Heme and iron are essential molecules for many physiological processes and yet have the ability to cause oxidative damage such as lipid peroxidation, protein degradation, and ultimately cell death if not controlled. Blood-sucking arthropods have evolved diverse methods to protect themselves against iron/heme-related damage, as the act of bloodfeeding itself is high risk, high reward process. Protective mechanisms in medically important arthropods include the midgut peritrophic matrix in mosquitoes, heme aggregation into the crystalline structure hemozoin in kissing bugs and hemosomes in ticks. Once heme and iron pass these protective mechanisms they are presumed to enter the midgut epithelial cells via membrane-bound transporters, though relatively few iron or heme transporters have been identified in bloodsucking arthropods. Upon iron entry into midgut epithelial cells, ferritin serves as the universal storage protein and transport for dietary iron in many organisms including arthropods. In addition to its role as a nutrient, heme is also an important signaling molecule in the midgut epithelial cells for many physiological processes including vitellogenesis. This review article will summarize recent advancements in heme/iron uptake, detoxification and exportation in bloodfeeding arthropods. While initial strides have been made at ironing out the role of dietary iron and heme in arthropods, much still remains to be discovered as these molecules may serve as novel targets for the control of many arthropod pests. Estimates suggest there are more than 1 million insect and arachnid species inhabiting planet Earth, approximately 14,000 of which have adapted the ability to feed on vertebrate blood  as well as in the cytoplasm of midgut epithelial cells or other tissues in the body (intracellular).Over time, hematophagous arthropods have honed their antioxidant systems to avoid oxidative stress  microplus, GST activity was confirmed as antioxidant in the eggs and larvae of engorged females. More specifically, GST enzymatic activity significantly increased and O2 consumption progressively increased during embryonic development and eventually peaked at hatch  catalyze the conversion of superoxide anions into hydrogen peroxide. Subsequently, catalase (CAT) and general peroxidases (POD) are responsible for detoxifying hydrogen peroxide   in R. prolixus , Dansa-Petretski et al.     and to a lesser extent unsaturated 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (uPC) produced brick shaped and blunt ended β-hematin crystals similar to those produced by plasma-fed or blood-fed R. prolixus midgut lipids   and heme crystal hemozoins in association with a perimicrovillar membrane (R. prolixus) have been documented as protective mechanisms against oxidative stress, I. scapularis ticks (vector of Borrelia burgdorferi)  is a semipermeable extracellular layer, which lines the midgut of most invertebrates Peters, . The perAnopheles gambiae there are cases of modified peritrophin-A domains (mPADs). The mPAD of AgAper25a and AgAper29 are characterized as having two additional cysteine residues, however, these two cysteine residues are not considered significant . Each CBD is comprised of cysteine residues that allow for disulfide bonds within the CBD, and the number of disulfide bonds within a CBD can range from three to five , there are partial PCDs that contain 67–70 amino acids . While the CBDIII contains 10 interspersed cysteine residues (similar to PCD), this domain is typically 110–117 amino acid residues long.While less common, CBDs can also be classified as Peritrophin-B domain (PBD) or Peritrophin-C domain (PCD) according to the number of cysteine residues. An individual PBD contains eight interspersed cysteine amino acid residues, and is 81–88 residues long  is a 275-amino acid glycoprotein that contains three chitin-binding domains and a mucin domain between CBD 1 and CBD 2   has utilized a similar methodology  gene which encodes STAT, the cytosolic component of the Janus kinase (JAK)/STAT pathway  heme transporter has been characterized in both humans and mice , which had a low sequence identity and homology to the C. elegans genes hrg-1 and hrg-4 and was found localized via palladium mesoporphyrin IX fluorescence in the vitelline and ovary regions of the females of the species. However, heme transport by Smhrg-1 was only identified in these organs indicating that the heme transport mechanism after digestion to the vitelline and the ovary regions is not due to Smhrg-1 but some other unknown mechanism. A Leishmania amazonensis homolog of the C. elegans heme transporter hrg-4, LHR1, was identified by Huynh et al. , separately or combined with CsA (an ABC inhibitor) confirmed an ABC transporter was responsible for heme transport after a bloodmeal, while an anti-PgP-1 antibody identified the membrane of digestive vacuoles to be its location in the cells. RNAi of the RmABCB10 transporter in female bloodfed ticks and Zinc protoporphyrin IX (ZnPp) fluorescence showed reduced ZnPp in the hemosomes, but more in the digestive vacuoles, which confirmed RmABCB10 as the transporter characterized above. Lara et al.  , iron and CO by heme oxygenase (HO)  as determined by ESI-MS , zip13, and mco1. Mvl is a homolog of the human natural resistance-associated macrophage proteins (Nramp). Both loss and gain-of-function mvl mutants in adult flies resulted in altered taste perception, particularly sugar and salt perception  were present suggesting anaNramp may not be regulated by cytoplasmic Fe concentration. Few arthropods have been characterized that contain h-Nramp homologs, those that do contain the gene require further study as the regulation method behind their iron transport is unidentified.Three genes in zip13), an iron efflux pump that moves iron from the cytosol to the ER/Golgi  to ferric iron (Fe3+) in the hemolymph allowing its binding to transferrin and similar iron binding proteins for transport in D. melanogaster. Lang et al. , when ferritin is upregulated during bacterial infection lending evidence that host-bacteria regulatory mechanisms involving ferritin production do exist in these organisms , a key component in molting, metamorphosis and vitellogenesis  is an essential transporter of heme to the embryos. RNAi mediated knockdown of RHBP did not alter fecundity, however the resultant eggs were not viable and were white in color. This is in contrast to the dark red embryos oviposited by controls. Taken together, the above studies shed light on the sheer importance of heme-binding and iron-binding proteins in terminal incorporation of heme and iron into the developing eggs of hematophagous arthropods, in particular ticks, which lack the de novo heme synthesis pathway. Whether corresponding heme carrier proteins are utilized by bloodfeeding arthropods that maintain the ability to synthesize heme remains unknown.Walter-Nuno et al.  found thD. melanogaster. While certain life history traits make D. melanogaster a powerful model organism, they do not bloodfeed, highlighting the need to perform direct experimentation on the bloodfeeding arthropods.Although many arthropods have evolved the ability to feed on blood, relatively little insight into the internal processing of blood meal heme and iron is available. Much of the available information regarding iron processing in arthropods, more specifically insects, is gleaned from Through this review, we provide a framework for future studies aimed at obtaining a better understanding as it relates to the fate of heme and iron processing in hematophagous arthropods Figure . With reGiven that catalase appears to be a key antioxidant in both midgut and ovarian oxidative stress avoidance for many hematophagous arthropods, further studies targeting this enzyme are needed to assess its potential for disrupting the midgut and ovarian oxidative balance on a larger scale when hematophagous arthropods take a blood meal.Heme aggregation/crystalization appears to be a common first line of defense in many hematophagous arthropods. Better understanding of this multifaceted protection playbook for each hematophagous arthropod can provide insight into key targets for breakdown of this system.In 2009, Dinglasan et al. set the stage for midgut PM protein composition exploration and discovery utilizing a highly sensitive MS-based approach. The success of this methodology was largely associated with the availability of an artificial protein-free meal. Such protein-free artificial meals for other hematophagous arthropods are needed to allow similar exploration of related and divergent PM structures.An. coluzzii and peritrophin-1 in I. scapularis). Further studies utilizing reverse genetic tools, such as RNAi and CRISPR/Cas9 are needed to determine the physiological function of PM peritrophins in medically important hematophagous arthropods.While peritrophic matrix peritrophins have been identified in many hematophagous arthropods, only two PM peritrophin has been knocked-down using RNAi (APER1 in Ae. aegypti) has been conducted -binding proteins (PCBP1-4) facilitate iron loading of ferritin. Homologues of these proteins in bloodfeeding arthropods could represent a missing link in iron homeostasis in these organisms.D. melanogaster and Ae. aegypti the mode of entry and recovery of iron from ferritin into cells and tissues remains uncharacterized.While ferritin or its substrate, dietary iron, has been localized in multiple tissues in both Transferrin, another conserved iron binding transport protein between vertebrates and arthropods, is found in many bloodfeeding arthropods. While transferrins have been shown to be upregulated after a bloodmeal or during pathogen infection, little is known about the biological role or specialization of these transferrin genes.Hemolymph heme-binding proteins  allow for proper transport of heme and iron to developing eggs. However, further studies are needed to determine if orthologs for these proteins exist in other hematophagous arthropods and their role in proper shuttling of heme to ovaries. Loss of any heme and iron shuttling processes can decrease egg viability, and ultimately be utilized in novel mosquito population control techniques.SW, HE, and ZA conceived the manuscript and edited the manuscript into its final form; SW and HE drafted the initial 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.


