Large-scale genetic sequencing studies have identified a wealth of genes in which mutations are associated with autism spectrum disorder (ASD). Understanding the biological function of these genes sheds light onto the neurodevelopmental basis of ASD. To this end, we defined functional categories representing brain development – (1) Cell Division and Survival, (2) Cell Migration and Differentiation, (3) Neuronal Morphological Elaboration, (4) Development and Regulation of Cellular Excitability, and (5) Synapse Formation and Function – and place 100 high confidence ASD-associated genes yielding at least 50 published PubMed articles into these categories based on keyword searches. We compare the categorization of ASD genes to genes associated with developmental delay (DD) and systematically review the published literature on the function of these genes. We find evidence that ASD-associated genes have important functions that span the neurodevelopmental continuum. Further, examining the temporal expression pattern of these genes using the BrainSpan Atlas of the Developing Human Brain supports their function across development. Thus, our analyses and review of literature on ASD gene function support a model whereby differences in brain development – from very early stages of macroarchitectural patterning to late stages of activity-dependent sculpting of synaptic connectivity – may lead to ASD. It will be important to keep investigating potential points of mechanistic convergence which could explain a common pathophysiological basis of ASD behind this disparate array of genes. Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by core deficits in social interaction, communication, repetitive stereotyped behaviors and restricted interests. The underlying pathophysiological basis of these deficits is unclear, yet it has been linked to a variety of overlapping mechanisms. Molecularly, there has been evidence of altered translation  and defiIt has become increasingly clear, nonetheless, that a large proportion of cases have a genetic origin . HoweverThe human brain consists of a complex cellular architecture of 86 and 85 billion neurons and glia, respectively, ,17 and tA list of ASD-associated genes was generated from the SFARI (Simons Foundation Autism Research Initiative) gene database, selecting for the highest confidence genes, classified as either “Syndromic” (Category S), “High Confidence” (Category 1), “Strong Candidate” (Category 2) or “Suggestive Evidence” (Category 3) as per SFARI criteria. As a comparison, and because little is known about the differential genetic contributions of ASD versus developmental disability (DD) genes common key features of ASD and DD, we also examined a list of high confidence DD-associated genes extracted from the United Kingdom-wide collaborative Deciphering Developmental Disorders study . From thA customized function in MATLAB R2015a was written to quantify the number of published articles found after searching for a specific set of keywords in the PubMed database. We first searched for the ASD or DD gene name only in order to select for genes whose search produced more than 50 published articles, yielding a total of 100 ASD and 100 DD genes Genes were then classified into categories that were defined to reflect the stages of brain development by searching for the gene name combined with the following keywords: (1) gene AND (mitosis OR apoptosis) for Cell Division and Survival, (2) gene AND (migration OR differentiation) for Cell Migration and Differentiation, (3) gene AND (dendrite growth OR axon growth) for Neuronal Morphological Elaboration, (4) gene AND  for Development and Regulation of Cellular Excitability, and (5) gene AND (synapse OR GABA OR glutamate) for Synapse Formation and Function.For each ASD and DD gene, to control for the amount the gene had been studied, the raw number of publications within each category was expressed as a fraction of the total number of article publications for that gene. Also for each ASD and DD gene, to control for the amount each category has been studied, the raw number of publications within each category was expressed as a fraction of the median number of publications for that category for a list of non-ASD-linked control genes. As our controls, we obtained a list of brain-expressed genes by selecting a random subset of genes from a list of brain-expressed genes obtained from the Human Protein Atlas gene database  combinedFinally, within each category, values were peak scaled. Genes were then clustered and sorted for easy visualization according to a customized nested sorting algorithm intended to integrate the strength of their association to the category they were most strongly associated with as well as the two flanking categories.vs. 1%) and cellular excitability .Using this system, we found that of the 100 well-studied ASD candidate genes, when normalizing to the random list of brain-expressed genes, 27% were most strongly associated with cell mitosis and survival, 40% with migration and differentiation, 4% with morphological elaboration, 13% with cellular excitability, and 16% with