups showed similar levels of improvement in suicidal and self-injurious behavior, BPD symptoms, and level of health-care utilization. Additionally, these gains were largely sustained at 2-year follow-up20.In addition to the stepped-care model, treatment approaches have been described that can be incorporated into the practice of community practitioners with little deviation from their typical work. One such treatment is general psychiatric management (GPM)In addition to standard psychotherapies for BPD and related conditions, some adjunctive treatments and technologically based interventions have been developed recently to help patients with BPD. These include two group-based adjunctive treatments with specific foci as well as web-based interventions.et al.21 developed a 14-week group therapy, called emotion regulation group therapy (ERGT), which was designed to be an adjunctive treatment for patients with BPD and recurrent deliberate self-harm (DSH). An initial evaluation of this treatment found that women with BPD and recurrent DSH who participated in ERGT show improvements on DSH, other self-destructive behaviors, emotion dysregulation, BPD symptomatology, depression, and quality of life post-treatment, and additional improvements were observed by 9-month follow-up. Changes in symptoms and problematic behaviors appear to be mediated by changes in emotion regulation22, lending support to the proposed mechanism. Furthermore, patients who exhibited greater severity in multiple relevant domains as well as greater levels of comorbidity showed a greater response to treatment, suggesting that the type of patient who is likely to be referred to ERGT is also likely to benefit from it23.Gratzet al.24 developed a module based on forgiveness to be added to outpatient DBT. In one study, participants who completed this module showed improvements in forgiveness and decreases in psychiatric symptoms and attachment insecurity during the module and up to 6 weeks after. Similar changes were not observed following the preceding module of distress tolerance.A different adjunctive treatment module was developed to enhance patients’ strengths. Specifically, Sandageet al.25, has also been shown to be beneficial for patients with BPD in a randomized controlled trial. This program included regular web-based assessments and the completion of an online curriculum about BPD. During the first 12 weeks of the study, patients who completed the psychoeducation program showed greater improvement in impulsivity and overall functioning when compared with participants who did not complete the program. During the maintenance phase of the trial , those who had been randomly assigned to read the curriculum showed greater improvements in all BPD symptom domains and overall severity of BPD symptoms compared with those who did not. Overall, these findings suggest that web-based psychoeducation is a promising and cost-efficient early intervention or adjunctive treatment for those newly diagnosed with BPD.A newly developed internet-based psychoeducation program, developed by Zanarini26, are currently being tested. This intervention will combine elements of psychoeducation with self-paced interventions/exercises and is intended for patients who are also receiving care as usual.Other web-based interventions, including the REVISIT trial, a web-based adjunctive treatment based on schema therapy27 and that symptomatology during this developmental period is qualitatively different from experiencing a tumultuous period of normal development. In a recent commentary, Chanen and Thompson28 stressed the importance of intervention in early stages of the disorder and advocated for a “clinical staging” approach in treating BPD in younger populations, like the stepped-care plan that has been evaluated in adults15: starting with simpler and more benign interventions in early stages of the disorder and increasing intensity if the disorder progresses.There has also been growing attention regarding earlier treatment of BPD, particularly in adolescence. There is ample evidence to suggest that the diagnosis of BPD is valid in adolescence29 has been developed for adolescents with repeated self-harm and suicidality. This treatment follows a format similar to that of standard DBT, and individual therapy is combined with skills group and skills coaching; however, families are included in skills groups, and there is a separate family therapy component. In one trial29, DBT-A was found to be superior to enhanced usual care in reducing self-harm, suicidal ideation, and depressive symptoms. Additionally, an adolescent form of MBT (MBT-A)30 was developed and tested for adolescents with self-harm. Like DBT-A, MBT-A is similar in approach to standard MBT but also includes a monthly family therapy component and has an explicit focus on impulsivity and affect regulation. Compared with treatment as usual, MBT-A was superior in reducing self-harm and depression30. Improved mentalization and reduced attachment avoidance appeared to be key mediating factors.Some full-package treatments initially developed and evaluated in adults with BPD have been adapted for adolescents with BPD. For instance, an adolescent form