We hypothesized that exploring the interplay between co-occurrence, mutual exclusivity, and functional interactions between genes will further improve our understanding of the disease and help to uncover new relations between cancer driving genes and pathways. To this end, we designed a general framework, BeWith, for identifying modules with different combinations of mutation and interaction patterns. We focused on three different settings of the BeWith schema: (i) BeME-WithFun, in which the relations between modules are enriched with mutual exclusivity, while genes within each module are functionally related; (ii) BeME-WithCo, which combines mutual exclusivity between modules with co-occurrence within modules; and (iii) BeCo-WithMEFun, which ensures co-occurrence between modules, while the within module relations combine mutual exclusivity and functional interactions. We formulated the BeWith framework using Integer Linear Programming (ILP), enabling us to find optimally scoring sets of modules. Our results demonstrate the utility of BeWith in providing novel information about mutational patterns, driver genes, and pathways. In particular, BeME-WithFun helped identify functionally coherent modules that might be relevant for cancer progression. In addition to finding previously well-known drivers, the identified modules pointed to other novel findings such as the interaction between NCOR2 and NCOA3 in breast cancer. Additionally, an application of the BeME-WithCo setting revealed that gene groups differ with respect to their vulnerability to different mutagenic processes, and helped us to uncover pairs of genes with potentially synergistic effects, including a potential synergy between mutations in TP53 and the metastasis related DCC gene. Overall, BeWith not only helped us uncover relations between potential driver genes and pathways, but also provided additional insights on patterns of the mutational landscape, going beyond cancer driving mutations. Implementation is available at  The genomic landscape of cancer obtained from large scale studies revealed mutational patterns such as mutual exclusivity and co-occurrences. Despite significant efforts, the understanding of the patterns harbored by specific genes and their interplay with functional interactions are still limited. Both mutual exclusivity and co-occurrence can arise due to several reasons. For example, two functionally interacting genes may dysregulate the same cancer related pathway when either of them is mutated, leading to mutually exclusive mutations. Alternatively, mutual exclusivity might reflect mutations specific to two different cancer types. Methods for joint analysis of co-occurrence, mutual exclusivity, and functional interaction relationships can lead to a better understanding of the causes and impacts of mutations in cancer. We report a new computational approach, BeWith, which identifies groups of genes (or gene modules) with coherent patterns within modules, but distinct properties among genes in different modules. The general formulation of our method allows us to investigate various aspects of the cancer mutational landscape, leading to uncovering relationships between mutated gene modules, cancer subtypes, and mutational signatures. PLOS Computational Biology Methods paper.”“This is a The analysis of the mutational landscape of cancer has been instrumental in studying the disease and identifying its main drivers and subtypes. In particular, mutual exclusivity of mutations in cancer drivers has recently attracted a lot of attention. This relation can help identify cancer drivers, cancer-driving pathways, and cancer subtypes –10. AlthImportantly, both properties can arise due to several different reasons, making the interpretation of the implied gene-gene relations challenging. Specifically, mutually exclusive mutations within functionally interacting genes may indicate that a mutation in either of the two genes dysregulates the same pathway. On the other hand, mutually exclusive mutations might also reflect a situation where mutations in two genes are associated with two different cancer types or subtypes. We have previously observed that within cancer type mutual exclusivity is more enriched with physically interacting pairs of genes compared to between cancer type mutual exclusivity . Thus, tAs with mutual exclusivity, co-occurrence of mutations might emerge due to a number of different causes. One of the most important cases is when simultaneously disabling two genes might be beneficial for cancer progression. Examples of such a scenario include the co-occurrence of TP53 mutation and Myc amplification ,17 or coGiven the diversity of reasons for observing the mutual exclusivity and co-occurrence relations, we hypothesised that jointly considering co-occurrence, mutual exclusivity, and functional interaction relationships will yield a better understanding of the mutational landscape of cancer. As a step in this direction, our goal was to develop a method to identify groups of genes (or gene modules) that show coherent patterns within modules, but distinct properties with genes outside modules. While many methods to identify cancer related modules exist, such modules are typically identified by focusing on the relationships of genes within a module. In particular, there have been several previous attempts to combine mutual exclusivity and functional interactions for module identification –3,19. HoTo address this challenge, we designed a general framework, named BeWith, for identifying modules with different combinations of mutation and interaction patterns. On a high level, BeWith tackles the following problem: given a set of genes and two types of edge scoring functions (within and between scores), find clusters of genes so that genes within a cluster maximize the “within” scores, while gene pairs spanning two different clusters maximize the “between” scores. We formulated the BeWith module identification problem as an Integer Linear Programming (ILP) problem and solved it to optimality. The flexibility of the ILP formulation allowed us to include additional constraints, such as module density, to enhance the module discovery process : Our first setting ( setting  uses funSetting 2 (BeME-WithCo): This setting searches for groups of genes that are co-mutated (within), exclusively compared to other groups (r groups . There aSetting 3 (BeCo-WithMEFun): Our third setting is specifically designed to identify co-occurring driver pathways. We seek modules displaying functional and mutual exclusivity relations inside a module and co-occurrence between modules  in their GBM data analysis , which makes sure that all the variables in the optimal solution are binary but reduces the running time , in which EME is a set of gene pairs that have significantly mutually exclusive mutations based on WeSME. A co-occurrence network GCO =  was computed with hypergeometric tests or WeSCO tests, depending on the setting. In addition, we utilized functional interaction information in some settings, obtained from the STRING database [We applied BeWith in three complementary settings for two TCGA datasets: somatic mutation profiles in breast cancer (BRCA) and endometrial cancer (UCEC). With somatic mutation profiles of 665 BRCA samples and 207 UCEC samples (after removing ultra mutated samples), we first computed their mutual exclusivity and co-occurring relationships for each gene pair and constructed networks retaining only significant relationships. To this end, we used WeSME and WeSCO, which are efficient, weighted sampling based methods for testing mutually exclusivity and co-occurrence respectively, taking into account mutation frequencies of cancer samples . Specifidatabase .wF(ij), wME(ij) and wCO(ij) for each pair are defined based on the protein functional interaction confidence scores and p-values from the mutual exclusivity and co-occurrence tests, respectively. The weights are set to be 0 if the edge does not exist. In the three BeWith settings we used different definitions of between and within functions, and slightly different variants of the constraints as described below.The weights This setting searches for functionally related groups of genes with potential relevance for cancer. In order to ensure that genes within each module are likely to be in the same pathway, we enforce functional edges within modules while penalizing functional interactions of genes from different modules. We also reward mutual exclusivity between modules, so our optimization function is:i in a module, it has at least some mutual exclusivity edge(s) with genes in other modules as in constraints  = wCO(ij) − wME(ij)). To capture co-occurrence modules that are biologically relevant, we reward mutual exclusivity relations between modules  = wME(ij) − wCO(ij)). The objective function is then defined as follows:In order to identify co-occurring modules, we perform BeWith enforcing the co-occurrence within a module but penalizing within module mutual exclusivity (EY = EME) among the modules.To strengthen the co-occurrence within each module, we enforce that each module has dense co-occurring interactions by the constraints . Similarwithin = wME(ij) − wCO(ij) + wF(ij)). Genes in different modules are rewarded for co-occurrence  = wCO(ij) – wME(ij) – wF(ij)). The objective function is then defined as:Complementing the above analyses we utilized BeWith to look for modules that contain mutually exclusive and functionally related gene modules that might co-occur with other modules. Specifically, we enforce mutual exclusivity while penalizing co-occurring mutations within modules  with genes in other modules (EY = ECO):To strengthen the co-occurrence between modules and mutually exclusivity within each module, we additionally required that for each gene S1 TextAdditional results and method description.(PDF)Click here for additional data file.


