Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often  exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely,  In 1838, the work of several scientists including Matthias Jakob Schleiden, Theodor Schwann and Rudolf Carl Virchow culminated in the so-called ‘cell theory', postulating that: (1) all living organisms are composed of one or more cells; (2) the cell is the basic unit of life; and (3) all cells arise from pre-existing, living cells.bona fide living organisms.5 However, the discovery that the giant Acanthamoeba polyphaga mimivirus can itself be infected by other viral species has casted doubts on this point.8 Thus, the features that underlie the distinction between a living and an inert entity remain a matter of debate. Along similar lines, defining the transition between an organism's life and death is complex, even when the organism under consideration is the basic unit of life, a cell. From a conceptual standpoint, cell death can obviously be defined as the permanent degeneration of vital cellular functions. Pragmatically speaking, however, the precise boundary between a reversible alteration in homeostasis and an irreversible loss of cellular activities appears to be virtually impossible to identify. To circumvent this issue, the Nomenclature Committee on Cell Death (NCCD) previously proposed three criteria for the identification of dead cells: (1) the permanent loss of the barrier function of the plasma membrane; (2) the breakdown of cells into discrete fragments, which are commonly referred to as apoptotic bodies; or (3) the engulfment of cells by professional phagocytes or other cells endowed with phagocytic activity.11A corollary of the cell theory is that viruses do not constitute 14 Indeed, it has been reported that engulfed cells can be released from phagosomes as they preserve their viability, at least under some circumstances.15 Thus, the NCCD recommends here to consider as dead only cells that either exhibit irreversible plasma membrane permeabilization or have undergone complete fragmentation. A compendium of techniques that can be used to quantify these two markers of end-stage cell death in vitro and in vivo goes beyond the scope of this review and can be found in several recent articles.25However, the fact that a cell is engulfed by another via phagocytosis does not imply that the cell-containing phagosome fuses with a lysosome and that the phagosomal cargo is degraded by lysosomal hydrolases.in vivo, for instance as a result of burns or traumatic injuries, it cannot be prevented or modulated and hence does not constitute a direct target for therapeutic interventions.28 Nonetheless, cells exposed to extreme physicochemical or mechanical insults die while releasing elevated amounts of damage-associated molecular patterns (DAMPs), that is, endogenous molecules with immunomodulatory (and sometimes cytotoxic) activity. Some DAMPs can indeed propagate an unwarranted cytotoxic response (directly or upon the involvement of innate immune effectors) that promotes the demise of local cells surviving the primary insult.31 Intercepting DAMPs or blocking DAMP-ignited signaling pathways may mediate beneficial effects in a wide array of diseases involving accidental (as well as regulated) instances of cell death.32Importantly, cell death instances can be operationally classified into two broad, mutually exclusive categories: ‘accidental' and ‘regulated'. Accidental cell death (ACD) is caused by severe insults, including physical , chemical  and mechanical  stimuli, is virtually immediate and is insensitive to pharmacologic or genetic interventions of any kind. The NCCD thinks that this reflects the structural disassembly of cells exposed to very harsh physicochemical conditions, which does not involve a specific molecular machinery. Although ACD can occur 33 Thus, the course of RCD can be altered by means of pharmacologic and/or genetic interventions targeting the key components of such a machinery. Moreover, RCD often occurs in a relatively delayed manner and is initiated in the context of adaptive responses that (unsuccessfully) attempt to restore cellular homeostasis.38 Depending on the initiating stimulus, such responses can preferentially involve an organelle, such as the reticular unfolded protein response, or operate at a cell-wide level, such as macroautophagy (hereafter referred to as autophagy).44 Thus, while ACD is completely unpreventable, RCD can be modulated  not only by inhibiting the transduction of lethal signals but also by improving the capacity of cells to mount adaptive responses to stress.50 Importantly, RCD occurs not only as a consequence of microenvironmental perturbations but also in the context of (post-)embryonic development, tissue homeostasis and immune responses.54 Such completely physiologic instances of RCD are generally referred to as ‘programmed cell death' (PCD)  involves a genetically encoded molecular machinery.h' (PCD) .9, 33execution is generally used to indicate the ensemble of biochemical processes that truly cause the cellular demise. Conversely, initiation is commonly used to refer to the signal transduction events that activate executioner mechanisms. Thus, the activation of caspase-8 (CASP8) in the course of FAS ligand (FASL)-triggered apoptosis is widely considered as an initiator mechanism, whereas the consequent activation of caspase-3 (CASP3) is categorized as an executioner mechanism (see below).57For the purpose of this discussion, it is useful to keep in mind the distinction that is currently made between the initiation of RCD and its execution. The term essential and accessory aspects of RCD, that is, between those that etiologically mediate its occurrence and those that change its kinetics or morphologic and biochemical manifestations.Here, the NCCD formulates a set of recommendations to discriminate between 20 In 1964, the American biologist Richard A Lockshin was the first to thoroughly describe the demise of intersegmental muscles in developing silk moths, a seminal contribution to the modern understanding of PCD.58 A few years later, in 1972, the Australian pathologist John F Kerr together with his Scottish colleagues Andrew H Wyllie and Alastair R Currie coined the term ‘apoptosis'  type I cell death (apoptosis), manifesting with the morphologic features described above; (2) type II cell death (autophagy), featuring an extensive vacuolization of the cytoplasm; and (3) type III cell death (necrosis), exhibiting neither apoptotic nor autophagic characteristics.60 Such a ‘visual catalog' has dominated the field of cell death research for decades. Nonetheless, the NCCD views it as an oversimplification and considers it rather misleading, for several reasons.The early classifications of cell death were purely morphologic, owing to obvious technical limitations.11 Along similar lines, apoptosis was misconceived as an immunologically silent, if not tolerogenic, cell death modality.62 It is now clear that PCD does not always manifest with an apoptotic morphotype and does not necessarily fail to induce inflammatory or immune responses. For instance, the degradation of Drosophila melanogaster salivary glands and larval midgut relies on PCD manifesting with a type II morphology,70 whereas the remodeling of bones at the growth plates is associated with type III-like features.71 Moreover, specific inducers are capable of promoting a variant of RCD that displays an apoptotic morphotype, yet is capable of activating adaptive immune responses.73 These observations indicate that morphologic and functional aspects of cell death are not necessarily linked to each other.First, when this classification was formulated, necrosis (as defined by morphologic features) was considered as a strict equivalent of ACD, whereas apoptosis (as defined by morphologic features) was viewed as the sole programmed subroutine of cell death.74 Indeed, necrosis can manifest with a stereotyped panel of features, including a generalized swelling of the cytoplasm, which acquires a translucent aspect, and organelles (oncosis), as well as a peculiar alteration of chromatin  and the nuclear membrane (dilatation).74 The evolution of the morphologic characterization of necrosis reflects the relatively recent discovery that RCD can also manifest with a necrotic aspect (see below).Second, the negative morphologic definition of necrosis as a cell death modality that fails to exhibit apoptotic or autophagic features has been reconsidered.75 Such an expression was coined based on morphologic considerations  only, but it soon became misused to imply that the molecular machinery of autophagy would actively contribute to the cellular demise.75 The NCCD strongly recommends the use of the expression ‘autophagic cell death' from a functional perspective only, that is, to indicate a cell death subroutine that is limited or delayed by the pharmacologic or genetic inhibition of the autophagic machinery (see below).76Third, the use of the term ‘autophagic cell death' has been a matter of intense debate.10 Moreover, several pharmacologic agents and genetic interventions designed to inhibit the execution of cell death often fail to do so when administered in a therapeutic (as opposed to prophylactic)77 manner, at least in the mammalian system, yet efficiently change its morphology.91 This applies to N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (Z-VAD-fmk) and (3S)-5--3-[[(2S)-3-methyl-1-oxo-2-[(2-quinolinylcarbonyl)amino]butyl]amino]-4-oxo-pentanoic acid hydrate (Q-VD-OPh), two broad-spectrum caspase inhibitors that have been widely investigated in the late 1990s as a means to mediate clinically relevant cytoprotection.95 Z-VAD-fmk and Q-VD-OPh prevent the appearance of several morphologic markers of apoptosis, including nuclear pyknosis and blebbing (which rely on caspases),97 yet fail to limit stimulus-dependent cell death if administered in therapeutic settings .100 Thus, caspase inhibition most often results in a shift from an overtly apoptotic to a mixed or necrotic cell death morphology.91 Conversely, both necrostatin 1 (Nec-1), a highly specific small inhibitor of the enzymatic activity of receptor-interacting protein kinase 1 (RIPK1), and geldanamycin, which targets heat-shock protein 90 kDa alpha (cytosolic), class A member 1 (HSP90AA1), have been demonstrated to shift the necrotic morphotype of RCD induced by tumor necrosis factor (ligand) superfamily, member 10  at slightly acidic pH to an apoptotic one.101 A similar morphologic shift has been observed in cells succumbing to tumor necrosis factor receptor superfamily, member 1A  ligation in the presence of geldanamycin102 and in the absence of RIPK1 or one of its downstream targets, that is, receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL).104 Also, cells exposed to DNA-alkylating agents in the presence of poly(ADP-ribose) polymerase 1 (PARP1) inhibitors die while manifesting an apoptotic, rather than a necrotic, morphology.106 Conversely, the introduction of