of DBT (DBT-A)31, which seeks to compare the efficacy of three types of early intervention models for adolescents and young adults with BPD: (1) the Helping Young People Early (HYPE) service model plus cognitive analytic therapy, (2) the HYPE service plus a control psychotherapy condition, and (3) a general youth mental health-care model plus control psychotherapy condition. Another ongoing trial 32 is testing a group-based MBT for adolescents.There are also multiple treatments that are in development and being tested to treat BPD in adolescents. These include the MOBY trial 33, treatments that are developed with common comorbidities in mind  or the evaluation of empirically supported treatments in patients with serious comorbidities  represent important advances in BPD treatments. Specifically, these studies suggest that, despite comorbidities, patients benefit from these treatments and should be viewed as good candidates for these therapies. That being said, many full-package treatments are costly in time and money, and patients may benefit from alternative models of treatment delivery or adjunctive treatments with specific targets. Thus, the further development of staged treatment models, adjunctive treatments, and technologically based interventions26 may be particularly useful when cost-efficient interventions are needed, patients are newly diagnosed or awaiting full-package treatment (or both), or a specific deficit needs to be addressed in the context of an ongoing treatment. Additionally, given the growing evidence that BPD is a valid diagnosis in adolescence, treatments oriented at younger populations32 constitute a necessary and important advance.Recent developments in interventions for BPD address specific needs for the treatment of this population. Specifically, given that comorbidities are common in these patients


An increasing number of studies have shown that bone plays an active role in regulating glucose metabolism, affects renal, and cardiovascular diseases and even influences the development of offspring. These novel findings have indicated that bone plays a much more important role in the human body than only providing physical support. However, further investigations of the mechanisms underlying the effects of bone are needed. Recently, extracellular vesicles (EVs) have received increased attention because they can transfer functional proteins, mRNAs, and miRNAs between cells/organs. After reviewing the existing evidence, we hypothesized that bone may be involved in interorgan communication via EVs. Further research exploring bone-derived EVs may facilitate the understanding of bone as a multifunctional organ. As research has progressed, the functions of various organs have been updated. The common function of bone is traditionally believed to be that of an effector. Bone is usually a target regulated by other organs for physiological function, and to an extent, bone acts as a recipient during physiological communication. However, recent studies have proven that bone not only acts as an effector but also regulates other organs by secreting biological molecules. For example, fibroblast growth factor 23 (FGF23) secreted from osteoblasts and osteocytes can regulate phosphate and mineral metabolism , 2. BoneRecent studies have found that various organ systems can exchange information between cells through extracellular vesicles (EVs), which is a new mechanism of communication between cells and organs. Studies have found that EVs from different sources are widely involved in endocrine, tumor microenvironment, and nervous system regulation –10. ThesRecent studies have found that bone could affect multiple organs and be involved in physiological and pathological changes by secreting biomolecules , 15. NotWith the deepening of research, investigators have paid attention to EVs as biomarkers in the early stage and have gradually discovered that EVs can stably transfer proteins and genetic material, which suggests that EVs may play a potential role in physiological regulation.More importantly, the discovery of circulating EVs has given researchers new inspiration. Fruhbeis et al. found that exercise triggered a rapid release of EVs into the circulation. The dynamics of EVs varied between cycling and running exercise. The results suggest that EVs released during physical activity may participate in long-distance signaling during exercise-mediated adaptation processes . GuescinIn addition to participating in energy metabolism, circulating exosomes have also been found to be widely involved in the physiological and pathological processes of the heart. Cheng et al. found that circulating myo-miRs are carried in exosomes and mediate functional crosstalk between the ischemic heart and the bone marrow (BM). They found a higher level of circulating exosomes in acute myocardial infarction (AMI) mice that could transfer myo-miRs into BM monocular cells (MNCs). The results also showed that AMI exosomes could mediate the downregulation of CXC chemokine receptor 4 (CXCR4) and contribute to BM progenitor cell mobilization . Howeverin vivo and suppress the secretion of proinflammatory cytokines in adipocytes in vitro  levels and through