Stem cells derived from human dental pulp tissue (DP-MSC) differ from the other mesenchymal stem cells prepared from bone marrow or adipose tissue due to their embryonic origin from the neural crest and are of special interest because of their neurotropic character. Furthermore, the therapeutic potential of DP-MSCs is realized through paracrine action of extracellularly released components, for which exosomes play an important role. In this review, we intend to explore the properties of these cells with an emphasis on exosomes. The therapeutic applicability of these cells and exosomes in dental practice, neurodegenerative diseases, and many other difficultly treatable diseases, like myocardial infarction, focal cerebral ischemia, acute lung or brain injury, acute respiratory distress syndrome, acute inflammation, and several others is concisely covered. The use of cellular exosomes as an important diagnostic marker and indicator of targeted cancer therapies is also discussed, while the importance of stem cells from human exfoliated deciduous teeth as a source of evolutionally young cells for future regenerative therapies is stressed. We conclude that exosomes derived from these cells are potent therapeutic tools for regenerative medicine in the near future as clinical administration of DP-MSC-conditioned medium and/or exosomes is safer and more practical than stem cells. The involvement of mesenchymal stem cells (MSCs) in the regeneration of damaged or aged tissues is well supported by current research. Furthermore, cell manufacturing procedures for obtaining high quality, bioactive MSCs from human bone marrow has been approved by the US Food and Drug Administration , while rDental pulp mesenchymal stem/stromal cells (DP-MSCs) are of particular interest because of their neurotropic character, which makes DP-MSCs and their exosomes particularly attractive as a new therapeutic tool for the alleviation of symptoms of neurodegenerative diseases and many other difficultly treatable maladies. Dental tissue-derived stem cells besides DP-MSCs include multiple types such as stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), and dental follicle progenitor cells (DFPCs) . All of Quantitative gene expression analysis of MSCs isolated from the umbilical cord (UC) and dental pulp (DP) generally reflects their biological functions. Genes related to cell proliferation, angiogenesis, and immune responses are expressed at higher levels in UC, whereas genes related to growth factors, receptor activity, and signal transduction are more highly expressed in DP . MSCs arDP-MSCs are known for their high proliferative potential, as demonstrated by their ability to be isolated and expanded from dental pulp tissue fragments that adhere to plastic tissue culture dishes. These tissue fragments can be transferred from one dish to another for 3 months with no interruption in cell proliferation. The cells outgrowing from the explants and the differentiation of DP-MSCs to three lineages in vitro are depicted in DP-MSCs have been intensively studied because the dental pulp is made of ecto-mesenchymal elements, categorizing DP-MSCs as neural crest-derived cells. Specifically, DP-MSCs from the dental pulp of third molar teeth or human stem cells from exfoliated SHEDs have unique neurogenic properties that could potentially be exploited for therapeutic use. Our comparison of DP-MSCs with bone marrow and adipose tissue-derived MSCs (AT-MSCs) revealed differences in the expression of pluripotent stem cell genes that reflect the ecto-mesenchymal origin of DP-MSCs . DP-MSCsThe dental-tissue-derived stem cells are isolated from specialized tissue and therefore could have the potential to differentiate into odontogenic cells. Out of all of them, DP-MSCs show the greatest potential to produce a comparatively high volume of mineralized matrix during in vitro experiments. However, Hung along with collaborators demonstrated that both implants of AT-MSCs and DP-MSCs were able to grow self-assembled, new teeth in adult rabbit extraction sockets with a high success rate. Furthermore, both dentinogenesis and mineral volume was enhanced when using AT-MSCs, suggesting that they might also be useful for regenerative dental therapies . The reaAlso, preclinical studies of the efficacy of stem cells on periodontal regeneration were recently comprehensively reviewed . In an eβ1 gave a similar effect in periodontal tissue regeneration in dogs [ε-caprolactone)-biphasic calcium phosphate scaffold constructs [It is more and more recognized that medicinal signaling realized by secreted bioactive compounds and exosomes induces regenerative processes. In fact, Caplan  suggeste in dogs . Furthernstructs . Huang enstructs .There are attempts to innovate classical endodontic therapies consisting of disinfecting and then sealing the endodontic space with dental stem cells. Recent findings suggest that regenerating fully functional pulp tissue may be an ideal therapeutic solution to maintaining a tooth defense system by detecting and helping to manage future injuries . Future A long-term goal of dentistry is to create functional biomimetic tooth buds for eventual tooth replacement in humans. The tooth is an ectodermal organ composed of soft connective tissues, a distinctive hard tissue, and additional tissues like peripheral nerve fibers and blood vessels. Tooth development is regulated by mutual interactions of mesenchymal and epithelial cells, and considering our present knowledge about exosomes as factors capable of transferring bioinformation between cells, their involvement in this natural process seems likely.To recapitulate organogenesis, it is required to have compartmentalization of epithelial and mesenchymal cells at a high enough cell density to mimic multicellular assembly conditions and epithelial-mesenchymal interactions leading to a bioengineered tooth germ. The tooth germ is capable of generating a structurally correct tooth in vitro and erupts successfully with the correct tooth structure when transplanted into the oral cavity. For example, whole-tooth restoration has been demonstrated both in a murine tooth-loss model  and whenDental pulp stem cells are obviously the most proper source of MSCs for tooth regeneration. Although these cells demonstrate high growth potential, the small size of dental pulp tissue is currently a limiting factor for the expansion of the large cell number needed for regenerative dentistry. Hung et al. tested the possibility of using AT-MSCs, which are an easier cell source than DP-MSCs for tooth regeneration. They showed that by using strictly controlled, side-by-side comparisons between the two types of stem cells that the expression patterns of gene markers were very similar. Then, they demonstrated that both implants of AT-MSCs and DP-MSCs were able to grow self-assembled new teeth in adult rabbit extraction sockets . This fiMesenchymal stem cell senescence is an adverse factor from the perspective of any cell-based therapies. Several studies have shown that senescence impairs the proliferation and differentiation potentials of DP-MSCs and that donor age is an important factor that affects their use for tooth regeneration. The age-associated changes of DP-MSC, determined by comparing isolated cells from young and aged dog teeth, were attributed to a decrease in the regenerative potential of resident stem cells . HoweverIt has been well documented that MSCs release rich secretomes containing massive amounts of cytokines, chemokines, and growth factors, together with extracellular nanoparticles. The most important nanoparticles in the medium conditioned by MSCs are exosomes. Exosomes are extracellular vesicles of 30–120 nm in diameter secreted by cells that act as messengers by communicating with other cells and carry intracellular sorted cargo that is encapsulated in a lipid bilayer-bound vesicle. While all cells purge intracellular waste by a release of exosomes, those formed in the cell by endocytosis include important informative molecules, like microRNAs, mRNAs, and overexpressed proteins in their cargo. A cell-derived lipoprotein coat of exosomes protects their cargo from degradation in the systemic circulation and determines their tropism. After internalization into recipient cells, MSC-exosomes have the ability to change them to specifically differentiated cells. This regenerative process starts by paracrine action of MSC secretome on intrinsic native cells of the recipient.Exosomes are released from all cells, and their internal cargo reflects the cell's status. Additionally, exosomes are capable of intercellular communication. For instance, exosomes derived from tumor cells signal cellular aggressiveness and carry information about resistance to previously used therapeutic treatments , thereby representing a useful marker for the selection of personally suited therapies, such as when tumor cell-derived exosomes were shown to determine organotropic metastasis, creating a premetastatic niche . ConsequExosomes were originally recognized as nanoparticles secreting cellular waste . HoweverHuman MSCs are widely available from various tissues and have been proposed as a promising cell replacement therapy for maladies of the geriatric population. Therapy for neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, spinal muscular atrophy, and Huntington's disease currently lack effective treatments. There is an increasing amount of evidence that MSC-conditioned medium containing exosomes, or the exosomes themselves, reiterates the regenerative induction capability of particular MSCs and retains the homing properties of their parent cells. The beneficial effect of exosomes derived from dental mesenchymal/stromal cells has been shown in neurodegenerative diseases, peripheral neural regeneration, myocardial infarction, focal cerebral ischemia, stimulation of angiogenesis, neurogenesis in the ischemic rat brain, acute lung injury, suppression of acute inflammation, and syndromes connected with immunosuppression or autoimmunity, and the list is likely not final.The importance of the paracrine action of components released from MSCs has been recognized, but the exact mechanisms behind these effects are presently unknown. Rich MSC secretome and a number of bioinformative compounds in an exosome's cargo are involved, but how the damaged cells choose the competent ones remains to be elucidated. Nevertheless, it is not exceptional in medicine to use therapeutically beneficial tools without exact knowledge of their actions. Additionally, MSC-derived exosomes have multiple advantages