a non-cleavable PARP1 variant appears to convert the apoptotic phenotype of cells succumbing to FAS ligation into a necrotic one.107 Possibly, this reflects the ability of PARP1, an NAD+-dependent enzyme initially characterized for its role in DNA repair and the DNA damage response,109 to provoke an abrupt decline in intracellular ATP levels (secondary to NAD+ depletion), hence blocking various morphologic manifestations of apoptosis.115 Such a morphologic shift, however, does not appear to stem from the inhibition of caspases, because neither the catalytic functions nor the activation of these proteases require ATP .119Fourth, many instances of RCD present both apoptotic and necrotic traits.bona fide cytoprotection, casting doubts on the actual value of morphology-based classifications of cell death.9In summary, the morphologic manifestations of cell death can easily be altered in the absence of 9 In substitution, the NCCD identified the main molecular events associated with specific cell death subroutines as well as the pharmacologic and/or genetic interventions that may be used to discriminate between various instances of cell death in experimental settings, in vitro and in vivo.9In 2012, the NCCD proposed to abandon the morphologic catalog of cell death instances in favor of a new classification based on quantifiable biochemical parameters.121 This notion began to emerge in the late 1980s,122 but became widely accepted only two decades later, owing to the milestone discoveries of Peter Vandenabeele,127 Jurg Tschopp128 and Junying Yuan,131 and to the characterization of the key role played by peptidylprolyl isomerase F  in necrotic instances of RCD.135 The identification of a genetically encoded machinery that promotes RCD with necrotic features generated an intense wave of investigation that has not yet come to an end.121Since then, our comprehension of specific RCD modalities has progressed significantly. Thus, while no paradigm-breaking discoveries have been made on the regulation and execution of caspase-dependent RCD instances (which most often display an apoptotic morphology), profound insights have been obtained into the mechanisms underlying cases of RCD that do not depend on caspases and generally manifest with necrotic features.51 Other events commonly associated with apoptosis, such as the exposure of phosphatidylserine on the outer leaflet of the plasma membrane, are indeed less universal and more context-dependent139 than previously thought.140 Apoptosis can be initiated by intracellular (intrinsic) or extracellular (extrinsic) stimuli. Intrinsic apoptosis critically relies on mitochondrial outer membrane permeabilization (MOMP), a process that results in the holocytochrome c (CYTC)-, deoxy-ATP- and apoptotic peptidase-activating factor 1 (APAF1)-dependent activation of caspase-9 (CASP9) and CASP3.144 MOMP obligatorily requires either of two Bcl-2 family members, namely, B-cell CLL/lymphoma 2 (BCL2)-associated X protein (BAX) and BCL2-antagonist/killer 1 (BAK1), whose pore-forming activity is inhibited (both directly and via indirect circuitries) by other components of the family, including BCL2 itself, BCL2-like 1  and myeloid cell leukemia 1 (MCL1).148 Importantly, the physical and functional interactions between pro- and antiapoptotic multidomain BCL2-like proteins are under the control of small components of the family known as BH3-only proteins, including (but not limited to) BCL2 binding component 3 , BCL2-like 11  and BH3-interacting domain death agonist (BID).151From a biochemical standpoint, apoptosis is defined as a caspase-dependent variant of RCD.β cells and multiple neoplastic cells) also involves MOMP, owing to the CASP8-dependent activation of BID.157 Whether the apoptotic response to extracellular cues requires MOMP or not reportedly depends on the expression levels of X-linked inhibitor of apoptosis (XIAP),159 an ubiquitin ligase with multipronged cytoprotective functions.161 High amounts of XIAP prevent indeed the direct activation of CASP3 by CASP8, a block that can be circumvented by the release of diablo, IAP-binding mitochondrial protein  and other XIAP inhibitors into the cytosol following MOMP.164Extrinsic apoptosis proceeds along with the activation of a CASP8/CASP3 signal transduction axis that in some cell types  agonists or type I interferons, which relies not only on RIPK3 but also on TLR adaptor molecule 1 .184 In the absence of RIPK1, TLR agonists and type I interferons trigger necroptosis even in caspase-competent cells,183 suggesting that RIPK1 can inhibit necroptotic RCD as induced by these stimuli at two distinct levels. Furthermore, RIPK1 tonically suppresses CASP8 and RIPK3 activation in developmental scenarios independent of its kinase activity.186 This explains why Ripk1−/− mice fail to survive to adulthood even in the absence of Fas (TNFRSF6)-associated via death domain (Fadd), which is required for CASP8 activation by extracellular cues, but mature normally in the absence of both Ripk3 and Fadd or Casp8.183 Other instances of necroptosis, such as those promoted by Z-DNA-binding protein 1 (ZBP1) in response to viral infection, appear to proceed independently of RIPK1.187 Upon phosphorylation by RIPK3, MLKL has a critical, non-redundant role in necroptosis.179 Phosphorylated MLKL forms indeed oligomers that translocate to cellular membranes (including the plasma membrane) and bind specific phospholipids, resulting in the loss of barrier function.188It recently became clear that the main players in the RCD subroutine commonly referred to as necroptosis, which we previously defined as a caspase-independent, RIPK1- and RIPK3-dependent lethal signaling pathway initiated by death receptors,189 Moreover, contrary to initial beliefs,190 the lethal activity of RIPK3 is not influenced by the absence of phosphoglycerate mutase family member 5 (PGAM5) and dynamin 1 like .176 Based on these results, the NCCD proposes here to redefine necroptosis as an RCD modality that critically depends on MLKL and on the kinase activity of RIPK1 (in some settings) and RIPK3. Of note, both RIPK1 and RIPK3 have been shown to regulate caspase activation, at least under some circumstances.193 Taken together, these observations suggest that the signal transduction cascades responsible for the initiation of apoptosis and necroptosis are highly interconnected.Recent data argue against an essential role for mitochondria in necroptosis. Indeed, parkin RBR E3 ubiquitin protein ligase (PARK2)-overexpressing cells depleted of the vast majority of mitochondria upon the induction of mitophagy  become resistant to inducers of MOMP-dependent RCD, but remain sensitive to TNFR1 ligation in the presence of Z-VAD-fmk .197 three key components of caspase-dependent RCD initiated by death receptors.203 Taken together with the notion that the absence of either Ripk3, Casp8 or Fadd fails to rescue Ripk1−/− mice from neonatal lethality,185 these results pointed to the existence of a switch mechanism that regulates cell fate upon TNFR1 ligation.205 Intriguingly, such switch may not operate in all cell types, as demonstrated by the fact that Ripk1−/− intestinal epithelial cells are fully rescued by the concomitant absence of Casp8 .Necroptosis is actively inhibited by a supramolecular complex containing CASP8, FADD and the long isoform of CASP8 and FADD-like apoptosis regulator ,206 In comparatively more physiologic conditions, however, the fate of cells exposed to death receptor ligands may be determined by the activation kinetics of mitogen-activated protein kinase kinase kinase 7 ,207 which normally initiates a cytoprotective response centered around the transcription factor NF-κB and autophagy,212 or by the availability of baculoviral IAP repeat containing (BIRC) family members,193 ubiquitin ligases with a central role in TNFR1 signaling.213 In line with this notion, cells treated with a SMAC mimetic (resulting in the depletion of BIRC2 and BIRC3) or a chemical TAK1 inhibitor (NP-009245) reportedly respond to TNFR1 ligation by activating caspases in a RIPK1-dependent manner.192 Taken together, these observations indicate that death receptors generate a lethal stimulus that can be propagated along several signal transduction cascades. Thus, caution should be used in evaluating necroptotic instances of cell death based on their sensitivity to Nec-1 only.Recent data obtained with genetically engineered RIPK1 and RIPK3 variants indicate that the catalytic pathways activated in response to death receptor ligation depend on the availability of CASP8, FADD and MLKL.214 At present, CYPD is the sole genetically confirmed component of the permeability transition pore complex (PTPC) in the mammalian system.217 The term PTPC generally refers to a supramolecular complex operating at the junctions between the inner and outer mitochondrial membrane to cause the so-called ‘mitochondrial permeability transition' (MPT), an abrupt increase in the permeability of the inner mitochondrial membrane to small solutes triggered by cytosolic Ca2+ overload or oxidative stress.221 Unlike MOMP,224 MPT seals the cell fate independently of caspase activation.226 Nonetheless, MPT-driven RCD can manifest with (at least some) morphologic features associated with apoptosis,228 corroborating the limited informative value of cell death classifications solely based on morphology. The NCCD recommends the use of the term ‘MPT-driven RCD' for instances of cell death whose course can be influenced with the genetic or pharmacologic inhibition of CYPD or other components of the PTPC. Of note, CYPD surely does not constitute the long-sought pore-forming unit of the PTPC, which most likely involves subunits of the so-called ‘ATP synthasome', the supramolecular complex that imports ADP and inorganic phosphate into the mitochondrial matrix, catalyzes ATP synthesis and exports ATP back to the mitochondrial intermembrane space (from where it can easily reach the cytosol).234 Perhaps, the central role of CYPD in MPT-driven RCD reflects its ability to control the Ca2+-buffering capacity of the mitochondrial network.236 This hypothesis has not yet been formally addressed.Another variant of RCD that often, although not always, manifests with a necrotic morphotype critically relies on CYPD.33 The main molecular features of parthanatos are the hyperactivation of PARP1 and the release of apoptosis-inducing factor, mitochondrion-associated, 1 (AIFM1) from the mitochondria.240 Interestingly, although TNFR1-driven necroptosis and parthanatos have been suggested to constitute completely independent RCD subroutines,241 this issue remains a matter of debate.242 Possibly, such a controversy originates from the ability of some insults to simultaneously trigger necroptosis and parthanatos, at least in some model systems.243 Ferroptosis has been defined as an iron-dependent form of RCD under the control of glutathione peroxidase 4 (GPX4).247 Both the pharmacologic and genetic inhibition of CYPD fail to prevent