that matching bone mass to body weight. The selective deletion of RANK from NPY neurons leads to a significant increase in fat mass and a decrease in whole body bone mineral density –42.As more and more bone-secreted factors were confirmed to be involved in interorgan communication, we can conclude that bone may actively participate in the regulation of physiological processes.However, there is no definitive evidence to date that bone-derived EVs could regulate remote organs. Various studies have found that bone secreted factors involved in interorgan communication could be transferred through EVs. Yi et al. found that OCN could be packed into exosomes and is able to be transferred to the aorta endothelial cells via exosome incorporation . PreviouBone is a dynamic organ that is constantly remodeled by the activities of several cells, including mesenchymal stem cells, osteoblasts, osteocytes, and osteoclasts. Many studies have found that bone-derived EVs play important roles in cellular communication in bone remodeling. In the bone-remodeling microenvironment, bone-derived EVs contain specific proteins and genetic material and are involved in regulating cell activity.−/− mice, a murine model characterized by the lack of osteoclasts. Osteoclasts in RANKL−/− mice were inactive due to the lack of pro-osteoclastic function. The osteoblast-derived EVs stimulated osteoclasts in RANKL/− mice. Osteoblast-derived EVs could recover this function by inducing TRAcp-positive cells (in vitro (in vitro and in vivo (Bidirectional osteoblast-osteoclast communication plays crucial roles in bone remodeling . Capparive cells , 47. On ve cells , 12. Mesve cells –51. Howeve cells , 53. Recve cells . Qin et in vitro . MSC-der in vivo . Likewis in vivo .Sato et al. compared osteocyte-secreted exosomes and circulating exosomes from osteocyte-less mice. The results indicated that ablation of osteocytes in mice alters the miRNA levels of plasma exosomes . This stIn conclusion, paracrine functions of bone-derived EVs are widely involved in intercellular communication and genetic material transmission in the bone microenvironment.There are still many key issues that need to be addressed to prove this hypothesis. First, current research has confirmed that bone secreted factors play a role in the regulation of brain, liver, muscle, pancreas, testis, and adipose tissue. Further research should begin with these known directions to explore whether bone-related EVs are involved in the regulation of these organs. Second, it is also important to identify the EV-mediated bone effects on the physiological functions of other organs. Current research has primarily explored the role of bone in endocrine and energy metabolism, but whether bone has effects on other systems, such as the immune system, requires further exploration.in vivo (in vitro or in vivo by injection of EVs harvested from cells in culture. The characteristics of bone-derived EVs in physiological states require further exploration. Further study should illuminate the life span, the concentration and the in vivo metabolism of bone-derived EVs in the bloodstream. As a result, these studies may not reflect the spatiotemporal properties or concentrations of EVs active in normal physiology in vivo. A direct demonstration that functional EV-mediated biological cargo transfer is the relevant mechanism in certain biological processes is still difficult to achieve (Another important problem is the lack of knowledge of bone-derived EVs. Most studies published thus far have analyzed mixed EV populations, and the most important step is to comprehensively compare the different subtypes of EVs . Furtherin vivo . Current achieve .Confirmation of EV-mediated interorgan communication of bone would help deepen the understanding of the communication networks between the skeleton and other systems. The results of existing research have shown that bone-secreted proteins could regulate interorgan communication. The growing awareness that bone is an active organ will broaden our understanding of the pathogenesis of bone in other systemic diseases. The interorgan communication function of bone mediated by EVs may become a therapeutic target for other diseases.Furthermore, confirming this hypothesis may expand our knowledge of EVs. Many studies have confirmed the widespread presence of EVs in body fluids. However, the biological functions of biofluid-derived EVs remain unclear. The current study mainly focused on EVs from body fluids for disease diagnosis, but their physiological functions still need to be revealed. In addition, the role of EVs is not only transferring functional proteins but also transferring genetic material. Confirmation of the regulation of bone-secreted EVs on multiple organs will help us better understand the transmission of genetic information. Meanwhile, as EVs have been acknowledged as natural targeted-drug carriers, EV-based nanotechnology has provided unprecedented opportunities to help develop EV-related therapeutics , 61. ResLZ and PT made major contributions to the conception of the work. YL and PY drafted the manuscript. ZG, MC, HL,YD, and YG revised 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.