synapse formation and function , Table 1(1)Early embryonic brain development begins in the first three weeks post-fertilization with neural induction. At this stage, the three germ layers – endoderm, mesoderm and ectoderm – have been formed and all major cells and tissues of the central and peripheral nervous system will derive from ectoderm.First, cells of the ectoderm differentiate into neural cells in the process of neural induction. Fibroblast growth factor (FGF), noggin, chordin and follistatin contribute to this differentiation by inhibiting the TGF-β family protein bone morphogenic protein 4 (BMP4) . In the Parts of the brain next develop functional identities that distinguish them from other regions through characteristic patterns of cellular phenotype, organization, and connectivity in the process of regionalization. The embryonic brain differentiates as such into the rhombencephalon (future hindbrain), which gives rise to the metencephalon and myelencephalon, the mesencephalon (future midbrain), and the prosencephalon (future forebrain), which gives rise to the optic vesicle, diencephalon and telencephalon . These regions are sculpted and maintained by a combination of key transcription factors which include OTX2, GBX2, FGF8, EN1 and a variety of Pax transcription factors ,25.Dorsoventral and rostrocaudal axes finally get specified in the process of nervous system patterning. This is orchestrated by several major developmental control gene groups whose spatially specific expression patterns govern proper neuronal differentiation and establishment of regionalized neuronal identity . The dorThe functional categorization algorithm yielded 27 out of 100 (27%) of the ASD-linked genes as being most closely linked to processes of cell division and survival. It is important to point out that of all the categories, functional classification of genes into this category is likely biased by the abundance of cancer literature. We controlled for this by normalizing values obtained for each category to the mean number of articles in each category for the list of control genes. However, if a particular gene has been studied more in the context of cancer than in the context of neuronal function, this may have biased the placement of a gene into this category. Further, while many genes may function in “cell division and survival”, they may later in development continue to function in the processes of “morphological elaboration” or “synapse formation”.However, several key pathways emerge as points of convergence for genes placed in this category. One main group of genes is involved in cellular growth through the PI3k/Akt/mTOR intracellular growth and cell cycle regulatory pathway. It is notable that 21.7% of genes in this category, or 5% of all genes, are linked to the mTOR pathway. These genes include the mTOR inhibitors phosphatase and tensin homolog (PTEN) and tuberous sclerosis 1 and 2 (TSC1 and TSC2) -34, as wAnother main group of genes is involved in the regulation of cell survival through the Wnt/GSK3β/β-catenin signaling pathway. Such genes include β-catenin (CTNNB1), a regulator of cell-cell adhesion and gene transcription, as well as dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), chromo-helicase domain protein 8 (CHD8), and ubiquitin protein ligase E3A (UBE3A). CHD8 inhibits β-catenin through direct binding  and DYRKA final group of genes is linked to the regulation of transcription and translation, a convergent mechanisms which re-emerges in analyses of genes linked to the other four defined developmental categories. Such genes include the transcriptional repressor CTCF, the nucleosomal DNA-binding protein HMGN1, the linker protein between gene transcription and histone ubiquitination WAC, and the histone deacetylase HDAC4 . The latDisruption of any of the ASD-linked genes involved in cell division and survival could either lead to overactive or underactive cell growth and proliferation, offsetting the tightly spatiotemporally regulated balance of developmental neurogenesis. This could induce hypertrophic, hyperplastic, hypotrophic or hypoplastic cell growth, leading to regionalized or generalized macrocephaly or microcephaly, both highly prevalent in ASD and both ultimately compromising brain function .(2)v1.2 Na+ channel expression [Once a cell has been born, it can either remain undifferentiated, giving rise to more cells, or differentiate into a neuron. This decision is regulated by several key factors that maintain a cell’s undifferentiated and self-renewable state. Tailless (TLX) is an orphan nuclear receptor that regulates embryonic and adult stem cell self-renewal by regulating cell cycle progression -53. It mpression ,60.Post-mitotically, neurons migrate away from their germinal zones to assume their final positions in the developing cortex where they will finish maturing and differentiating. Cortical excitatory pyramidal projection neurons migrate radially along a fibrous pathway formed by radial glial cells which extend from the subventricular zone (SVZ) to the surface of the developing cerebral cortex. These migrate in a layered inside-out