ferroptosis as triggered by erastin, a small molecule that is selectively lethal for cancer cells expressing oncogenic variants of Harvey rat sarcoma viral oncogene homolog (HRAS).248 This suggests that ferroptosis and MPT-driven RCD constitute independent variants of RCD. Of note, erastin inhibits system xC−, an heterodimeric antiporter of the plasma membrane that normally exchanges intracellular glutamate for extracellular cysteine, resulting in glutathione depletion and iron-dependent accumulation of reactive oxygen species.247 A similar cascade of events contributes to  the death of neurons exposed to glutamate. This necrotic instance of RCD has previously been referred to as oxytosis.249 Besides inhibiting system xC−, glutamate can trigger MPT-driven RCD upon the hyperactivation of ionotropic receptors, a neurotoxic process commonly known as excitotoxicity.251Two forms of RCD other than necroptosis and MPT-driven RCD have recently attracted attention as potential targets for the development of cytoprotective interventions, namely ‘parthanatos' and ‘ferroptosis'.76 The NCCD recommends using this term only for RCD instances that can be influenced by the pharmacologic or genetic interventions targeting at least two distinct components of the molecular machinery for autophagy.76 While autophagy accompanies RCD in a vast number of pathophysiologic settings,252 it truly contributes to the cellular demise only in a few of them.259 Beth Levine's laboratory has recently discovered a bona fide instance of autophagic cell death that relies on the plasma membrane Na+/K+-ATPase, and dubbed it ‘autosis'.255 Of note, autosis occurs not only in vitro, in cells exposed to cell permeant autophagy-inducing peptides, but also in vivo, in the brain of rats subjected to an ischemic insult.255 It remains to be determined whether all cases of autophagic cell death require the Na+/K+-ATPase or not. If so, the terms ‘autosis' and ‘autophagic cell death' would be synonymous. If not, autosis would constitute a special instance of autophagic cell death.260Caspase-unrelated variants of RCD include ‘autophagic cell death', which (among other processes) is biochemically associated with the lipidation of microtubule-associated protein 1 light chain 3  and the degradation of sequestosome 1 .in vitro and in vivo), Z-VAD-fmk and more specific CASP3 inhibitors administered as therapeutic (as opposed to prophylactic)77 interventions fail to significantly limit primary RCD, in spite of the fact that they efficiently limit caspase activation.265 In some of these scenarios, RCD overtly manifests with alternative biochemical processes, including RIPK1, RIPK3 or PARP1 activation, and (at least in part) can be influenced by agents that interfere with these pathways, including Nec-1, 3-aminobenzamide (a PARP1-targeting agent) and necrosulfonamide (an inhibitor of human MLKL).264 However, the proportion of cells eventually succumbing to RCD does not change. The depletion of BIRC2/BIRC3, RIPK1, RIPK3 or MLKL, and the administration of NP-009245, Nec-1 or geldanamycin (which indirectly destabilizes RIPK1)266 reportedly changes the kinetics of necroptosis and its biochemical profile, that is, it allows for caspase activation, yet fails to block the cellular demise.267 Along similar lines, 3-aminobenzamide and 4-amino-1,8-naphthalimide (another PARP1 inhibitor) can convert the cytotoxic response to alkylating DNA damage or TNFR1 ligation from a caspase-independent one to apoptosis, manifesting with caspase activation.107Importantly, a growing body of evidence indicates that the pharmacologic or genetic inhibition of the processes that are commonly considered as essential for cell death execution often does not avoid the demise of mammalian cells, but rather alters its kinetics and biochemical (and morphologic) manifestations. Thus, in many experimental paradigms . Two mutually exclusive models can be put forward to explain how adaptive stress responses promote RCD when unsuccessful. First, a ‘conversion model' postulates that RCD-inhibitory signals cease at some stage of the adaptive response and are replaced by RCD-promoting ones. Second, a ‘competition model' hypothesizes that RCD-inhibitory and -promoting signals coexist and counteract with each other starting from the detection of microenvironmental alterations, but at some stage the latter predominate over the former  the removal of the initiating stimulus (when possible), (2) the repair of molecular and/or organelle damage, and (3) eventually, the re-establishment of physiologic conditions.e former . Althougirreversibly and causally seal the cell fate, and the term ‘propagation' to indicate the processes that link primary (stimulus-dependent) RCD to the stimulus-independent initiation of a secondary RCD wave, including the release of DAMPs and the consequent inflammatory response (Ppif (the CYPD-coding gene),274Ripk3278 and Mlkl,177 as well as to the administration of chemical CYPD inhibitors 274 and Nec-1.277 Conversely, pharmacologic and genetic interventions expected to interrupt RCD at late steps of the process (when cells are commonly considered as committed to die) generally fail to confer significant long-term cytoprotection in mammalian models, casting doubts on the actual etiological value of these steps for RCD. Thus, Casp3−/−, Casp9−/− and Apaf1−/− mice display a consistent hyperplasia of the central nervous system associated with reduced amounts of PCD in specific cerebral areas, resulting in embryonic or perinatal lethality.284 However, the neuronal phenotype of Casp3−/− mice does not develop in all genetic backgrounds,286 and the penetrance of the perinatal lethality associated with the Apaf1−/− genotype is incomplete .287 Moreover, the developmental death of interdigital cells  occurs close to normally  in mice bearing a homozygous loss-of-function mutation in Apaf1, and in mice exposed to broad-spectrum caspase inhibitors.80 In these settings, however, the demise of interdigital cells cannot be detected by the terminal-deoxynucleotidyl-mediated dUTP nick end-labeling (TUNEL) assay, measuring caspase-dependent DNA fragmentation.80 Conversely, the simultaneous ablation of Bax and Bak1288 or Bcl2l11 and Bmf (encoding two BH3-only proteins involved in MOMP initiation)291 truly prevents the programmed demise of several cell types, causing their persistence throughout adult life.293 These observations are compatible with the hypothesis that the phenotype associated with some defects in the molecular cascades linking MOMP to caspase activation originates from a delay, rather than from bona fide inhibition, of PCD. Moreover they suggest that RCD, be it programmed or caused by microenvironmental perturbations, can only be avoided by interventions that target upstream steps of the process.Based on this conceptual construction, the NCCD recommends to use the term ‘initiation' to indicate the RCD-causing events that are reversible, that is, that do not irrevocably commit cells to die as they occur when adaptive responses are still operational. In addition, we encourage the use of the expression ‘execution' for referring to the processes that response . The blo298 traumatic events300 and ischemia/reperfusion injuries of the central nervous system, heart and kidney.304 Nonetheless, Z-VAD-fmk and similar compounds inhibit not only several caspases but also a wide panel of non-caspase proteases that participate in the initiation of RCD, such as calpains.306 Moreover, CASP3, caspase-6 (CASP6) and caspase-7 (CASP7)  have been involved in feedforward circuitries that amplify lethal cues leading to MOMP,310 implying that their inhibition may also counteract the initiation of RCD. Finally, in models of this type it is difficult to discriminate between the primary wave of RCD  and the delayed, secondary demise of parenchymal cells caused by DAMPs (directly or upon the establishment of inflammation).313 The cytoprotective effects that Z-VAD-fmk-like chemicals exert in similar scenarios, which are most reliably evaluated by histological determinations or functional tests, might therefore reflect their ability to block the initiation of DAMP- and inflammation-driven secondary RCD rather than the execution of stimulus-induced, primary RCD. In line with this notion, consistent cytoprotection has also been achieved in vivo by means of anti-inflammatory agents, even when these compounds do not directly influence RCD.317 Taken together, these observations reinforce the notion that caspases may not mediate RCD but simply accelerate its course, at least in the mammalian system.Caution should also be taken in inferring the actual etiological value of caspases in RCD based on the therapeutic administration of Z-VAD-fmk or other broad-spectrum caspase inhibitors. Caspase blockers have indeed been associated with (at least some degree of) cytoprotection in rodent models of various human diseases linked to the excessive loss of parenchymal cells. These pathologies include, but are not limited to, neurodegenerative disorders,Parp1−/− genotype and the administration of (relatively unselective) PARP inhibitors have been associated with bona fide cytoprotection in rodent models of ischemia/reperfusion injury and retinal degeneration.321 These observations suggest that PARP1 and/or other members of the PARP family also participate in the initiation of RCD. Alternatively, the inhibition of PARP1 may limit the release of DAMPs or the consequent inflammatory response, at least in some pathophysiologic settings. Further experiments are required to clarify these possibilities.Apparently at odds with the role of PARP1 in the execution of parthanatos, both the bona fide executioners of RCD, that is, the processes that directly drive cells across the boundary between life and death are less characterized, less inhibitable and perhaps more homogeneous than previously thought.322 In line with this theoretical construction, here the NCDD proposes to use the term ‘initiation' to refer to all the steps in the RCD cascade that are reversible, that is, which occur before cells make an irrevocable commitment to die. An attentive reinterpretation of the literature suggests that actual cytoprotection can only be achieved with pharmacologic or genetic interventions that inhibit or outcompete lethal signals at this stage, when adaptive responses to stress are still operational. Interestingly, some cells manifesting biochemical and morphologic features associated with late-stage RCD  appear to recover  and replicate, a process that has been dubbed ‘anastasis' .324 This suggests that the actual point of no return in the signal-transduction cascades leading to RCD may exhibit at least some degree of context dependency.As discussed above, the processes that until now were thought to mediate RCD most often do not causally underpin the cellular demise but represent biochemical manifestations of it. A growing body of data indicates indeed that the in vitro 24–96 h after stimulation, whereas