over cell therapy, because exosomes are stable in the systemic circulation without losing function, and in comparison to stem cells, they exhibit a superior safety profile.Potential, broad therapeutic applications of microvesicles, especially exosomes, was overviewed in the position paper of the International Society for Extracellular Vesicles . In ParkMany preclinical and clinical studies have been published showing the positive effect of cell transplantation in Parkinson's disease and peripheral neural regeneration . DP-MSC Exosomes derived from DP-MSCs have been shown to suppress experimentally induced acute inflammation in mice and reduced induced edema to similar levels seen with prednisolone as a positive control. However, the effects of prednisolone were more prominent at the early stages, while exosomes exerted their suppressive effects gradually and at later time points . The theRecent developments in peripheral nerve regeneration have shown that human gingiva-derived mesenchymal stem cells are easily induced into neural progenitor-like cells. These cells, when transplanted to the injury site, promoted recovery in the crush-injury model of rat sciatic nerve. The molecular mechanism behind this regeneration was the expression of the antagonistic myelination regulators c-Jun and Krox-20/EGR2 in Schwann cells . Very reSong et al. compared the therapeutic effects of transplanted human DP-MSCs and human BM-MSCs in a stroke model of rats. Either human DP-MSCs or BM-MSCs were intravenously injected into rats after middle cerebral artery occlusion and showed improved functional recovery and reduced infarct volume versus control rats. Furthermore, the DP-MSC-treated group showed greater reduction in infarct volume than BP-MSCs . MSC exoMSCs recognize a tumor as a nonhealing wound . As suchDiffuse intrinsic pontine gliomas (DIPGs) are fatal tumors found in the pons of young children and do not respond to standard therapies like radiotherapy or chemotherapy. However, nanomedicine may offer novel therapeutic options for the treatment of DIPGs. Multiple nanoparticle formulations capable of traversing the blood-brain barrier into the pons like Qdots, gold nanoparticles, dendrimers, and liposomes have been studied as a potential tool for therapeutic intervention . For exa


We present a case of a 67-year-old man with stage III chronic kidney disease, uncontrolled diabetes mellitus, coronary artery disease, and high surgical risk who presented with two episodes of acute coronary syndrome attributed to in-stent restenosis (ISR) associated with heavily calcified lesions. In this case, we were able to improve luminal patency with orbital atherectomy system (OAS); however, withdrawal of the device resulted in a device/stent interaction, causing failure of the device. Given limitations in current evidence and therapies, managing ISR can be a technical and cognitive challenge. Balloon expansion of the affected region often provides unsatisfactory results, possibly related to significant calcium burden. OAS could be an efficacious way of reestablishing luminal patency in ISR lesions, as these lesions are often heavily calcified. ISR is a pathological development of neointimal hyperplasia resulting in the progressive loss of luminal area. Recent studies have suggested that early phase neointimal hypertrophy is in part due to calcification. These lesions are more robust and may contribute to stent under expansion if treated with balloon angioplasty alone , 2. The A 67-year-old male with coronary artery disease, New York Heart Association Class III diastolic heart failure, paraplegia, diabetes mellitus, and stage III kidney disease presented with a high-risk, non-ST segment elevation myocardial infarction (NSTEMI) and worsening renal failure. The patient had presented six months earlier with an NSTEMI attributed to in-stent restenosis. Angiography at that time revealed calcified lesions, in addition to a 90% in-stent restenosis within the left anterior descending artery (LAD). These lesions were treated with primary balloon angioplasty and the placement of two drug-eluting stents within the culprit region of the LAD. The patient was subsequently discharged on dual antiplatelet therapy.His most recent presentation was characterized by features of NSTEMI including typical cardiac chest pain, elevated troponins, and worsening renal function. The patient initially received goal-directed medical therapy and was monitored for a few days until his renal function recovered enough to permit iodinated contrast administration. Subsequent cardiac catheterization revealed disease in the right coronary artery of 65% distal and 90% bifurcating lesions, 90% small circumflex, and 90% in-stent restenosis of the mid LAD . CardiotThe percutaneous coronary intervention was performed using a 6-French guide catheter and the Diamond Back orbital atherectomy device . The lesAfter withdrawal of the device from the catheter system, it was observed that the orbital head was enveloped with wires that had been severed from the braided driveline. The procedure was further complicated by embolization into the femoral artery of the distal segment of the ViperWire  that occOver the past several years, innovations in interventional techniques and devices have afforded physicians the ability to address complex coronary interventions with improvements in clinical outcomes. Despite these advancements, highly calcified coronary lesions still remain a complex clinical entity that present challenges for current coronary devices and techniques. In addition to providing technical challenges, calcified lesions are associated with poorer clinical outcomes including increased frequency of myocardial infarction and in-stent restenosis, which are likely related to issues of poor stent expansion and wall approximation. Indeed, stent under expansion, asymmetric expansion, and stent malposition are frequently found in the intravascular ultrasound (IVUS) evaluations of heavily calcified plaques . IndicatUse of OAS in the management of in-stent restenosis is off-label in the United States. To our knowledge, there are no studies or case reports documenting the use of OAS in ISR, although there is some data demonstrating both the safety and efficacy of a rotational atherectomy device used in infrainguinal ISR lesions .n = 10) or RA (n = 10), OA was associated with deeper dissections, particularly in plaques composed of comparatively more lipid and less calcium. In this small study, there was also a signal toward improved stent expansion following OA; in comparison with RA-treated patients, OA-treated patients had a significantly lower incidence of stent strut malapposition . Whether this distinction will lead to long-term reductions in target lesion failure remains to be confirmed by a prospective, randomized controlled trial. Multicenter Prospective Study to Evaluate Outcomes of the Moderate to Severely Calcified Coronary Lesions (MACE) is needed, which will make available observational data about the real-world application, comparative safety, efficacy, and costs of different treatment strategies for de novo calcified lesions, including OA and RA [Use of the Jetstream® rotational atherectomy device in the infrainguinal vessels demonstrated favorable patency rates and was not associated with adverse outcomes, such as stent perforation or device/stent interactions . To dateA and RA , 8.The patient in our case represented a difficult situation because he had multiple medical ailments prohibiting surgical revascularization and had also endured a previous NSTEMI attributed to in-stent restenosis in a calcified artery. As he was a high surgical risk and rotational atherectomy has been proven efficacious in the treatment of infrainguinal ISR, OAS seemed to be a viable option. OAS did improve luminal patency; however, as noted, the procedure was prematurely aborted due to device failure attributed to stent/device interaction. The embolization of the distal end of the wire was likely related to a fracture of the wire occurring from initial device failure. It has been claimed that the device failure can be attributed to the orbital head skirting behind a stent strut .This could suggest that the workhorse wire initially entered behind a strut with initial cannulation of the vessel. We suggest that using an IVUS with the workhorse wire would allow for visualization of the wire's course and prevent inadvertent deflection of the wire behind the strut. Secondly, IVUS is considerably more accurate than angiography for coronary artery calcification location and permits accurate determination of the calcification burden and vascular dimension , 7, 8.


Robust evidence has demonstrated that cannabis use increases the risk to develop psychotic disorders. However, a limited number of studies have investigated if and how cannabis use influences psychopathology profiles at first episode psychosis (FEP).Based on the evidence that dopamine dysfunction contributes to explain positive symptoms in psychosis, and that the main cannabis’ psychoactive component, Δ9-Tetrahydrocannabinol (THC), modulates the dopamine system, we hypothesise that: 1) positive symptoms at FEP are more common among psychotic patients who used cannabis compared with never users; 2) this association is a dose-response relationship.We analyzed a sample of 1130 FEP patients as part of the EUGEI study, recruited across six countries. The MRC Socio-demographic Schedule was used to collect sociodemographic information. Psychopathology was assessed with the OPerational CRITeria (OPCRIT), and symptom items were analyzed using Mplus to estimate a multidimensional model of psychosis. The Cannabis Experience Questionnaire modified version (CEQmv) was administered to collect information on cannabis, and different patterns of use were computed based on frequency of consumption and type of cannabis, as a proxy of exposure to THC.The lifetime rate of cannabis use was 63%. Fifty-five percent of cannabis users consumed mostly high-potency cannabis, and 46% showed a daily frequency. Mixed-effects linear regression revealed that frequency of cannabis use was associated with the positive symptom dimension score. Daily users of high-potency cannabis presented with the strongest association , even after gender, age, ethnicity, other drug use, and study site were controlled for.Our results show that patients with a history of daily use of high potency cannabis express more positive symptoms at psychosis onset, even after taking into account other substance use and relevant sociodemographic factors.