Oral infections occur frequently in humans and often lead to chronic inflammations affecting the teeth , the gingival tissues surrounding the teeth , and the tooth-supporting structures . At least four basic pathogenic mechanisms have been proposed that involve oral inflammations in the pathogenesis of atherosclerosis: (1) low level bacteremia by which oral bacteria enter the blood stream and invade the arterial wall; (2) systemic inflammation induced by inflammatory mediators released from the sites of the oral inflammation into the blood stream; (3) autoimmunity to host proteins caused by the host immune response to specific components of oral pathogens; (4) pro-atherogenic effects resulting from specific bacterial toxins that are produced by oral pathogenic bacteria. In this narrative review, we summarize published experimental evidence related to these four mechanisms and discuss their impact on the pathogenesis of atherosclerosis. Atherosclerosis is a chronic inflammatory condition affecting arterial blood vessels ,2. Due tAbout 50% of MIs occur without significant previous symptoms and despite significant progress in the acute care of MI patients, the 4-week lethality after the first MI could not be lowered significantly below 50% , accountFour basic pathogenic mechanisms have been proposed that involve oral inflammations in the pathogenesis of atherosclerosis: (1) low-level bacteremia by which oral bacteria enter the blood stream and invade the arterial wall; (2) systemic inflammation induced by inflammatory mediators, which are released from the sites of the oral inflammation into the blood stream; (3) autoimmunity to host proteins which results from the host immune response to specific components of oral pathogens; (4) pro-atherogenic effects resulting from specific bacterial toxins that are produced by oral pathogenic bacteria.In this narrative review, we summarize published experimental evidence related to these four mechanisms and discuss the impact in view of the pathogenesis of atherosclerosis. Since this topic is rather broad, a comprehensive consideration of all relevant publications in this area was beyond the scope of this article. Hence, we focused mainly on recent studies published in peer-reviewed high impact journals and we do not claim the completeness of the cited literature.Oral infections occur frequently in humans and often lead to chronic inflammations affecting the teeth (caries), the gingival tissues surrounding the teeth (gingivitis and endodontic lesions), and the tooth-supporting structures (periodontitis)  and atheromas (48% positive) [Streptococcus mutans can invade vascular endothelial and smooth muscle cells in vitro and, thus, may be able to trigger endothelial dysfunction which could in turn promote atherosclerosis [Bacteria from endodontic lesions, such as ositive) . The corclerosis .Oral infections, including gingivitis, periodontitis, and endodontic lesions consistently elevate systemic levels of C-reactive protein (CRP), which is a sensitive biomarker for systemic inflammation. One of the first studies published by Boucher et al.  showed hOral inflammations increase the circulating levels of many other inflammatory markers and cytokines in addition to CRP  [Porphyromonas gingivalis, one of the pathogens causing periodontitis, expresses a prokaryotic PAD enzyme, which citrullinates not only some of its own bacterial proteins but also a number of host proteins  [Autoantibodies to citrullinated proteins represent another autoimmune mechanism potentially involved in the pathogenesis of atherosclerosis . Proteinse (PAD) ,58. Humase (PAD) . Healthyimentin) . It was imentin) ,61,62,63imentin) ) and a simentin) .Aggregatibacter actinomycetemcomitans  was recently shown to secrete 179 proteins, including cytolethal distending toxin, leukotoxin A (LtxA) and macrophage infectivity protein [Aggregatibacter actinomycetemcomitans can induce hypercitrullination of a large number of proteins in host neutrophils [Porphyromonas gingivalis were shown to secrete up to 200 proteins, including gingipains, agglutination proteins, P. gingivalis PAD, and receptor antigens [Patients with chronic oral inflammations are exposed to a complex mixture of bacterial components. Lipopolysaccharide (LPS) is an endotoxin produced by Gram-negative bacteria, which has important pro-inflammatory effects . It coul protein . Leukotx protein . When ad protein . In additrophils . The porantigens . Rgp andantigens .The presented findings support the argument that chronic oral inflammations likely affect multiple pathways involved in atherosclerosis, and that all four basic mechanisms that were proposed in this context are important. None of these mechanisms is specific for oral inflammations. However, because of their high prevalence and chronic nature, it cannot be excluded at this stage that these inflammations have a profound population-based impact on the atherosclerosis-related disease burden.The well documented enrichment of oral bacteria or their DNA in atherogenic lesions likely has profound implications. Bacteria and their DNA trigger the innate immune system by activating pattern-recognition receptors (PRRs), such as Toll-like receptors, or TLRs, and NOD proteins, which recognize so-called “pathogen-associated molecular patterns (PAMPs)” and activate multiple pro-inflammatory signaling pathways . In addiThe well-documented induction of systemic inflammation by chronic oral inflammations implies that the affected individuals are at