the most reliable assessment of RCD in vivo is based upon histologic determinations or functional tests performed days, if not weeks or months, after such experimental interventions. In the former scenario, investigators can collect valuable kinetic data but are unable to estimate the true long-term survival of cells exposed to perturbations of homeostasis. In the second scenario, it is difficult to discriminate between the interruption of primary RCD and the inhibition of DAMP-driven inflammatory reactions and secondary RCD. This may have profound mechanistic and therapeutic implications. Indeed, retarding the demise of a cell that has already committed to die in an irreversible manner, and the biochemical manifestations of such death, may have limited cytoprotective effects for the cell in question, but may impact on the emission of DAMPs and hence significantly influence RCD propagation. Thus, considerable degrees of cytoprotection might be attained by means of agents that interrupt lethal cues at (or before) RCD initiation  combined with strategies that inhibit propagation . The superior beneficial effects of cyclosporin A may indeed stem from its ability to inhibit MPT-driven RCD and simultaneously exert a robust anti-inflammatory activity.326In the vast majority of scientific reports, RCD is measured 327 circumstantial evidence points to dropping ATP levels, which at some point abolish the activity of all ATP-dependent enzymes  and a compromised redox balance (which inactivates various enzymes and causes oxidative molecular damage to organelles and membranes) as central players in the execution of RCD  may causally underpin RCD in all its manifestations. Further experiments are required to explore these possibilities.If caspases and other enzymes commonly thought to mediate RCD in mammalian cells only underpin its manifestations, what are the true causes of cell death? Although the concentration of ATP is preserved (or even increases to some extent) in the course of adaptive stress responses,n of RCD .328 AlteCaenorhabditis elegans and D. melanogaster are truly protected by Z-VAD-fmk and by the genetic inhibition of caspase orthologs and other proteins involved in the postmitochondrial phase of apoptosis.335 This may indicate that the signal transduction cascades underlying RCD are interconnected in a different manner in mammals and non-mammalian organisms. Alternatively, the actual requirement of caspases for (at least some instances of) RCD might have been concealed by the evolutionary expansion of the caspase family. Both the human and murine genome encode indeed 14 distinct caspases,337 and it seems unlikely that Z-VAD-fmk and other pharmacologic or genetic interventions may simultaneously inhibit all of them in an efficient manner.At odds with mammalian models, Until these uncertainties have been resolved, the NCCD recommends that investigators focus on essential aspects of cell death; first of all its actual occurrence. It appears indeed that measuring the functional status or subcellular localization of RCD-relevant proteins including (but not limited to) caspases, RIPK1, RIPK3, MLKL, CYPD, PARP1 and GPX4, can provide insights into the mechanisms that accompany (and regulate the kinetics of) cellular demise, but not into those that truly push cells beyond the point-of-no-return separating life and death. Precisely defining where this border stands from a bioenergetic and metabolic perspective may facilitate the development of novel and efficient cytoprotective agents for clinical use.


Adolescent obesity has tripled in the past two decades, and adolescents with disabilities, specifically autism spectrum disorders (ASD), may be at greater risk for obesity due to the behavioral, physical, and psychosocial complications related to their disorder. This case study reports the effects of video game playing on an obese adolescent with ASD and illustrates the use of a multiple baseline single subject design. Over 12 weeks, the participant played inactive (6 weeks) and active video games (6 weeks) on the Wii console. Physiological data were evaluated weekly at home. Stress and anxiety were measured via the Stress Survey Schedule for Individuals with Autism and Other Pervasive Non-Developmental Disorders (SSS) and the Behavior Assessment System for Children Second Edition (BASC-2) pre- and postintervention. The Therapy Attitude Inventory (TAI) was used to determine parental perception of video game playing as a socially valid intervention to reduce stress and anxiety. Results demonstrated that active video game playing slowed and/or reduced weight and BMI with minimal changes to waist-to-hip ratios, triceps skinfolds, and stress and anxiety. This study demonstrates how alternative methods for physical activity may be used to improve health outcomes of overweight/obese adolescents with ASD and suggests directions for future research. Adolescent obesity has tripled over the past two decades across all race and ethnic groups, genders, and socioeconomic classes creating an epidemic and public health crisis . Indeed Children with developmental disabilities, such as those with autism spectrum disorders (ASD), may be particularly vulnerable to the development of obesity due to the behavioral, physical, and psychosocial complications related to their disorder , 7. IndeASD presents unique challenges for researchers and warrants novel interventions that can be tailored to meet the needs of individuals to address the challenge of obesity. Many caregivers of adolescents with ASD are often focused on problematic ASD features, with less concern for adverse health behaviors and weight , aberranSince nearly three-quarters of obese adolescents with ASD continue the obesity trend into adulthood, many develop comorbidities that are beyond the scope of previous interventions . AdditioN = 322) and were sedentary video gamers in order to evaluate the effects of physically active video games on participants' weight, body composition, physical activity, and fitness. Results revealed that the intervention group had a slight change in body mass index (BMI) from baseline (p = .02), a reduction in body fat (p = .02), and an increase in active video game play time (p < .01). In contrast, adolescents in the control group continued to gain weight playing inactive video games. These findings indicate that creative interventions have the potential to slow weight gain, an important component in reducing obesity.One such creative approach is the use of electronic video games as an intervention to treat obesity in adolescents with ASD. Maddison et al.  conductePhysical activity interventions represent an effective method to induce weight loss in adolescents with ASD. Active video gaming could have an effect on obesity in male adolescents with ASD since 41% of their free time is spent playing video games  and theyThere are no studies to date that have examined the effects of video game playing on obese adolescents with ASD. Thus, the purpose of this case study was to determine the feasibility and effectiveness of active video game playing for an obese adolescent with ASD.2, as defined by the CDC, and had other medical diagnoses such as Attention Deficit/Hyperactivity Disorder (ADHD) and Obsessive Compulsive Disorder (OCD). His daily medication regimen included Intuniv, 3 mg per day, Clonidine, 0.15 mg at night, Prozac, 20 mg per day, Trazadone, 75 mg at night, Clemastine, 0.67 mg in the morning and 4.02 mg at night, probiotics, and a multivitamin. He lived with his mother and a sibling and had weekend visitation with his father. JD was enrolled in public school and did not participate in any extracurricular activities. He typically stayed home and socialized with his family and a neighbor boy who is four years younger than JD. His diet consisted of two bowls of cereal or muffins for breakfast, pizza or sandwich with chips for lunch, and chicken nuggets, pasta, or pizza for dinner. Although his family offered healthier selections, he consistently chose these items.The Institutional Review Board at University of Florida in Gainesville approved this work. The participant, “JD,” was a Caucasian male adolescent with mild-to-moderate ASD, diagnosed at the age of 5, and was 15 years of age. JD's BMI classified him as obese, 28.7 kg/mJD and his parents were told that they were participating in a video game playing intervention to learn more about exercise via active video game playing and weight in adolescents with ASD. JD and his mother were blinded to the study hypotheses and were told not to change any habitual physical activities or eating habits over the course of the study.The active video game playing was completed using the Wii console and accessories needed. JD was given gaming options rated “E” for everyone for the Wii console. He chose an inactive and an active game. Baseline data were collected for four weeks prior to any gaming. JD's physiological data  were collected during baseline and gaming weeks while gaming data (activity steps and playing time) were collected each week during interventional weeks of the study. JD also kept a daily food and activity log during all weeks of the study. After collection of baseline data, JD was instructed to play the chosen inactive video game for four or more days a week for a minimum of 30 minutes each day for a total of six weeks. Then, he played an active video game for four or more days a week for a minimum of 30 minutes each day for a total of six weeks.JD was weighed at the same time of day using the same digital scale. Weight was obtained once a week during baseline and intervention phases in his home. JD wore similar clothing for weight assessment during the entire study.2). Once the BMI was calculated, his weight status was determined using an age- and sex-specific percentile for children aged 2–19 years. Overweight was defined as “a BMI at or above the 85th percentile and lower than the 95th percentile for children of the same age and sex” and obese as “a BMI at or above the 95th percentile for children of the same age and sex” [Body mass index (BMI) was calculated using JD's height and weight as defined by the CDC  and obtaand sex” .Waist-to-hip ratio was measured in inches using the same measuring tape and collected weekly during baseline and intervention phases. The ratio provides an indication of the overall health risk indicating low, moderate, or high risk for developing diseases such as cardiovascular disease or diabetes. Ratio norms are different for girls and boys, thus removing gender bias. Waist-to-hip ratio was obtained by measuring around the smallest part of the waist  and the widest part of the hips without pants or shorts but over underpants.Anthropometric data, triceps skinfolds, were measured on the right arm using Harpenden calipers. Measurements were obtained weekly during baseline and intervention phases. Triceps skinfolds have been shown to more adequately correlate body fat percentages for children and adolescents