Traditional Chinese medicines (TCMs) have been used in China for more than two thousand years, and some of them have been confirmed to be effective in cancer treatment. Protein kinases play critical roles in control of cell growth, proliferation, migration, survival, and angiogenesis and mediate their biological effects through their catalytic activity. In recent years, numerous protein kinase inhibitors have been developed and are being used clinically. Anticancer TCMs represent a large class of bioactive substances, and some of them display anticancer activity via inhibiting protein kinases to affect the phosphoinositide 3-kinase, serine/threonine-specific protein kinases, pechanistic target of rapamycin (PI3K/AKT/mTOR), P38, mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) pathways. In the present article, we comprehensively reviewed several components isolated from anticancer TCMs that exhibited significantly inhibitory activity toward a range of protein kinases. These components, which belong to diverse structural classes, are reviewed herein, based upon the kinases that they inhibit. The prospects and problems in development of the anticancer TCMs are also discussed. The protein kinase family encompasses all enzymes in the human body that catalyze the transfer of a phosphate group from a high energy molecule such as adenine triphosphate (ATP) to a specific amino acid in a protein. The human genome codes for more than 500 different protein kinases, which are divided into different families according to their selectivity for substrates  in the United States and used successfully in the treatment of human cancers clinically. In recent years, an increasing number of novel compounds have been isolated from Traditional Chinese Medicines (TCMs), and many of them have been reported to possess potent anticancer activity via inhibition of protein kinase mediated signaling pathways, including PI3K/AKT/mTOR, P38 MAPK, and ERK Figure . In thisMAPK, a serine/threonine specific protein kinase, regulates a variety of biological processes including cell survival, proliferation, differentiation, and apoptosis through downstream signal transduction cascades  Franquet is used in TCM to treat swollen skin, tuberculosis and abscess of the lung. Tubeimoside-1  Gaertn (tartary buckwheat) has been widely used as an important folk medicine in China as a nutritional food. Studies have shown that the herb has multiple benefits including antioxidant, antitumor, antihypotensive, hypoglycemic, and hypolipidaemic effects  Gaertn. Recent study revealed that tatariside G notably inhibited cell viability and induced apoptosis in human cervical cancer HeLa cells through both activation of p38/JNK phosphorylation and inhibition of Akt phosphorylation  and has been widely used alone or in combination with other herbal ingredients for cancer treatment over centuries in China. An injectable formulation of toad venom called cinobufacin (Huchansu) was developed to treat various solid tumors in China in 1990's. Clinical studies have shown that cinobufacin significantly increased the antitumor efficacy of docetaxol or cisplatin in the combination therapy. Decreased toxicity and improved life quality were also observed in the clinical trial with cancer patients  is derived from the dried skin secretions of giant toads  Benth, commonly known as “Guang-huoxiang” in China, is a TCM herb widely used to treat gastrointestinal diseases in many Asian countries  belongs to the family of tyrosine kinase receptors that include erbB2 , erbB3 (HER3), and erbB4 (HER4). EGFR once combined with EGF can promote the related genes in the cell nucleus, leading to cell proliferation. EGFR is commonly highly expressed in a variety of malignant tumors  is believed to possess the activity of nourishing vitality and is widely used in China for patients with poor health condition. Ginsenoside Rg3  has been extensively used to treat several types of chronic inflammatory diseases in TCM  is used to treat diarrhea and ep-igastric pain in TCM. Recent study showed that one of the components myrislignan (Figure in vitro and in vivo (Lu et al., The seed of n Figure  isolatedPeganum harmala L. is used to treat cancer in TCMs and Uygur medicine. A novel compound called 3α-acetoxy-27-hydroxyolean-12-en-28-oic acid methyl ester (OA, Figure 50 value of 269 μM against A549 lung cancer cells (Guo et al., The discussed components here are isolated from TCMs and their modes of actions are summarized in Table in vitro and in vivo. However, the isolated bioactive component, Oleanolic acid, displays much weaker antitumor activity compared to the crude extract in the nude mouse models of xenografted human tumors, suggesting synergistic antitumor effects exist among the components in the extract of clove bud (Liu et al., In recent years, TCM has gained increasing acceptance and attention worldwide, and is recognized as a rich source for drug discovery and development. TCM is widely used in China to reduce the side effects of chemotherapeutic drugs and improve the outcome of conventional treatment. It should emphasize that in some cases, the mixed extracts of TCM display more potent antitumor effects than the single individual component and exhibited synergistic effects with most TCM preparation. Our recent study revealed that the crude extract of clove bud can induce cell death via apoptotic pathway and inhibit the growth of cancer cells both in vivo. It is likely that the efficacy of TCM in cancer treatment may lead to novel strategies in fight against various cancers.Although, chemotherapy is acknowledged as one of the most effective therapeutic methods for cancers in late stage, it faces serious side effects and drug resistance. TCM provide a novel strategy for cancer therapy. However, the underlying mechanisms of most TCM have not been elucidated yet. On the other hands, numerous components isolated from TCMs possess poor bioavailability; novel approaches, including chemical modification, nanotechnology etc. should be employed to improve their efficacy ML, GZ are Equal contributors and co-first authors. ML, GZ, YZ, and XFL consulted literature about Targeting protein kinases with components from Traditional Chinese Medicine. ML, GZ wrote the review. XKL, SC edited and revised the manuscript critically for important intellectual content.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.


Sargassum muticum from both, its native (Japan) and invasive (Germany) range, b) the two non- or weak invasive species Sargassum fusiforme and Sargassum horneri from Japan, and c) Fucus vesiculosus, a native brown alga from Germany. Crude and surface extracts and lipid fractions of active extracts were tested against common fouling organisms and zygotes of a dominant competing brown alga. Extracts of the native brown alga F. vesiculosus inhibited more bacterial strains (75%) than any of the Sargassum spp. (17 to 29%). However, Sargassum spp. from Japan exhibited the strongest settlement inhibition against the diatom Cylindrotheca closterium, larvae of the bryozoan Bugula neritina and zygotes of the brown alga F. vesiculosus. Overall, extracts of S. muticum from the invasive range were less active compared to those of the native range suggesting an adaptation to lower fouling pressure and competition in the new range resulting in a shift of resource allocation from costly chemical defence to reproduction and growth. Non-invasive Sargassum spp. from Japan was equally defended against fouling and competitors like S. muticum from Japan indicating a necessity to include these species in European monitoring programs. The variable antifouling activity of surface and crude extracts highlights the importance to use both for an initial screening for antifouling activity.Competition and fouling defence are important traits that may facilitate invasions by non-indigenous species. The ‘novel weapons hypothesis’ (NWH) predicts that the invasive success of exotic species is closely linked to the possession of chemical defence compounds that the recipient community in the new range is not adapted to. In order to assess whether chemical defence traits contribute to invasion success, anti-bacterial, anti-quorum sensing, anti-diatom, anti-larval and anti-algal properties were investigated for the following algae: a) the invasive brown alga  Increased human travel and trade result in an unprecedented movement of organisms from all phyla across the globe. Today, invasive species represent one of the greatest threats to food security, human and animal health, and biodiversity . ParticuAttempting to explain the dispersal success of certain invasive species, research on chemical defence properties increasingly focuses on comparative studies of invasive and native plants . The ‘‘nNovel metabolites might protect introduced algae from being overgrown, a process known as epibiosis or biofouling , thus, pSome algal metabolites are known to inhibit bacterial quorum sensing (QS), a densitiy dependent microbial cell-to-cell communication , 18. InhSargassum muticum (Yendo) Fensholt 1955 is a well-defended member of the Sargassaceae family and a rich source of phenolic compounds, which are known to possess, amongst others, deterrent characteristics against e.g. herbivores and larval settlement  measurement of algal tissue.(XLSX)Click here for additional data file.