increased risk of CVD. It could be demonstrated that elevated concentrations of CRP, IL-6 and fibrinogen are associated with increased 10-year risk of CVD ,80. RecePorphyromonas gingivalis. Humoral and cellular immunity against HSP60 is a normal feature of healthy humans, which participates in the protection against microbial infections [Activation of the HSP60 autoimmunity mechanism has been firmly established to operate in patients with periodontitis infected with fections . If endofections . Since tfections .Porphyromonas gingivalis GroEL, has so far not been addressed. Since many studies have shown an association between CVD and endodontic lesions [Whether the HSPs of endodontic pathogens elicit a similar immune response as was demonstrated for  lesions ,93,94,95Citrullinated proteins, especially fibrinogen, were identified in atherosclerotic plaques , suggestProfound technological advances in areas such as genomics, proteomics and metabolomics have recently led to the identification of a vast number of factors that are secreted by oral pathogens (“secretome”), some of which might influence the host immune system and the pathogenesis of atherosclerosis. These factors warrant further characterization with respect to their specific effects. This fascinating area of research has a high potential to identify factors that may lead to the development of drugs.In a scientific statement published in 2012, the American Heart Association concluded that “observational studies supported an association between periodontitis and atherosclerosis independent of known confounders” . However


Despite causing considerable damage to host tissue at the onset of parasitism, invasive helminths establish remarkably persistent infections in both animals and plants. Secretions released by these obligate parasites during host invasion are thought to be crucial for their persistence in infection. Helminth secretions are complex mixtures of molecules, most of which have unknown molecular targets and functions in host cells or tissues. Although the habitats of animal- and plant-parasitic helminths are very distinct, their secretions share the presence of a structurally conserved group of proteins called venom allergen-like proteins (VALs). Helminths abundantly secrete VALs during several stages of parasitism while inflicting extensive damage to host tissue. The tight association between the secretion of VALs and the onset of parasitism has triggered a particular interest in this group of proteins, as improved knowledge on their biological functions may assist in designing novel protection strategies against parasites in humans, livestock, and important food crops. Upon infection, helminth parasites establish an intricate relationship with their host. Helminths cause considerable damage during host invasion, migration through host tissues, and feeding on host cells , 2, but Excretory/secretory (ES) products are regarded as the tools employed by helminth parasites to control host defense responses. Recently, it was shown that ES products of helminth parasites reflect their diversity in lifestyles and hosts and therefore have little in common between plant and animal parasites . HoweverThe expression of VALs is specifically up-regulated during parasitic phases of the life cycle of helminths, which could point to a role in host–parasite interactions –9. The pRecent reports have shed new light on structural properties, biochemical modes of action, and functions of secreted VALs of parasitic helminths. However, most of these reports have been published in specialized journals dedicated to either medical and veterinary biology or plant pathology. Here, we present an interdisciplinary review of the latest findings from phylogenetic analyses, x-ray crystallography, and functional studies on VALs from parasitic helminths. The aim of this review is to explore conserved mechanisms underlying the role of VALs as modulators of host responses during parasitism with an emphasis on their potential impact on common concepts in plant pathology and human and/or animal parasitology.Ditylenchus destructor and Bursaphelenchus xylophilus), animal-parasitic nematodes , and free-living nematodes (Panagrellus redivivus) clustered together  to over a hundred (Ancylostoma caninum). Gene family expansion also does not seem to be linked to a particular lifestyle or host organism. Altogether, our phylogenetic analysis revealed no clear link between overall sequence diversity in VAL genes in nematodes and parasitism in plant and animals.Multiple lineages within the nematode phylum have independently evolved the ability to parasitize plants or animals. Modelling the evolutionary history of nematode VALs, based on overall sequence diversity, may reveal links between patterns of diversification within this protein family and particular lifestyles or host organisms. To this end, we generated a Bayesian tree of available VAL sequences from plant- and animal-parasitic nematodes as well as their free-living close relatives from the distal end of the nematode tree  10]. Fi. Fi10]. together  insets. Oo-ASP-1 from Ostertagia ostertagi ) and information on lipid binding, interactions and function are given when available. PDB, Protein Data Bank; RCSB, Research Collaboratory for Structural Bioinformatics; VAL, venom allergen-like protein.Collection of helminth VALs as discussed in this review with current nomenclature, structural information (including RCSB PDB reference [(XLSX)Click here for additional data file.