while height and weight are better instruments for total body fat percentages .Stress and anxiety were measured using the Stress Survey Schedule for Persons with Autism and Other Developmental Delays (SSS ) and BehParental satisfaction with the video game playing intervention was measured by the Therapy Attitude Inventory (TAI ). The TAJD and his mother provided assent and written informed consent, respectively. A pretreatment session lasted approximately one hour in which basic information, such as demographic and medical information, was collected. Following the pretreatment assessment, JD completed four baseline assessments for a level of experimental control to demonstrate change as a result of treatment. Each baseline session lasted approximately 30 minutes and consisted of weekly measurements of weight, BMI, waist-to-hip ratio, and triceps skinfolds. JD's mother also completed stress and anxiety surveys during the first session.Following baseline sessions, JD was enrolled in treatment. The protocol involved 6 weeks of inactive video game playing for four days a week and a minimum of 30 minutes each day, followed by 6 weeks of active video game play for the same amount of time and days. Video games selected for the study were rated E (Everyone) by the Entertainment Software Rating Board and were considered suitable for ages of 6 years and older. Selection was based on current popularity, activity levels, and review of market ratings. During video game play, JD wore a Fitbit, a wireless tracker in a wristband that tracks steps taken, distance traveled, calories burned, and very active minutes, to ensure video game play as well as documenting video game play on their activity logs. The Fitbit was chosen for its convenience of wear, wrist only, and its proven reliability and validity of measuring step counts and energy expenditure in previous research –24. MeasAfter completing all phases, JD's mother completed the stress and anxiety surveys as well as the TAI to determine social validity of the intervention and their perception of its effectiveness. Furthermore, feedback regarding barriers to performing the intervention and the feasibility of continuing a similar treatment to address obesity and health issues were discussed.2 and in phase B a variation of only 0.30 to 0.40 kg/m2 , internalizing problems , behavioral symptoms index , and adaptive skills . The BASC-2 preintervention T scores that were classified as at-risk were externalizing problems (composite score) and adaptive skills (adaptability). Postintervention results for the SSS only had minimal changes that did not result in a difference in categorical classification. Postintervention BASC-2 results for JD did not result in any changes in T scores. The TAI results for JD revealed a score of 42 out of 50, demonstrating that the parents perceive active video game playing as an effective intervention in reducing stress and anxiety.Preintervention results of stress and anxiety for JD demonstrated moderate-to-severe anxiety with the SSS instrument in the following categories: changes, unpleasant experiences, sensory/personal, food related, and rituals. Results for moderate anxiety were in the categories of anticipation, positive experiences, and social/environment. The preintervention results for the BASC-2 were noted to have clinically significant JD had reductions in weight after the introduction of active video game playing; however, there was an overlap of data between phases. For example, JD's waist-to-hip ratios between finishing phase B and starting phase BC were the same measurement. Interestingly, JD gained weight after the introduction of the intervention designed to increase physical activity. As reported on weekly food logs and verbally by his mother, JD increased his food intake during the first few weeks of increased physical activity, which may account for the increase in weight gain. Furthermore, foods chosen to offset the caloric expenditure were the typical energy dense favorites: chicken nuggets, pizza, and hot dogs. An additional explanation for the weight gain after the introduction of the active video game may be an increase in muscle mass. However, after these first few weeks, JD continued to reduce his weight as well as BMI, even if minimally. Although JD's overall weight increased, the active video game playing slowed the progression of weight gain during phase BC. BMI also decreased due to changes in weight and height during the intervention. Similar to Maddison et al. , these rStress and anxiety results had minimal changes in pre- and postintervention phases demonstrating that physically active video game playing did not reduce stress or anxiety in all areas. Although these findings were counter to expectations, research has shown that approximately 40% of children under the age of 18 with a diagnosis of ASD also had at least one comorbid anxiety disorder . Thus, sAlthough the results from the case study are preliminary, there is a need for future research to address limitations and replicate findings. A clear limitation of this work is that it describes only a single participant and that these findings may not be generalizable. However, single subject design serves a distinct purpose in that it allows for exploration of individual patient characteristics on treatment outcomes and an opportunity to manipulate variables during intervention. Additionally, the present study results may have been influenced by expectations; that is, JD and his family were aware of active video game playing even though they were blinded to the hypotheses. This may have contributed to positive findings as JD and his mother may have consumed improved food choices and/or less food to produce weight loss. However, results obtained by multiple baseline design suggest that positive outcomes are less likely to explain treatment effects since changes were directly related to the intervention. The length of the intervention may be a limitation because the physiologic data of waist-to-hip ratio and triceps skinfold measurements may not demonstrate changes over short periods of time. Another possible limitation is that the stress and anxiety measures were based on parent report which has the potential for parental bias and may lack sufficient objectivity. Finally, JD demonstrated overlap of data between phases B and BC which may create difficulty in establishing a true functional relationship of variables. Therefore, study replication with extended baseline assessments and intervention phases is needed to determine if there is true cause and effect. Further research, including study replication and randomized controlled trials with larger samples of adolescents of both genders and varied ethnicities, is needed to determine if the treatment effects of active video game playing are robust and generalizable.The case study report describes a protocol that can be replicated and provides preliminary evidence for the effectiveness of active video game playing with an overweight/obese adolescent with ASD. As noted previously, there are no studies yet published regarding the use of active video game playing in this population, a major gap in extant literature. Given the accumulating evidence for the effectiveness of physical activity in treating typically developing obese adolescents, it is critical that future research explores alternative solutions for physical activity among obese adolescents with ASD, for example, the active games used in this study.


PLXND1 and REV3L represent totally unrelated pathways involved in hindbrain development: neural migration and DNA translesion synthesis, essential for the replication of endogenously damaged DNA, respectively. Interestingly, analysis of Plxnd1 and Rev3l mutant mice shows that disruption of these separate pathways converge at the facial branchiomotor nucleus, affecting either motoneuron migration or proliferation. The finding that PLXND1 and REV3L mutations are responsible for a proportion of MBS patients suggests that de novo mutations in other genes might account for other MBS patients.Möbius syndrome (MBS) is a neurological disorder that is characterized by paralysis of the facial nerves and variable other congenital anomalies. The aetiology of this syndrome has been enigmatic since the initial descriptions by von Graefe in 1880 and by Möbius in 1888, and it has been debated for decades whether MBS has a genetic or a non-genetic aetiology. Here, we report  lt has been debated for decades if there is a genetic aetiology underlying Möbius syndrome, a neurological disorder characterized by facial paralysis. Here Tomas-Roca et al. use exome sequencing and identify de novo mutations in PLXND1 and REV3L, representing converging pathways in hindbrain development. HOXB1 has been associated with an autosomal recessive form of an MBS-like syndrome, with limited phenotypic overlap with typical MBSde novo mutations as the underlying mechanismMöbius syndrome (MBS) MIM 157900) is a rare congenital cranial dysinnervation disorder characterized by non-progressive facial palsy and impairment of ocular abduction, due to paralysis or weakness of the facial (n7) and abducens n6) nerves, and frequently other cranial nerves57900 is 2345 nerves, 101112PLXND1 at chromosome 3q21-q22   and the other in the PLXND1 gene   identified in patient 3   is a c.501delA frameshift mutation in exon 2 (NM_001101357)  (REV3L messenger RNA obtained from Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCLs) of patient P5. Sequencing of these products revealed aberrant REV3L transcripts lacking either exon 9 or exons 9 and 10, in addition to decreased levels of normal REV3L transcript  , one in _015103) . SPO11 i studies . Sanger atient 3  is unlik1101357) . Patient1101357)  carries _002912) . Aberrananscript .CCDC160, PLXND1 and REV3L by Sanger sequencing in a cohort of 103 MBS patients, including some HCFP patients who were not previously tested for PLXND1  and P10 . The mutation in P9 affects a conserved arginine residue in the GTPase-activating protein domain of the protein. The silent mutation in P10 is at a leucine residue in the IPT/TIG domain  that is characteristic for plexin proteinsde novo mutations in REV3L were identified in patients P11 and P12. A de novo missense mutation c.1160A>G was identified in patient P11, predicting an amino-acid substitution p.Glu387Gly. In addition, patient P12 carried a nonsense mutation , which is predicted to result in a loss-of-function allele.Next, we screened ND1 ref. . The scrng REV3L . The PLXPLXND1, REV3L and CCDC160 truncating variants in NHLBI Exome Sequencing Project  and a low residual variant intolerance score for these genes of −1.30 (4.88th percentile), −2.1 (1.55th percentile) and 0.014 (54.98th percentile), respectively, are consistent with PLXND1, REV3L and CCDC160 being intolerant to loss-of-function mutationsREV3L and PLXND1 are highly conserved throughout evolution (p.Glu387Gly (P11), p.Asn1895Lys (P1), p.Arg1485Pro (P9) and