Alcohol and nutrition have the potential to interact at multiple levels. For example, heavy alcohol consumption can interfere with normal nutrition, resulting in overall malnutrition or in deficiencies of important micronutrients, such as zinc, by reducing their absorption or increasing their loss. Interactions between alcohol consumption and nutrition also can affect epigenetic regulation of gene expression by influencing multiple regulatory mechanisms, including methylation and acetylation of histone proteins and DNA. These effects may contribute to alcohol-related organ or tissue injury. The impact of alcohol–nutrition interactions has been assessed for several organs and tissues, including the intestine, where heavy alcohol use can increase intestinal permeability, and the liver, where the degree of malnutrition can be associated with the severity of liver injury and liver disease. Alcohol–nutrition interactions also play a role in alcohol-related lung injury, brain injury, and immune dysfunction. Therefore, treatment involving nutrient supplementation  may help prevent or attenuate some types of alcohol-induced organ damage. The mericans  highligh disease . The benDecreased risk of ischemic stroke ;Protection against type 2 diabetes ;Decrease in rheumatoid arthritis ;Improved cognition ;Decreased progression of liver disease to fibrosis in obese individuals ; andImproved renal function .Indeed, moderate alcohol consumption may be associated with an overall modest survival benefit .Moderate alcohol consumption also has been shown to decrease biomarkers of inflammation, such as C-reactive protein, and reduced inflammation could be one unifying mechanism underlying alcohol’s protective effects . On the Alcohol and nutrition can interact at multiple levels. For example, alcohol metabolism can result in the generation of reactive oxygen species, which can deplete endogenous nutritional antioxidant stores and contribute to oxidative stress. Heavy alcohol consumption also can cause poor intestinal absorption of certain nutrients  or increase nutrient losses . Moreover, nutrition can have a far-reaching impact through altering epigenetic mechanisms, such as methylation and acetylation of DNA and associated proteins. Finally, the degree of alcohol-related malnutrition can be associated with the severity of organ injury . This article reviews how nutritional alterations may predispose to alcohol-induced organ injury and how nutritional supplementation may prevent and/or treat alcohol-induced organ injury. The article specifically highlights the effects of certain alcohol–nutrient interactions, with a focus on zinc and linoleic acid, and their impact on epigenetics and selected organ injury.From a nutrition perspective, alcohol is a significant source of calories, but these can be considered “empty” calories—that is, they contain few micronutrients, such as vitamins and minerals, normally found in most food sources . The maiThe issue of alcohol as a nutrient becomes more prominent when dealing with people with AUD and those with alcohol-induced organ injury. Analyses of the nutritional status of people with AUD admitted to treatment programs found that these individuals often consumed 35 to 50 percent of their total calories as alcohol, and some exhibited inadequate micronutrient intake and micronutrient serum levels . HoweverZinc is an essential trace element required for normal cell growth, development, and differentiation, including such processes as DNA synthesis, RNA transcription, and cell division and activation. It is a critical component of many proteins/enzymes, including zinc-dependent transcription factors. Zinc deficiency or altered zinc metabolism is frequently observed in heavy alcohol drinkers and may result from decreased dietary intake, increased urinary excretion, abnormal activation of certain zinc transporters, and induction of hepatic metallothionein . Zinc deZinc supplementation has been documented to block or attenuate experimental organ injury and dysfunction in the gut, liver, lung, and brain through multiple pathways. Thus, zinc may strengthen the integrity of the intestinal wall by stabilizing tight junctions, reduce transfer of toxic bacterial molecules  into the blood, lower the levels of metabolic toxins such as ammonia in the blood, decrease production of inflammation-promoting  cytokines, reduce oxidative stress, and attenuate apoptotic cell death  . The dosThe critical role for specific types of dietary fat  in intestinal and liver injury has been demonstrated and extensively studied in preclinical animal models of alcohol feeding using various sources of dietary lipids. Experimental evidence has shown that dietary saturated fats (SFs) attenuated, and unsaturated fats (USFs) enhanced, alcohol-induced liver damage . Thus, iFurther analyses focused on the role of different types of dietary polyunsaturated fatty acids (PUFAs) in alcohol-induced gut and liver injury. There are two major families of dietary PUFAs— omega-6 [ω-6] and omega-3 [ω-3] PUFAs—each of which includes numerous related metabolites. It has been demonstrated that linoleic acid, an ω-6 PUFA [18:2ω-6], is required for the development of experimental alcohol-induced intestinal and liver injury and that the severity of alcoholic liver disease (ALD) is correlated with the amount of linoleic acid in the diet . ConversIn virtually every cell type, epigenetic mechanisms—that is, modifications to the genetic material that do not alter the DNA sequence—play a critical role in both the physiologic and pathologic regulation of gene expression. These mechanisms, which involve chromatin remodeling initiated by posttranslational modifications of histones and changes in DNA methylation status, can activate or deactivate gene transcription. The proteins that are involved in posttranslational histone modifications and DNA methylation changes require a variety of cofactors, including acetyl coenzyme A, S-adenosylmethionine (SAM), nicotinamide adenine dinucleotide, and zinc . A persoIt is becoming increasingly evident that histone-associated epigenetic modifications, such as histone acetylation and methylation, play a significant role in the regulation of gene expression and development of alcohol-induced organ pathology, such as liver disease and immune dysfunction . In partInvestigation of the dietary influences on epigenetic processes has revealed a direct link between SAM, which serves as the primary biological methyl donor, and DNA methylation changes that epigenetically influence gene expression . In geneExcessive alcohol consumption can decrease SAM levels via multiple mechanisms, such as reduced folate levels and inhibition of key enzymes in one-carbon metabolism. The reduced SAM levels lead to aberrant DNA methylation patterns and pathogenic alterations in gene expression . ImportaThe intestinal mucosa plays a critical role in preventing passage of toxins from the intestine into the bloodstream, as well as in immune function, detoxification, and metabolism. The importance of the gut in alcohol-mediated multiorgan pathology is becoming increasingly recognized. Clinical and experimental data have demonstrated that the gut-derived bacterial product, lipopolysaccharide, also referred to as endotoxin, plays a crucial role in the development and progression of alcohol-induced organ injuries, including ALD. Significantly increased endotoxin levels in the blood  have been found in patients with different stages of ALD, including fatty liver, hepatitis, and cirrhosis .Multiple mechanisms contribute to alcohol-associated endotoxemia, including alcohol-mediated alterations in the composition of the bacterial population of the gut   and incrThe animals that received EtOH+ USF showed increased gut permeability and elevated endotoxemia compared with those that received EtOH+SF  figure .Compared with EtOH+SF, a chronic EtOH+USF diet triggered an intestinal proinflammatory response characterized by increased levels of several cytokines, including tumor necrosis factor-α and monocyte chemoattractant protein-1. In addition, the intestinal mucus layer and antimicrobial defenses were altered .Intestinal inflammation was positively correlated with the EtOH+USF-triggered disruption of the intestinal tight junctions . Even inUnlike EtOH+SF, dietary EtOH+USF caused alterations in gut microbiota . In summary, an important component of alcohol-induced organ inflammation/injury arises in the gut and may be modified by nutrition.Patients with severe alcoholic hepatitis almost invariably demonstrate some form of malnutrition. Probably the most detailed information concerning malnutrition in ALD comes from two large studies from the Veterans Health Administration (VA) Cooperative Studies Program in patients with alcoholic hepatitis , 1995a,bInitial interest in nutrition therapy for ALD was stimulated by Enteral nutrition supplements also have been shown to improve nutritional status and immune function in outpatients with alcoholic cirrhosis as well as to reduce hospitalization. The concept of an outpatient late-evening snack (prior to bedtime) was established after studies demonstrated altered energy metabolism in people with liver cirrhosis. These patients exhibit depleted hepatic glycogen stores, which force the body to depend on fat and protein stores, leading to catabolism during an overnight fast. A randomized controlled trial demonstrated that provision of a late-evening nutritional supplement (compared with daytime supplements) over a 12-month period could improve body protein stores in patients with cirrhosis. The nighttime snack resulted in body protein accrual equivalent to about 2 kg of lean tissue sustained over 12 months, whereas this benefit was not observed with daytime snacks. Thus, late-evening snacks are valuable nutritional interventions in outpatients with alcoholic cirrhosis .Many types of nutritional supplements have yielded positive effects in animal models of ALD, especially antioxidants. However, human studies using specific nutrients or combination therapy are limited and generally have shown equivocal or negative results. Larger, well-designed studies are required.Chronic alcohol abuse alters the phenotype of the lung and makes it more susceptible to subsequent challenges, such as bacterial infection and acute lung injury. One of the mechanisms that contribute to increased susceptibility to infection and injury is alcohol-induced oxidative stress. Oxidative stress is defined as an imbalance between oxidants and antioxidants, and the way cells sense and respond to such an imbalance is a key determinant of disease initiation/progression or resolution. Oxidant-sensing and -signaling pathways rely primarily on proteins with reactive thiol-containing cysteine residues. The reactivity of a given protein thiol can be fine tuned by its local redox environment—that is, by the ratio of reduced versus oxidized molecules in the cell. This redox environment largely is controlled by two low-molecular-weight thiol-disulfide redox couples: one composed of the amino acid cysteine (Cys), which is the reduced partner of the pair, and its disulfide cystine (CySS), which serves as the oxidized partner. The other redox pair comprises glutathione (GSH) as the reduced partner and its disulfide GSSG as the oxidized partner. The two pairs are related but have different roles. Cys is one of the three component amino acids making up GSH, so it is not surprising that they share similar chemical properties. However, these redox control systems are compartmentalized; GSH/GSSG provides control mechanisms within cells and in the lung-lining fluid, whereas Cys/CySS predominates in the extracellular fluids of plasma and interstitium. The extracellular Cys/CySS redox state has been shown to have a direct effect on the production of two important proinflammatory cytokines, namely production of transforming growth factor β by lung fibroblasts  and inteAccumulating evidence suggests that the Cys/CySS and GSH/GSSG redox couples can be controlled by the diet. Dietary supplementation with the cysteine precursors N-acetylcysteine or procysteine has been used extensively to counteract the effects of oxidative stress. Although the effects of these cysteine precursors usually are attributed to