We have recently identified metastatic-initiating cells, and described how they specifically rely on fatty acid uptake and lipid metabolism to promote metastasis. This intriguing finding indicates that external influences, such as those derived from our diet, exert a strong influence on tumour progression, and that such dietary factors could be therapeutically modulated if understood. In this News and Thoughts, I will comment on recent findings regarding how and why lipid metabolism modulates the behaviour of metastatic cells, and how this knowledge can be harnessed to devise new and specific anti-metastatic therapies.The identity of the cells responsible for initiating and promoting metastasis has been historically elusive. Consequently, this has hampered our ability to develop specific anti-metastatic treatments, resulting in the majority of metastatic cancers remaining clinically untreatable. Furthermore, advances in genome sequencing indicate that the acquisition of metastatic competency does not seem to involve the accumulation of  However, recent evidence suggests otherwise. For instance, careful Figure 1). This has allowed us to start identifying the intriguing characteristics that distinguish metastatic from non-metastatic cells. Specifically, we have found that: i) metastasis-initiating cells remain relatively quiescent in the primary tumor, but become highly proliferative during the initial stages of metastatic colonization; ii) upon orthotopic transplantation, metastasis-initiating cells initiate primary lesions as efficiently as their previously identified primary tumor-initiating counterparts, yet differ in that they are exclusive in their ability to generate metastasis; iii) metastatic-initiating cells express high levels of the fatty acid translocase CD36, and are characterized by a distinct lipid metabolic signature related to lipid uptake, fatty acid beta-oxidation (i.e. withdrawing energy from lipids), lipid biosynthesis, and lipid storage; iv) the metastatic potential of metastatic-initiating cells is boosted by a high-fat diet or exposure to high concentrations of palmitic acid. Consequently, the predisposition of tumors to develop metastasis might be directly linked to the content of fat in our diet; v) importantly, metastatic-initiating cells are therapeutically sensitive to CD36 inhibition, which almost completely abolishes their metastatic potential in orthotopic models of melanoma, and oral, breast, and ovarian cancers (so far tested) without significantly affecting their ability to generate primary tumors, and without overt systemic side effects; and vi) high expression of CD36+ correlates with poor prognosis in melanoma, and ovarian, hepatocellular, breast, head and neck, lung, pancreatic and bladder cancers, suggesting that these cells operate in many different types of tumors 13Figure 1). Of note, although the signature of CD36+ cells does not seem to be strongly associated with the expression of epithelial to mesenchymal transition (EMT) genes, fatty acid uptake by CD36 and the fatty acid binding proteins FABP1 and FABP4 induce EMT in liver cancer cells, thereby increasing their invasive capacity 13As a first step towards addressing these critical questions, we have recently identified a population of metastasis-initiating cells in several types of human tumors, and have determined that these are highly sensitive to dietary fatty acids de novo DNA methyltransferase Dnmt3a upregulate the expression of the lipid signature associated with CD36+ metastatic-initiating cells, and are consequently much more predisposed to generate metastatic squamous tumours 1911The results described above indicate that metastatic cells are very sensitive to lipid metabolism, unearthing a previously overlooked link between how much fat, and the type of fat, we eat and metastasis. However, many aspects of this metabolic switch remain unknown. First, why do metastatic-initiating cells respond to certain fatty acids such as palmitic acid, and how does this metabolism mechanistically affect their ability to colonize distant organs? Although the precise answer is unknown, it is likely that cancer cells require large amounts of energy to withstand the selective pressure against colonizing distant sites. For instance, highly aggressive triple negative breast cancer cells boost mitochondrial fatty acid beta-oxidation to obtain elevated amounts of acetyl-CoA that feed into the TCA cycle, thereby generating large quantities of ATP 1617i.e. metabolic plasticity) to the systemic and local changes they encounter as they egress the primary lesion and reach a distant site. For instance, as tumours grow, their capillary network becomes unstructured and generates areas of low oxygen, a phenomenon known as hypoxia. In addition, as metastatic cells arrive to distant organs, they initially lack a proper vasculature ensuring the oxygen supply necessary for tumour re-growth. Interestingly, in certain tumours, hypoxia can promote using alternative carbon sources to glucose, such as acetate, to sustain fatty acid synthesis. This lipid metabolic switch is associated to activation of the synthase ACSS2, whose expression is specifically increased in metastatic cells Besides our little knowledge of the cell intrinsic energetic and signalling changes lipid metabolism triggers in cancer cells to facilitate metastasis, our understanding of the factors that drive this metabolic switch is also still quite limited. One possible source of lipid metabolism rewiring could come from the fact that metastatic cells might adjust their metabolic state (Besides hypoxia-driven lipid metabolism, metastatic cells might obtain fat as an energy source by hijacking it from the cells they interact with during the metastatic journey. Two interesting examples support the notion of lipid metabolic coupling between cancer cells