p.Leu1006Leu (P10)) (The absence of u (P10)) .de novo mutations in the same gene in a cohort of 103 individuals was calculated using the Poisson test and is 0.0001217 for REV3L  and 9.34e−05 for PLXND1 . The presence of a mutation in the other allele in these patients was excluded by sequencing the coding exons of PLXND1 and REV3L. Screening of CCDC160 gene in our cohort of male MBS patients did not reveal additional de novo mutations.The probability of identifying multiple PLXND1 encodes a protein of 521 amino acids and is a member of the plexin family of proteins. Plexins bind to semaphorins (Semas), a large family of extracellular, secreted and membrane proteins26PLXND1 is expressed in the vascular system2829PLXND1 in central nervous system development, we compared morphologic structures of the embryonic brain of wild-type (wt), heterozygous and homozygous PlexinD1 knockout mice at E16.5  to Benzo[α]pyrene diolexpoxide (BPDE), an agent that induces DNA damage that mimics bulky endogenous nucleotide lesions. Rev3l heterozygous MEFs displayed a slight, but significant, decrease in cell survival  in the initial cohort of eight MBS patients. This result is of general interest, as several studies have suggested previously that vascular disruptions during the first trimester of pregnancy rather than genetic mechanisms are the leading cause of MBS567de novo mutations in coding and noncoding regionsde novo mutations were identified in PLXND1 and three in REV3L in our cohort suggests a low frequency of mutations in these genes as a cause of MBS, implying genetic heterogeneity and intrauterine insults as causative events in other MBS patients. We expect that the implementation of whole-exome sequencing and whole-genome sequencing of parent–patient trios in a diagnostic setting will further dissect the aetiology of MBS.Exome sequencing has detected causative de novo mutations  are given in the We identified eight isolated Möbius patients in this study. They were the only affected individuals in their families. These patients (patients 1–8) were selected from a cohort of patients with the pathognomonic signs of MBS6Exome sequencing was performed on DNA from peripheral blood, using the SureSelect Human All Exon v2 50 Mb Kit  followed by multiplexed analysis on a SOLiD 4 System sequencing slide  as described previouslyde novo mutations as the underlying cause of MBS syndrome, we excluded all variants inherited from either parent in the two trios  and in our in-house database  encompassing the candidate variants. To exclude additional changes in either REV3L, PLXND1 or CCDC160, we designed primers for the amplification of all exons and intron–exon boundaries of REV3L (Genbank ID NM_002912), PLXND1 (Genbank ID NM_015103) and CCDC160 (Genbank ID NM_NM_001101357). Sequences of primers are shown in Primer sequences were designed using Primer3 software  according to the manufacturer's protocols. RNA was reverse transcribed using the iScript cDNA synthesis kit  according to the manufacturer's protocol. Complementary DNA (cDNA) was purified with NucleoSpin extract II (Qiagen) according to the manufacturer's protocol.EBV-LCL cells were harvested by centrifugation at 6,000http://frodo.wi.mit.edu/)  as compared with the normal distribution of the five controls. We used an α-level of 0.05, because only one gene was assessed.SYBR green-based real-time QPCR expression analysis was performed on an Applied Biosystem Fast 7,500 System machine. Primer sequences around exon 10 were designed using Primer3 software (it.edu/) . PrimersREV3L. The first pair of primers was designed surrounding the canonical splice-site mutation (c.1096+1G>A) in exon 10, and a control pair of primers was designed within exon 14  for analysis. Plxnd1 null and wt mouse embryos (E16.5) were harvested from timed matings. Noon was designated E0.5. Embryos were genotyped as describedPlxnd1 mouse work was performed according to the University of Pennsylvania animal care guidelines.Rev3l heterozygous and Rev3l-deficient mice and embryos were described beforeRev3l heterozygous mice were mated to generate male and female embryos and newborn mice for analysis. Rev3l heterozygous and wt mouse embryos (E16.5) were derived from timed matings. Noon was designated E0.5. Embryos were genotyped as describedC57BL/6 wt, Plxnd1 wt, seven Plxnd1 heterozygous and seven Plxnd1 homozygous embryos at E16.5  at 4 °C for 24 h. Fixed brains and heads were embedded in paraffin and they were sectioned into 16-μm slices using a microtome.We analysed two different stages to compare phenotypic aberrations. Five wt and five Plxnd1 wt, seven Plxnd1 heterozygous and seven Plxnd1 homozygous embryos at E16.5, five Rev3l wt and five Rev3l heterozygous embryos at E16.5  obtained from the Developmental Studies Hybridoma Bank . Sections were washed in PBS and then treated with 0.1% hydrogen peroxide in PBS for 1 h in the dark to inactivate endogenous peroxidase activity. After several rinses in PBT (PBS with 0.2% Triton X-100), sections were blocked with 0.5% goat serum, 0.2% BSA and 0.2% Triton X-100  in PBS for 4 h and then incubated overnight at 4 °C with monoclonal antibody anti-Islet-1 (1:50 in blocking solution). Subsequently, sections were incubated with biotinylated goat anti-mouse secondary antibody , and then with streptavidin/horseradish peroxidase complex . All antibodies were diluted in the same blocking solution as the primary antibody. The histochemical detection of the peroxidase activity was carried out using 0.03% diaminobenzidine and 0.005% H2O2. After immunoreaction, the sections were mounted, dehydrated and then coverslipped with Eukitt .Two Plxnd1 wt, five Plxnd1 heterozygous and five Plxnd1 homozygous mice at E16.5 and five Rev3l wt and five Rev3l heterozygous P0 newborns, and we counted separately the number of motoneurons from the right and left hindbrain sides (around 12 sections each side) . For the counting we used the open-source ImageJ Cell Counter softwaret-test, using Graphpad Software .We counted the total number of motoneurons presents in the facial motor nucleus in paraffin sagittal sections processed with Nissl staining. We used five Rev3l wt and five Rev3l heterozygous mice at E16.5 were used. This embryonic stage was selected because the calcification of the cranial bones is not completed yet allowing the sectioning. For hindbrain volumes comparison, around 80 slices of each of the five Rev3l wt and five Rev3l heterozygous brains at P0 were used. After alignment of all images from each brain respectively, we highlighted the areas of interest in each section to measure their volumes. The boundaries of the hindbrain were delineated according to the Allen Developing Mouse Brain Atlas from rhombomere r1 until r11, including the cerebellum.We performed volume rendering of image stacks on serial light microscopic sections of 16 μm using the open-source ImageJ TrakEM2 ref. . For theRev3l survival assays were conducted with Rev3l+/+, Rev3l+/− and Rev3l−/− MEFs. In each well of a six-well plate, 0.5 × 106 exponentially growing cells were seeded in MEF medium  and cultured overnight. Then, the cells were exposed to 0–300 nM BPDE  that was dissolved in anhydrous tetrahydrofuran  for 60 min in serum-free medium at 37 °C. The cells were trypsinized and seeded in MEF medium at clonal densities in 9-cm dishes. After culturing for 12–14 days at 37 °C, cell clones were fixed, stained with methylene blue and the number of cell clones was counted (n=3). The number of clones after mock treatment was set at 100%. The data were obtained with two and three independent Rev3+/+ and Rev3+/− MEF lines, respectively, and one Rev3−/− MEF line.4 exponentially growing cells per well in a 24-well plate and culturing overnight in MEF medium at 37 °C. The cells were mock treated with the solvent THF or exposed to 500 nM BPDE in serum-free medium for 15 min at 37 °C. The medium was removed, cells were washed once with 150 mM NaCl and pulse labelled with [3H]thymidine  in MEF medium for 15 min at 37 °C. MEF medium with radioactive thymidine was removed and the cells were cultured in MEF medium supplemented with 10 μM cold thymidine. At different times after pulse labelling, the local unwinding of DNA ends of elongating replication forks, the separation of single-stranded DNA and double-stranded DNA (dsDNA) using hydroxylapatite columns and the determination of radioactivity in the fractions containing single-stranded DNA and dsDNA was performedn=3). The percent of maturation of replicons was calculated by the following equation:  × 100%. The data were obtained with single Rev3+/+ and Rev3−/− MEF lines and two Rev3+/− MEF lines.Progression of replicons using the Alkaline DNA unwinding assay was determined following seeding of 5 × 105 exponentially growing cells were seeded in MEF medium and cultured overnight. Then, the cells were mock treated with the solvent THF or exposed to 500 nmol BPDE for 15 min in serum-free medium at 37 °C. After rinsing the cells with PBS, cells were cultured in MEF medium. At different times after treatment, up to 12 h, cells were lysed using Laemmli buffer. The proteins were separated by SDS–polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes . The membranes were incubated overnight at 4 °C with antibodies specific for γH2AX (S129) , phosphorylated Chk1 , phosphorylated Rpa32 , total Rpa  and β-actin . After incubation with appropriate secondary antibodies conjugated to peroxidase (Bio-Rad), proteins were visualized using enhanced chemoluminescence detection. Uncropped scans with indicated size markers are shown in In each well of a six-well plate, 2.5 × 10The probability of finding two mutations in a cohort of 103 in each gene was calculated by the Poisson test. We took into account the presence of two alleles and estimated gene mutation rates based on recent exome sequencing datat-test. The significance of differences of the hindbrain volume and subarachnoid space between the Rev3l heterozygous and the wt mice was calculated by the comparative t-test. Significance in sensitivities of MEF lines to BPDE and in replication fork progression after exposure to BPDE was determined using the unpaired t-test, σ=0.90. A P value smaller than 0.05 was considered to be statistically significant (*), and P values smaller than 0.01 (**) or 0.001 (***) were considered highly significant.Statistical significance of differences in motoneuron counts between the wt mice and the mutant mice were determined by the unpaired How to cite this article: Tomas-Roca, L. et al. De novo mutations in PLXND1 and REV3L cause Möbius syndrome. Nat. Commun. 6:7199 doi: 10.1038/ncomms8199 (2015).http://www.ebi.ac.uk/ega/) under accession numbers EGAS00001001250.The novel exome sequencing data generated in this study has been deposited at the European Genome-phenome Archive (Supplementary Figures 1-5, Supplementary Tables 1-6 and Supplementary References