enhanced GSH synthesis, they also are effective even when given in combination with a GSH-synthesis inhibitor  . Recent Zinc deficiency, particularly within immune cells in the lungs , also contributes to increased susceptibility to bacterial infection in chronic alcoholics . StudiesPrenatal alcohol exposure can result in a range of detrimental effects, including damage to the developing brain, that are collectively known as FASD. Early autopsy studies, as well as more recent magnetic resonance imaging studies in both animal models and humans have revealed a variety of brain abnormalities, including reduced brain size  and anomalies of specific brain structures  after prenatal alcohol exposure . These eAvylocus  and morphological abnormalities . In addiAvylocus . Moreoveand PUMA .It also is well known that nutritional deficiencies contribute to the pathogenesis of FASD and to ethanol-induced damage to the developing brain. Heavy maternal alcohol consumption results in deficiency in nutrients that are critical for fetal development and maternal health, including vitamins A and D, thiamin, folate, and zinc . MoreoveMaternal nutrient supplementation may decrease the risk of FASD and serve as a potential intervention for FASD. Some nutritional interventions target oxidative stress. For example, antioxidant supplements, such as vitamins C and E, can reduce oxidative stress, cell death, and behavioral impairments in animals prenatally exposed to ethanol. Studies in the adult brain have demonstrated that ethanol-induced neuro-inflammation and degeneration can be countered by dietary DHA. Similarly, an ω-3-enriched diet that contains 24.6 percent DHA has been shown to reduce ethanol-induced oxidative stress in the developing brain , consistent with the relationship between dietary fat and organ injury discussed earlier. Other nutritional interventions may work through epigenetic modulations. Supplementation with nutrients that act as methyl donors, including folic acid and choline, may modulate epigenetic profiles and alter the expression of genes important for neurodevelopment. Thus, prenatal folic acid supplementation attenuated ethanol-induced malformations, growth retardation, and neuronal loss , whereasExcessive alcohol consumption has deleterious effects on the immune system. Several clinical and experimental studies have suggested that long-term alcohol use can lead to the dysregulation of both cell-mediated and humoral immunity . EpidemiA number of experimental animal models of ethanol abuse have established that chronic alcohol administration decreases the absolute numbers of CD4+ T cells in the thymus, spleen, lymph nodes, and periphery, as well as the immune function of these cells . SimilarIn subjects with AUD, the combined effects of alcohol metabolism and compromised nutrition led to major nutrient disturbances, including deficiency of the critical nutrient metabolite, SAM. Studies found that levels of SAM as well as of methionine adenosyltransferase (MAT II), the enzyme that converts methionine to SAM, were markedly reduced in cultured CD4+ cells exposed to alcohol. This resulted in a significant upregulation of expression and activity of several enzymes involved in apoptosis, leading to increased apoptotic cell death . MoreoveOverall, these findings have begun to provide critical molecular insights into epigenetic mechanisms underlying the alcohol- and nutrient (SAM)-status–induced immunotoxicity in human CD4+ T cells. Because there currently is no Food and Drug Administration–approved therapy for the treatment of immune suppression associated with chronic alcohol abuse, these findings have the potential to facilitate the development of nutrient (SAM)-based therapy in alcoholic patients.Alterations in nutrition and nutrient metabolism are common in chronic alcoholics and may contribute to alcohol-induced organ injury. Conversely, nutritional supplementation may prevent the development or attenuate the progression of alcohol-induced organ injury. Nutritional supplements may alleviate a nutrient deficiency or act as pharmacologic agents. Such nutrients also may have epigenetic effects. Nutritional supplementation as a therapy is especially attractive because there are currently no Food and Drug Administration–approved therapies for most forms of alcohol-induced organ injury and nutrient supplements are readily available.


Autophagy is a catabolic process by which cells can dispose of damaged content and intracellular microorganisms. Recent evidence implicates autophagy as a crucial repair process necessary to recover from critical illness-induced organ failure. Withholding parenteral nutrition in the acute phase of critical illness activates autophagy and enhances recovery. Several registered drugs have autophagy-stimulating properties, but all lack specificity and none has been investigated in critically ill patients for this purpose. In critical illness, a variety of stressors may induce cellular damage and lead to organ failure. After treatment of the primary condition leading to intensive care unit (ICU) admission and general ICU care, the majority of patients will recover. However, despite maximal care, approximately 10–30% of patients do not swiftly recover and enter a phase of prolonged critical illness, characterized by a prolonged dependency on vital organ support and associated with a high risk of mortality and long-term debilities . The undAutophagy, literally meaning “self-eating”, is a catabolic process by which intracellular content is delivered to the lysosome via an intermediate organelle, the autophagosome . AutophaEarly observational studies attributed sepsis-induced organ damage to the concomitant appearance of autophagosomes. However, without interfering with the process, causality is unclear. Recently, a considerable number of studies have provided clear evidence of potentially insufficient autophagy activation in relation to the degree of cellular stress. A pioneer study identified hallmarks of insufficient autophagy activation in liver and muscle of prolonged critically ill patients, with accumulation of autophagic substrate in the presence of a reduced number of autophagic vacuoles . SimilarThe necessary activation of autophagy in response to severe stress may be affected by artificial feeding and insulin therapy. Human and animal studies have demonstrated significant autophagy suppression by early parenteral nutrition (PN) in critical illness, which coincided with more organ damage and—in patients—a prolonged dependency on vital organ support , 14. AmoHence, autophagy activation emerges as a potentially important therapeutic target in critical illness. Withholding PN in the acute phase of critical illness was found to stimulate autophagy and improve organ recovery . However


Missed opportunities and barriers to vaccination limit progress toward achieving high immunization coverage and other global immunization goals. Little is known about vaccination practices contributing to missed opportunities and barriers among private healthcare providers in Africa.Service Provision Assessments (SPA) of representative samples of health facilities in four African countries  in 2010-2015 were used to describe missed opportunities and barriers for vaccination in public, private for-profit, private not-for-profit and faith-based facilities. Data included vaccination practices, observations during sick child and antenatal visits, and exit interviews following sick child visits.Haemophilus influenza type b antigens) daily ranged 0-77% across countries and facility types. Less than 33% of for-profit facilities in any country offered measles vaccination daily. A minority of public or private providers assessed the child’s vaccination status during a sick child visit , or offered tetanus toxoid during antenatal visits . Very few providers discussed the importance of newborn vaccination.Data from 3,219 health facilities, 11,613 sick child visits and 8,698 antenatal visits were included. A smaller proportion of for-profit facilities offered child vaccination services  than did public facilities . The proportion of facilities offering pentavalent vaccine (diphtheria, pertussis, tetanus, hepatitis B and Substantial missed opportunities for, and barriers to, vaccination were identified across this representative sample of health facilities in four African countries. Strategies are needed to ensure that private and public providers implement practices to minimize barriers and missed opportunities for vaccination. The Glo system” .Little is known about the characteristics of private sector delivery of immunization services in LMICs, including how private and public sector practices differ along lines of service quality and adherence to national immunization policies . The curThe GVAP calls on all countries to reach ≥ 90% national coverage and ≥ 80% coverage in every district for all vaccines in the country’s national immunization schedule by 2020 . Of partMinimizing missed opportunities for vaccination (MOV) is one important strategy to reach and sustain ≥ 90% coverage. MOV occur when an individual presents for any health service and is not provided all vaccinations for which they are eligible. Every preventive and curative health service encounter is an opportunity to vaccinate an eligible individual. MOV are common; a 2014 review in LMICs concluded that MOV occurred in 32%–46% of public health service encounters , 8, inclStrategies for reducing MOV include screening for vaccination status during visits to health facilities for mild illness, ensuring children receive all recommended vaccinations during a single visit, and using facility-based child vaccination registers to help track when individuals are due for vaccination . Both prHealth sector surveys such as Service Provision Assessment (SPA) surveys provide an opportunity for insight into MOV across private and public sectors. SPA surveys are conducted every 3-7 years in LMICs to assess availability, delivery, and quality of services in a representative sample of health facilities. This paper assesses potential barriers and MOV in public and private  health facilities in four African countries that conducted SPA surveys during 2010-2015: Kenya, Tanzania, Senegal and Malawi.SPA surveys collect information on facility-based health services in developing countries using service readiness indicators developed by WHO and the United States Agency for International Development (USAID) , 12. TenTypically, SPA surveys collect data from a nationally representative sample of 400-700 formal sector health facilities . In KenyGenerally, SPA surveys include 10-15 survey teams, each comprised of 3-4 surveyors. During the facility visit, the facility manager, person in-charge of the facility, or the most senior health worker is interviewed using the facility inventory questionnaire to obtain information on availability of health services, resources for these services and procedures followed for providing these services.Surveyors also observe client-provider consultations for a single day using standardized observation protocols for antenatal care, curative care for sick children, and other services , 13. SPAService observations are coupled with exit interviews using standardized questionnaires to document the client’s experience with the services received. For sick child visits, the child’s caregiver  participates in the exit interview. The surveyor reads a list of common topics associated with client dissatisfaction, asking if each topic posed a major or minor problem, or no problem during the visit.Health facility type was defined by operating authority categories used in SPA surveys: 1) public; 2) private for-profit (for-profit), 3) private not-for-profit/non-governmental organization (not-for-profit); and 4) private mission/faith-based organization (faith-based). In Tanzania, parastatal facilities were categorized as private not-for-profit facilities per Tanzania Ministry of Health definitions .SPA surveys conducted during 2010-2015 in African countries  were included in the analyses, using facility-level (inventory) and individual-level (observation protocol and client exit interview) questionnaire data -16, 18. In the Kenya SPA (2010) survey, 703 facilities were randomly sampled from