and their local cellular environment. For instance, metastatic ovarian cancer cells absorb lipids from the ommentum fat layer, one of the first sites they colonize, thereby ensuring a plentiful supply of energy required for surviving the colonization process 24http://www.who.int/mediacentre/factsheets/fs311 . The phenomenon is occurring worldwide, and even in countries that have historically favored a Mediterranean diet, underscoring the fact that eating habits have dramatically changed with industrialization.In conclusion, the ability of metastatic cells to engage in a high lipid metabolic state when confronted with different conditions and environments, strongly contributes to tumour progression. We find these findings very interesting scientifically, albeit socially worrying considering the high amount of added fat that is consumed regularly through processed food in established and newly industrialized countries. The percentage of clinically obese and overweight people has nearly doubled in the last 30 years, with more than 600 million adults considered obese, and 41 million children under the age of 5 overweight or obese, in 2014 (in vivo pre-clinical assays of metastasis, and clinical trials, will undoubtedly provide a deeper understanding of the relevance, and clinical potential, of lipid metabolism regarding the idiosyncrasies of metastatic-initiating cells.On the other hand, and notwithstanding these worrying figures, the connection between lipid metabolism and metastasis might in fact offer new therapeutic avenues to prevent and treat metastatic cancers. First, public awareness can and should be increased to highlight the importance of drastically reducing the amount of added fat in the food we consume on a daily basis. As an example of the benefits of weight loss, a recent study shows that whereas obesity boosts lung metastasis of breast cancer cells by recruiting neutrophils to the pre-metastatic niche in the lungs, a loss of weight is sufficient to reverse this effect 28


Obesity and associated comorbidities, including non-alcoholic fatty liver disease (NAFLD), are a major concern to public well-being worldwide due to their high prevalence among the population, and its tendency on the rise point to as important threats in the future. Therapeutic approaches for obesity-associated disorders have been circumscribed to lifestyle modifications and pharmacological therapies have demonstrated limited efficacy. Over the last few years, different studies have shown a significant role of intestinal microbiota (IM) on obesity establishment and NAFLD development. Therefore, modulation of IM emerges as a promising therapeutic strategy for obesity-associated diseases. Administration of prebiotic and probiotic compounds, fecal microbiota transplantation (FMT) and exercise protocols have shown a modulatory action over the IM. In this review we provide an overview of current approaches targeting IM which have shown their capacity to counteract NAFLD and metabolic syndrome features in human patients and animal models. NAFLD has emerged as the most common chronic liver disorder in Western countries, with an estimated prevalence of between 10 and 30% in USA and Europe and about 25% worldwide  and viruses. The IM bacteria comprise two predominant bacterial phyla: Firmicutes and Bacteroidetes which reach as much as 90% of total bacterial population, although Proteobacteria, Verrucomicrobia, Actinobacteria, Fusobacteria, and Cyanobacteria are also represented  is a complex ecosystem where 10Bacteroidetes and increased Firmicutes has been indicated as a main feature of obesity-related intestinal dysbiosis in humans  mice unveiled an overrepresentation of Firmicutes and reduction of Bacteroidetes . Moreover, this study reported a higher prevalence of Gram-negative bacteria and reduced diversity at phylum level in NAFLD patients  in NAFLD patients  in a TLR-independent signaling pathway , and fatty acid synthase (FAS)   or physical exercise protocols Figure .Lactobacillus, Streptococcus, and Bifidobacterium genera are the most frequently used probiotics due to the growing body of literature supporting their health-promoting effects and because all of these integrate the normal human gut microenvironment . Bifidobacterium abundance was restored to control levels in the probiotic treated group while Gram-negative bacteria enhanced by HFD (Escherichia/Shigella and Desulfovibrionaceae) were reduced. In a previous similar study the same strain also induced an increase in Bifidobacterium spp. in HFD-fed animals, while the opposite was true for Enterobacteriaceae  mice  supplementation  corroborated the bifidogenic effect of prebiotics in obese humans  achieved a reduction of AST levels and showed a tendency to lower ALT and triglycerides  diet with polyphenols tends to lower the Helicobacter pylori has been related to NAFLD. There are studies reporting a decline in Helicobacter genus or H. pylori using flavonoids   was used as a therapy for the first time in present-day medicine in 1958 in order to treat pseudomembranous colitis due to Intestinal microbiota involvement in other pathologies points to FMT as an alternative treatment for a wide range of diseases, including extraintestinal pathologies. Metabolic disorders are possible future targets of FMT , including the presence of dysbiosis. A recent study has shown that FMT prevents alcohol induced liver injury along with modulation of the IM . At genus and specie levels Lactobacillus was increased in obese rats after exercise and six species  were more abundant in this group more than the others. The study into hypertensive condition is also relevant being hypertension a manifestation associated with NAFLD. Hypertensive rats displayed a particular microbiota and it was also susceptible to be modified by exercise, exhibiting greater Allobaculum