Cryptococcus neoformans, is a common opportunistic neural infection in immunocompromised individuals. Cryptococcus meningitis is associated with fungal burden with larger capsule size in cerebrospinal fluid (CSF). To understand the role of CSF constituents in capsule enlargement, we have evaluated the effect of artificial CSF on capsule induction in comparison with various other capsule inducing media. Two different strains of C. neoformans, an environmental and a clinical isolates were used in the present study. While comparing the various capsule inducing media for the two different strains of C. neoformans, it was observed that the capsule growth was significantly increased when grown in artificial CSF at pH 5.5, temperature 34°C for ATCC C. neoformans and 37°C for Clinical C. neoformans and with an incubation period of 72 h. In addition, artificial CSF supports biofilm formation in C. neoformans. While investigating the individual components of artificial CSF, we found that Mg2+ ions influence the capsule growth in both environmental and clinical strains of C. neoformans. To confirm our results we studied the expression of four major CAP genes namely, CAP10, CAP59, CAP60, and CAP64 in various capsule inducing media and in different concentrations of Mg2+ and Ca2+. Our results on gene expression suggest that, Mg2+ does have an effect on CAP gene expression, which are important for capsule biosynthesis and virulence. Our findings on the role of Mg2+ ion as a signal for capsule induction will promote a way to elucidate the control mechanisms for capsule biosynthesis in C. neoformans.Cryptococcal meningitis caused by  Cryptococcal meningitis is a common, opportunistic neural infection in immunocompromised individuals . It has Cryptococcus neoformans has multiple virulence and predilection factors for invasion of central nervous system including capsule, melanin, phospholipase, mannitol, urease and proteinases . The cultures were maintained on potato dextrose agar slants and 20% glycerol stocks at –80°C.Two C. neoformans were cultured in PDB at 37°C for 24 h. Cells were washed with sterile PBS three times and resuspended in PBS. Cells were adjusted to 0.1 OD600 which is equivalent to ∼107 CFU/ml and were inoculated in the following media: PDB, DMEM, MOPS [3-(N-morpholino)propanesulfonic acid], ACSF, ACSF+ 10% heat inactivated FCS. The media were maintained at different pH  and temperatures . The capsule sizes were measured at three time intervals of 48, 72, and 96 h. Duplicates were maintained for the study  in 10 ml ddH2O; Stock III: 0.022 g CaCl2 (2 mM) in 10 ml ddH2O; Stock IV: 0.0120 g MgSO4 (1 mM) in 10 ml ddH2O; Stock V: 0.218 g NaHCO3 (26 mM) in 10 ml ddH2O]. All the 10 ml stocks were added in flask and made up to 100 ml with ddH2O to avoid precipitation, finally 0.1% glucose was added , 0.0186 g KCl (2.5 mM) in 10 ml double distilled water; Stock II: 0.015 g Naas added .2 (2 mM), MgSO4 (1 mM), and phosphate buffer (1.25 mM)] were tested for the induction of capsule growth in the presence of 0.1% glucose.The influence of individual components of ACSF  × 100}. Ten cells were measured for each determination and average was calculated  and 37°C  in a rotary shaker at 150 rpm (to early stationary phase). The cells were collected by centrifugation and adjusted to 0.1 OD600 with each media . The cells were then inoculated on glass coverslip for 72 h at 34°C (ATCC C. neoformans) and 37°C . The biofilm was then washed thrice with PBS, to remove non-adherent cells and media from the cover slips and stained with India ink. The stained biofilms were then observed using Trinocular microscope (Nikon) at 40×. The experiments were done twice  PDB; (ii) PDB + Mg2+; (iii) MOPS; (iv) MOPS + Mg2+; (v) DMEM; (vi) DMEM + Mg2+; (vii) ACSF; (viii) Ca2+ + 0.1% glucose; (ix) Mg2+ + 0.1% glucose; (x) Mg2++ Ca2+ + 0.1% glucose. Based on the dose dependent studies of Mg2+, 2 mM Mg2+, and 0.5 mM Mg2+ were selected for ATCC C. neoformans and clinical C. neoformans, respectively. After 48 h incubation time, C. neoformans were harvested and washed in chilled PBS. The RNA was extracted using RNAeasy mini kit .For transcriptional pattern analysis, the ATCC cDNA synthesis was performed in a total volume of 50 μl including 2–10 μg of RNA sample with one step RT-PCR Kit . PCR amplifications were made from templates (1:20) with target primers (2:20), and (10:20) Taq DNA polymerase master mix . Quantification of the transcript levels was performed using the ImageJ software, normalizing against β-actin.2 . The protocols of the PCR were as follows: initial denature at 95°C for 10 min, followed by 40 cycles at 95°C for 30 s, annealing at 57°C (β-actin and CAP59), 55°C (CAP10), 60°C (CAP60), or 54°C (CAP64) for 30 s, and extension at 72°C for 30 s. Primer pairs for PCR are listed in Table 1. These primer pairs were confirmed to amplify the genome DNA fragments of both ATCC and clinical strains  > ACSF+FCS (63%) > MOPS (62%) > DMEM (61%) > PDB (55%) and ACSF (68%) > ACSF+FCS (51%) > DMEM (49%) > MOPS (40%) > PDB (37%) for ATCC C. neoformans and Clinical C. neoformans, respectively. In both strains the capsule was induced by ∼32% when compared with the control media . Statistical analysis confirmed that ACSF was the best among the tested media  for both the strains in ACSF with pH 5.5. A prominent increase in capsule size than the cell size for ATCC C. neoformans was noticed. Whereas, for other media, the capsule and cell size were relatively equal with capsule induction in the range of 34–54%. The ambient pH for capsule induction was observed to vary with the media types which are shown in Figures 3 and 4. As suggested by previous reports the effect of pH on capsule growth might be due to the alterations in cell wall stability, protein stability, morphogenesis, nutrient uptake . From the results obtained it is clear that temperature does have an effect on capsule growth. However, we could not conclude whether an increase or decrease in temperature is influencing the capsule formation. ACSF induced capsule growth at 30, 34, 37°C but significant induction was at 34°C for ATCC C. neoformans and 37°C for clinical C. neoformans. However in our study, temperature has an effect on cell and capsule volume, postulating that the effect of physiological factors varies with type of strains used for the study.Similar to pH, the effect of temperature required for capsule induction was studied  and pH (5.5). Thus it is clear that capsule induction appears to vary with respect to changes in media, temperature and pH. However, longer incubations did not significantly change the capsule size in both strains.We then investigated the effect of incubation period for capsule induction in C. neoformans shows that capsule growth was significantly increased when grown in ACSF at pH 5.5, temperature 34°C for ATCC C. neoformans and 37°C for Clinical C. neoformans and with an incubation period of 72 h. Using this information, we have made an attempt to understand the effect of individual components of ACSF on capsule induction.In summary, comparison of various capsule inducing media for the two different strains of 2 (2 mM), MgSO4 (1 mM), and phosphate buffer (1.25 mM). When each individual component of ACSF was supplemented with 0.1% glucose, we noticed Mg2+ as the prominent capsule inducing factor in both strains. The order of capsule induction for different salts for clinical C. neoformans is Mg2+ (58%) > Mg2++ Ca2+ (52%) > Ca2+ (48%) > PO43-(46%) > glucose (46%). For ATCC C. neoformans, Mg2+ (59%) > Mg2++ Ca2+ (57%) > Ca2+ (54%) > PO43-(52%) > glucose (50%) and no capsule induction was observed in KCl for both the strains . So we increased the pH of the KCl containing medium for capsule synthesis. Surprisingly capsule induction was observed at higher pH (pH 7.5). The effect of ACSF ions on capsule formation were studied by comparing the cell size and capsule thickness. From the results we obtained, it is very clear that C. neoformans when grown in the medium containing Mg2+ ion, show increased capsule production even though the cell size was relatively small. Whereas in case of effect of ions like K+, PO43-, the cell size was greater than capsule. In the presence of Ca2+, the cell and capsule thickness were equal, whereas a significant induction was observed when Ca2+ medium was supplemented with Mg2+. The effect of Mg2+ ion in capsule induction is clearly evident in Figure 5. A similar kind of results was observed with clinical C. neoformans. However, in both the strains, Mg2+ significantly induced to capsule growth under any of the conditions tested, like different pH , temperature , induction period . Statistically Mg2+ was found to enhance capsule compared with other salts. Through One-way ANOVA analysis Mg2+ was found to be the best salt compared to other ACSF salts , CaCl.5 mM, CaC. neoformans capsule growth with some salts like NaCl, MgCl2, CaCl2, phosphate buffer, MOPS, FeS04,  in capsule assembly and capsule growth  were supplemented in MOPS, PDB, and phosphate buffer. As expected, the medium supplemented with Mg2+ produced larger capsule sizes than the control . The dose-dependent studies clearly demonstrate that capsule induction by Mg2+ is dose responsive. The optimum concentration required, however, varied between the strains. Dose-dependent analysis of Mg2+ on capsule thickness showed that 2.0 mM Mg2+ gave the best result for ATCC C. neoformans. With increase in Mg2+ concentration, there was a small decrease in capsule thickness, and a similar kind of result was observed for clinical C. neoformans at 0.5 mM Mg2+. This concentration is very similar to the concentration of human CSF . C. neoformans capsule induction was observed in microscopic analysis for both ATCC and clinical strains, microscopic images showed capsule induction in different media and ions . The One-way ANOVA confirmed that 2.0 mM and 0.5 mM Mg2+ concentration is required for significant capsule induction in ATCC C. neoformans and clinical C. neoformans, respectively  on gene expression shows that different media were able to stimulate the expression of CAP10, CAP59, and CAP60, whereas none of the media were able to stimulate the expression of CAP64. These results were similar for both environmental and clinical isolates of C. neoformans.To look specifically at capsule biosynthesis genes induced by Mg2+ in the media lead to enhanced expression of the gene when compared to Mg2+-free media, with best expression seen with MOPS + Mg2+ in the case of environmental isolate. However, with clinical isolate, presence of Mg2+ resulted in lower expression of CAP10 gene when compared to Mg2+-free media. Presence of Ca2+ did not have any significant effect on CAP10 expression in both isolates. For both environmental and clinical isolates of C. neoformans, ACSF did not promote significant CAP10 expression.With regard to CAP10, gene, presence of Mg2+ in the media leads to increase in expression of the gene in MOPS and DMEM for both environmental and clinical isolates. Presence of Mg2+ in PDB did not induce significantly higher CAP59 