a master list of 6,192 facilities; due to non-response, 695 facilities were included in the final dataset . For theFor Kenya, Tanzania, and Senegal, sample strata were created by crossing region and facility type. When a single unit stratum was present, adjacent strata from the same region were collapsed to calculate sampling errors. This was done separately for facility- and individual-level analyses since some facilities did not have any individual-level observations, resulting in a greater number of single unit strata than at the facility level. At the individual-level, facility was treated as the primary sampling unit. Since Malawi conducted a census of facilities, facility-level observations were not stratified. Individual-level observations were stratified by facility.Each individual-level sick child observation and exit interview was merged with the inventory questionnaire data for the facility at which that observation was conducted. For questions regarding TT receipt or counseling about newborn vaccination, each antenatal care observation was merged with the inventory questionnaire data for the facility at which that observation was conducted.Haemophilus influenza type b antigens) was used as a proxy for general vaccination service delivery. Daily vaccination was defined as provision of vaccination at least 20 days per month. For assessment of caregiver perception of service quality and satisfaction, data from the sick child exit interviews were used. For measured wait times, data from sick child visit observations were used. Percentages were not calculated for facility types within a country if there were ≤ 5 facilities or interviews within that facility type.For availability of vaccination services, supply of immunization cards, and service fees, data from the facility inventory questionnaire were used. Pentavalent vaccine . We used data from the provider’s response when asked We used the survey command in STATA 12, with the provided survey weights from the SPA survey dataset for our analyses, accounting for stratification and clustering as described above. We calculated weighted proportions for each indicator of interest across all facilities in each country and by facility type. We conducted chi-square tests to assess whether the frequency of an indicator significantly differed across the four facility types, and t-tests for pairwise analyses comparing public facilities with each of the other three facility types when the chi-square test indicated overall significance. We used a p-value threshold of ≤ 0.05 to assess statistical significance. For Malawi facility-level indicators only, no statistical tests of significance were conducted since the total number of facilities per strata were not available to calculate a finite population correction to ensure a proper estimate of variance for each indicator.The study included data from 3,219 surveyed facilities in the four countries . In KenyMost facilities in the four countries provided sick child and antenatal care; a lower proportion of for-profit and not-for-profit facilities than public facilities provided antenatal care . We inclIn all countries, higher proportions of public  facilities offered childhood immunization services than did for-profit  facilities . In TanzAmong for-profit facilities, 8%-33% (country range) offered measles vaccine daily compared to 13%-63% that offered pentavalent vaccine daily . A majorThe proportion of facilities that reported charging fees for immunization visits varied among countries. In Tanzania and Malawi, few facilities of any operating authority type  charged fees for immunization visits . In KenyThe proportion of providers who were observed reviewing the child health card for any reason during a sick child visit varied widely by country and facility type . A lowerDuring sick child visits, few providers reported administering vaccinations or referring the child for vaccination . Rather,In each country, ≤ 51% of providers in any facility type administered or prescribed TT to pregnant women . OverallCompared to other facility types, observed mean and median wait times for sick child visits were the shortest in for-profit facilities in each country except Senegal. For example, median wait times were 15, 30 and 10 minutes in for-profit facilities, compared to 30, 60, and 80 minutes in public facilities in Kenya, Tanzania and Malawi respectively (data not shown). In Kenya and Malawi, approximately 25% of caregivers visiting public facilities reported that wait times were a major problem, the highest proportion among the facility types .In Kenya, Tanzania and Malawi, a higher proportion of caregivers at public facilities reported major problems with availability of medicine compared to caregivers at for-profit or faith-based facilities . Only a The majority  of clients in each country reported being very satisfied with services, regardless of facility type. The proportion of caregivers not satisfied with services was low , but in Kenya and Malawi it was higher in public facilities than in for-profit or faith-based facilities. A notable proportion of caregivers sought sick child services at a facility other than the one closest to their home. Of those, more sought care at for-profit facilities  or faith-based facilities , compared to public facilities. The most common reasons for not visiting the facility closest to their residence included the closer facility being too expensive (country range 5%-20%), having a bad reputation , or having no medication , or the caregiver received a referral to the farther clinic .This study was the first to use SPA surveys to compare factors related to vaccination barriers and MOV across public and private health facilities. Data were drawn from nationally representative surveys, whose large sample sizes improve generalizability of results. The high facility response rates (99%) and the use of standardized questionnaires across countries enabled transnational comparisons. We observed differences in barriers to vaccination by facility type, including less availability of child immunization services in for-profit compared to public facilities in all countries, a higher proportion of for-profit facilities charging fees than public facilities in Kenya, and higher proportions of caregivers reporting problems with wait times and unsatisfactory care in public facilities than in for-profit facilities in Kenya and Malawi. We identified common missed opportunities across facility types, including infrequent vaccination screening and vaccine administration during sick child visits, and infrequent TT administration and counseling about newborn vaccination during antenatal care visits. This study helps to fill a knowledge gap about vaccination practices in the private sector and identifies potential opportunities to increase access to and utilization of immunization services across all facility types.Fees for immunizations, whether official or informal, create an important barrier to vaccinations , and theWait times were reported as a major problem across all types of facilities in all four countries. Long wait times are often cited as a barrier to health care seeking , 20. PriDespite problems that caregivers reported with services they received, the majority, across all facility types, reported satisfaction with services, similar to findings from other studies that reported relatively high levels of client satisfaction with the quality of primary health care services received , 22. CliA significant proportion of providers across countries and facility types did not screen sick children for eligible vaccinations, and vaccination was uncommon during sick child visits. Immunization histories of the sick children and their illness severity are unknown, so it is not possible to ascertain the proportion of children eligible for vaccination who were not screened. A recent study in Dominican Republic  found thAdditionally, we identified a potential MOV specific to measles vaccination; the majority of surveyed facilities, particularly for-profit facilities, reported providing measles vaccination less frequently than pentavalent vaccination. This practice may stem from the requirement that a measles vaccine vial be discarded within six hours of being opened, unlike pentavalent vaccine which can be stored for later use. Providers may be reluctant to open a multi-dose measles vaccine vial when only a few children present for vaccination; instead the child is told to return on a special measles vaccination day , 26. TurPregnant women receiving antenatal care often did not receive TT. Thus, most providers missed an important opportunity to protect neonates from tetanus at birth. TT vaccination contributes significantly to the elimination of neonatal tetanus. Across all facility types, there is a need to educate or re-orient providers on the importance of TT for pregnant women. Antenatal visits also provide an opportunity to increase awareness about newborn immunization, yet our results indicate such education rarely occurred. It is important for policy makers and service providers to make use of the opportunity that antenatal visits offer to improve maternal and child health. Further, an additional MOV for TT vaccination for women exists when women bringing their infants for routine immunization or sick child visits are not screened and vaccinated as indicated.Some facilities did not provide sick child or antenatal care on the day of the surveys, so observations and exit interviews were not conducted there. The characteristics of these facilities are unknown. However, of facilities that provided sick child or antenatal care, the majority contributed sick child observations (85%) or antenatal care observations (74%), likely minimizing potential bias. Variations in country context relative to health systems (including private provider practices) and health care seeking behavior may complicate country comparisons and limit generalizability to other LMICs.We used exit interviews following sick child visits to assess caregiver perceptions of care. Assessments following immunization visits may have yielded more specific information about provider immunization practices, but were not available in SPA surveys. The method of conducting exit interviews at the health facility may predispose clients to give socially desirable answers, resulting in higher reported satisfaction, but the addition of structured observation may mitigate some of this potential bias. Our analysis focused on generating hypotheses for future studies and was not intended to assess causal associations. To do so would require study designs incorporating collection and analysis of applicable covariate data alongside exposure and outcome data. Not all countries participate in SPA surveys and they are infrequent, limiting broader analysis of SPA survey data.Despite substantial progress in increasing the number of children vaccinated worldwide, over a third of all countries have not yet met the Global Vaccine Action Plan 2011–2020 target of 90% national DTP3 coverage . We founThe African region has the lowest immunization coverage of all WHO regions (76% DPT3 coverage in 2015);Reducing missed opportunities for and barriers to vaccination can improve immunization coverage and reduce immunity gaps;There is limited information about immunization practices in private health facilities; very few studies have compared immunization practices in public and private facilities in low and middle income countries.Service provider assessments provide useful large-scale and representative information about immunization in both public and private health facilities, and enabled identification of substantial missed opportunities and barriers to vaccination in four African countries;A smaller proportion of for-profit health facilities than public facilities in four African countries offer childhood immunization services. Less than a third of for-profit facilities offered measles vaccination daily;A minority of public and private providers in four African countries assessed immunization status of children during sick child visits, offered tetanus toxoid vaccine to pregnant women, or discussed the importance of newborn vaccination during antenatal visits.The authors declare no competing interest.