and less Aggregatibacter and Sutterella after training. Finally, in a study with genetically diabetic and non-diabetic mice, forced running 5 days a week for 6 weeks correlated with lower Bacteroides/Prevotella spp. and Methanobrevibacter spp.  seems to have similar or even greater metabolic effects than continuous aerobic training in diet-induced obese mice (Wang et al., Bacteroidetes and greater Firmicutes. Akkermansia muciniphila increased both in athletes and in low BMI controls. Moreover, microbiota of active lifestyle women differs from those with a more sedentary pattern, with enhanced detection in the former of potentially beneficial bacteria like Bifidobacterium spp., Roseburia hominis, A. muciniphila, and F. prausnitzii (Bressa et al., The impact of exercise on gut microbiota has also been shown in several human studies which identify a particular microbiota associated to individuals with divergent degrees of physical activity. Different microbiota composition was found between professional athletes and healthy controls, showing higher bacterial richness in athletes vs. controls (Clarke et al., Bacteroidales S24-7, as well as butyrate-producing Firmicutes families Clostridiaceae, Lachnospiraceae, and Ruminococcaceae. Finally, intestinal health promoting bacteria like A. muciniphila are associated to exercise and active lifestyle in humans (Clarke et al., In order to address the relationship between exercise-induced changes in microbiota and metabolic effects in the host, exercise impact on gut barrier should be considered. Several studies include evaluation of intestinal tissue after exercise in animal models. Exercise exerts protective effect on intestine epithelium counteracting the morphological changes associated to HFD and reducing inflammation (Campbell et al., in vivo models, but evidence from clinical trials is scarce. Moreover, only the most recent animal studies have carried out microbiome analysis to show that the results are linked to IM modulation. Furthermore, the findings obtained from these experiments need to be taken skeptically, due to the conflicting results. For this reason, it is too early to ensure the effectiveness of any of these methods. It is important to remark that different strategies are not mutually exclusive and maybe a synergistic combination of them could make the difference. In conclusion, intestinal microbiota modulation emerges as a potential therapeutic option for obesity-associated NAFLD. However, its application in clinical practice requires further investigation due to the lack in studies into humans.Intestinal microbiota dysbiosis and related gut-liver axis activation are now accepted as a critical process in NAFLD development. Due to the absence of a gold standard treatment for fatty liver disease, development of novel therapeutic strategies is a priority to manage NAFLD patients. Different approaches have been made to modulate intestinal microbiota: pre/probiotics administration, FMT, and lifestyle modification including physical exercise interventions. All of these strategies lead to NAFLD and associated metabolic disturbances remission in DP wrote the manuscript. DP, EN, SM-F, JG-G, MG-M, and SS-C discussed the literature and figures, contributed to the intellectual input, and edited the manuscript. All authors read and approved the final version.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.


Superresolution microscopy based on localisation is usually performed in a buffer containing enzymatic oxygen scavenger, which facilitates reversible photoswitching of the dye molecules. This makes correlative fluorescence localisation and atomic force microscopy (AFM) challenging, because enzymatic oxygen scavenging interferes with the AFM cantilevers. Here we report on the blinking kinetics of a new red cyanine dye, iFluor-647, which is similar to the Alexa-647 dye commonly used for superresolution microscopy, but with brightness and blinking properties which are superior to Alexa-647 in a buffer without enzymatic oxygen scavenger. We measure the blinking behaviour of iFluor-647 in buffers with and without enzymatic oxygen scavenger with different thiol concentrations. We then apply this dye for correlative localisation and atomic force microscopy in a buffer without enzymatic oxygen scavenger, which allows acquisition of AFM and superresolution images without buffer change. The thiol is thought to form a covalent bond with the dye molecule, forming the dark state of cyanine dyes . Be. Be20]. In this work, we have tested a new red cyanine dye, iFluor-647, and compared the results with Alexa-647, the most commonly used dye for STORM imaging. We found that iFluor-647 molecular brightness is slightly higher than Alexa-647, particularly in an MEA only buffer, and the brightness of both fluorophores decreases with increasing MEA concentration , with thWe have applied the iFluor-647-phalloidin conjugate for correlative AFM+STORM imaging of actin fibres in fixed HeLa cells in an MEA only buffer without enzymatic oxygen scavenger . The use5We present an easy and straightforward method for correlative AFM+STORM imaging of fixed samples using iFluor-647 dye in a simple buffer containing the thiol MEA but no enzymatic oxygen scavenger. The brightness and blinking characteristics of iFluor-647 in an MEA only buffer are comparable to the popular STORM dye Alexa-647 in a buffer containing enzymatic oxygen scavenger, yielding good quality STORM images. Unlike buffers containing enzymatic oxygen scavengers, the use of MEA only buffer allows long term imaging over many hours and is compatible with AFM imaging such that no buffer change is required between the imaging modalities, simplifying the process and eliminating artefacts in correlative AFM +STORM imaging.onlineSupplementary material for this article is available Supplementary Information