gene expression in both isolates. Interestingly, CAP59 gene in environmental isolate showed higher expression in ACSF media when compared to clinical isolate.For CAP59 expression, again presence of Mg2+ in the case of MOPS alone. For this isolate, presence of Mg2+ in PDB, and DMEM produced significantly lower levels of CAP60 gene expression. In the case of environmental isolate, presence of Mg2+ had a stimulating effect only in the case of PDB, while with MOPS and DMEM, the expression looked almost similar in both Mg2+-free and Mg2+-containing media. But for both isolates, ACSF induced a strong expression of CAP60 gene.Unlike CAP59, CAP60 gene expression in clinical isolate was enhanced in the presence of Mg2+, and the reasons for this are unclear. The environmental and clinical isolates displayed differences in expression of capsular polysaccharide under different growth conditions. Interestingly, our study demonstrates that the virulent genes (CAP genes) are highly expressed in the presence of Mg2+ ions in both clinical and environmental isolates. Together these results suggest that, Mg2+ does have an effect on CAP gene expression, which are important for capsule biosynthesis and virulence.Interestingly, for both isolates, CAP64 gene expression was not detected in any of the media, including with MgCryptococcus is becoming quite common . In a similar kind of study, C. neoformans strain B3501 was observed to form a strong biofilm in minimal medium containing glucose  to assess the potential of ACSF salts in inducing capsule formation. Similar studies were done previously by some authors to find the significant role of Mg2+ + Ca2+, PO43-, FeSO4, and NaCl in capsule formation. PO43- was found to be an essential mineral for nutrient uptake by C. neoformans for capsule growth  shows that the expression of CAP10, CAP59, and CAP60 was enhanced in the presence of Mg2+ for both the isolates, suggesting that Mg2+ concentration might be critical in regulating capsule synthesis and virulence of C. neoformans in vivo, and the exact concentrations of this important divalent cation in blood and CSF could influence the pathogenicity of C. neoformans during active infection. However, this remains a critical parameter to be assessed in meningitis patients to correlate with treatment outcome. Our findings on the role of Mg2+ ion as an inducing factor for capsule induction  could open up new avenues for targeting the capsule biosynthesis pathway in C. neoformans.We have extended our study to find the effect of pH and temperature on capsule induction. Though there are reports on the effect of pH and temperature on capsule induction, we also tried similar kind of experiments as the strains selected for the present study are different from those used by other authors. As also suggested by previous reports we could not conclude the role of pH in capsule induction . The infe growth , and Mg2assembly . WhereasMg2+ ion . Even thucidated . Pathogerophages . Mutatiorophages . Howeverrophages . The inchickness . Apart fAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Wuchereria bancrofti and the intensive movement of immigrant workers from endemic areas, Thailand has implemented two doses of 6 mg/kg diethylcarbamazine (DEC) with interval of 6 months to prevent IBF. In areas where immigrants are very mobile, the administration of DEC may be compromised. This study aimed to evaluate DEC administration and its barriers in such areas.Migration plays a major role in the emergence and resurgence of lymphatic filariasis (LF) in many countries. Because of the high prevalence of Imported Bancroftian Filariasis (IBF) caused by nocturnally periodic A cross-sectional study with two-stage stratified cluster sampling was conducted. We selected Myanmar immigrants aged >18 years from factory and fishery areas of Samut Sakhon Province for interview with a structured questionnaire. We also interviewed health personnel regarding the functions of the LF program and practice of DEC delivery among immigrants. Associations were measured by multiple logistic regression, at P <0.05.DEC coverage among the immigrants was 75 %, below the national target. All had received DEC only once during health examinations at general hospitals for work permit renewals. None of the health centers in each community provided DEC. Significant barriers to DEC access included being undocumented , unemployed , daily employed , short-term immigrant  and living in a fishery area . Incorrect perceptions about the side-effects of DEC also obstructed DEC access for Myanmar immigrants. All positive LF antigenic immigrants reported visiting and emigrating from LF-endemic areas.Hospital-based DEC administration was an inappropriate approach to DEC delivery in areas with highly mobile Myanmar immigrants. Incorporating health-center personnel in DEC delivery twice yearly and improving the perceptions of DEC side effects would likely increase DEC coverage among Myanmar immigrants. Culex quinquefasciatus), may result in the emergence of LF in these areas [Wuchereria bancrofti among Myanmar immigrants over recent decades [Thailand is a low-endemic area for lymphatic filariasis (LF). LF cases have only been reported among Thai residents in one southern province, and the country is in the process of verifying the elimination of the disease . Howeverse areas –6. High  decades , 7 has s decades . Multipl decades –14 and m decades . Due to  decades , 14, two decades –14.To reduce the sources of LF infection effectively, DEC coverage among eligible immigrants must be >90 %, while the prevalence of MF antigen must be <1 % . The MoPTherefore, this study aimed to evaluate biannual DEC administration and to identify barriers to DEC access among Myanmar immigrants in a metropolitan area. The findings of this study may provide insights regarding the situation of IBF control in areas where Myanmar immigrants are highly mobile and improve the effectiveness of DEC administration and IBF prevention & control programs in metropolitan areas.A cross-sectional study, using both quantitative and qualitative methods, was conducted in a metropolitan area with large seafood-processing and fishing industries, in Samut Sakhon Province, Thailand Fig. . Samut SThis study used semi-structured questionnaires to interview Myanmar immigrants and dialog guidelines to interview local health personnel responsible for the LF program. Four Myanmar translators, fluent in Thai and Burmese, were trained to translate during the dialog and interview. Local health personnel were asked questions related to the functions of the LF program and practice of DEC delivery among immigrants. Percentage and mean (SD) were used to describe the data. Chi-square test and multiple logistic regression were used to calculate odds ratios (OR) and identify barriers to DEC access. Significance was set at 5 % of alpha, with 95 % confidence level. The research protocol was approved by the Ethics Committee of the Faculty of Public Health, Mahidol University (COA. No. MUPH 2013–133).P <0.001) (Table P = 0.038). The proportion of DEC accessibility significantly differed between married, single and widowed (P = 0.034). Of those employed monthly, 88.3 % had accessed DEC compared with 64.4 % of unemployed (P < 0.001). A lower reported DEC intake was observed among immigrants who recently arrived in Thailand (≤12 months) than immigrants affirming longer lengths of stay (P = 0.009). Finally, respondents living in fishing areas tended to access DEC at a significantly lower proportion (69.2 %) than those living in factory areas (81.2 %). Sex and educational status were not significantly associated with different proportions of DEC accessibility  and undocumented immigrants significantly differed (5.1 %) , while unemployed and daily employed participants had substantially greater obstacles to DEC access compared with participants employed monthly . Short term Myanmar immigrants, and those living in fishery areas were more likely to suffer obstacles to DEC access than other groups  after adjusting for age, sex, educational status and marital status , compared with factory areas (3.9 %). The low DEC coverage might have resulted from DEC-administration practices, namely, DEC was provided by two general hospitals during health examinations for the renewal of work permits. Even though health centers were designated one of the two strategies to deliver bi-annual DEC to immigrants according to national guidelines , 15, no Undocumented immigrants commonly accessed healthcare services at general hospitals less because of their illegal status –19. AddiThe suboptimal DEC administration may not interrupt IBF transmission in areas with high population movement from endemic areas. Local health personnel, responsible for the LF program, face a great challenge. They must disseminate health information to undocumented immigrants and their employers that multiple DEC treatment is a key factor to prevent LF transmission and benefits the health of their employees. The high movement of Myanmar immigrants has suggested the need to strengthen the surveillance system for LF at border checkpoints and immigration stations regarding the national policy for border health prevention and control .No immigrant who received the first round of DEC received a second dose. The Thai helminthiasis-control DOTS policy requires Myanmar immigrants to ingest a single dose of 300 mg DEC and 400 mg albendazole in front of the nurse during physical examination for work permit renewal. This drug combination completely eradicates both microfilariae and macrofilariae , 10, 21.Factors associated with DEC inaccessibility included being undocumented, employed daily, unemployed, short-term immigration and living in a fishery area. Being an undocumented immigrant was strongly associated with impeded DEC access. This finding may be related to undocumented immigrants’ fear of accessing healthcare services because of their undocumented status –20. A siThis study found that hospital-based administration of DEC is inappropriate in areas with high proportions of undocumented immigrants and highly mobile populations. Thailand has a well-developed healthcare infrastructure. At lower levels of health services delivery such as health centers personnel work actively on disease prevention and control and generally know the community context rather well. Health-center personnel can identify the homes of all community members and easily deliver DEC to undocumented immigrants –25. In TOur study revealed that the IBF component of the Thai National Program to Eliminate Lymphatic Filariasis in study site did not achieve the desired goals as hospital-based DEC administration did not reach undocumented and short term Myanmar immigrants. Moreover, such a strategy was unable to provide DEC to Myanmar immigrants bi-annually, according to policy. To increase DEC coverage, health center-based DEC administration is suggested as a more effective way of reaching undocumented immigrants.