manner such that the youngest neurons are closest to the surface of the cortex. Successive generations of cortical neurons migrate up through the already established layers to reside in the next most superficial layer until all layers have been established . The maiOur functional categorization algorithm revealed 40% of our ASD-linked genes to be most closely linked to the processes of differentiation and migration. This disproportionately high fraction may reflect that the concept of neuronal differentiation is often used to encompass more discrete categories such as morphological elaboration, regulation of excitability, and synaptogenesis. A close look at these genes reveals that most of them are involved in regulating cytoskeletal dynamics and cytoskeletal binding to extracellular matrix (ECM) as well as broadly regulating transcription and translation. A subset of these is also linked to neither of these processes directly.Regulators of cytoskeletal dynamics and cytoskeletal binding to ECM include dystrophia myotonica protein kinase (DMPK), as well as dystrophin (DMD), integrin subunit beta 3 (ITGB3), reelin (RELN) and protocadherin 19 (PCDH19), the latter four of which mediate binding of the cytoskeleton to the ECM. Since regulation of neuronal morphology via cytoskeletal modulation and adherence of neurons to the ECM are crucial for neuronal differentiation and migration, disruptive mutations in any of these genes would result in aberrant differentiation and migration with cumulative downstream consequences on neuronal circuit formation and their information-encoding capacity.Genes regulating transcription include transcription factors TCF7L2 (TCF4), BCL11A, homeobox A1 (HOXA1), LXM1B, paired box 5 (PAX5), T-Box brain 1 (TBR1), myelin transcription factor 1-like (MYTL1), and forkhead box protein P2 (FOXP2). Genes regulating translation that are involved in chromatin remodeling and post-translational modifications that affect protein function include methyl CpG binding protein 2 (MECP2), euchromatic histone-lysine N-Methyltransferase 1 (EHMT1), chromodomain helicase DNA binding protein 2 (CHD2), lysine demethylase 5B (KDM5B), and lysine demethylase 6B (KDM6B). Disruption in the regulation of transcription and translation is an emergent theme in all developmental categories, the cumulative effects of which can be disastrous on the countless downstream processes regulated by these genes.Finally, an interesting example of a gene linked to neither of the above mechanisms is disrupted in schizophrenia 1 (DISC1). DISC1 inhibits GSK3β activity, resulting in reduced β-catenin phosphorylation and reduced activation of the Wnt/β-catenin signaling cascade ,67. ResuAs such, loss-of-function mutations in any of the ASD-linked genes involved in cell differentiation and migration could lead to deficits in proper neuro- and cytoarchitectural development and the brain function these enable.(3)In concert with and following differentiation and migration, neurons begin to mature and establish appropriate synaptic connections. They first begin to extend axons out to appropriate targets and subsequently develop dendrites and elaborate their arbors. Such axo-dendritic guidance and connectivity relies on a substantial number of well-coordinated and interacting intrinsic and extrinsic molecular and cellular events.Axons may need to travel far from their somas to reach their target destinations, making important navigational choices through complex terrain along their way. They do so in response to both intrinsic and extrinsic cues, by adhering to each other in fascicles, adhering to proteins expressed in the ECM or on the surface of other cells, and by sensing diffusible molecules that create attractant or repellent gradients in target brain regions. Neural cell adhesion molecule (N-CAM) facilitates fasciculation and intracellular signaling. Pathways activated upon this type of homophilic cell adhesion molecule (CAM) binding are calcium-dependent and may involve a variety of kinase signaling pathways ,72. GrowDendrites must grow too to meet their presynaptic counterparts and provide the neuromorphological framework necessary for the initiation of synaptogenesis. The specific size and shape of a growing dendrite will influence which and how many inputs a neuron receives, with larger dendrites being connected to more axons ,76. DendThere are a number of secreted factors that further modulate dendritic elaboration. In addition to their aforementioned role in cell survival, neurotrophins such as NGF, BDNF, NT-3 and NT-4 control dendritic elaboration via signaling downstream of tyrosine receptor kinases (Trk) -87. It iRegulation of axonal and dendritic growth and branching patterns involves the cooperation of many different types of signals, the downstream effectors and mechanisms of which remain to be fully elucidated. Whether these different processes are highly interwoven and convergent or allow for several parallel mechanisms, insufficiency in a critical gene will result in disastrous consequences. Our functional categorization algorithm most closely linked 4%  of our ASD-linked genes to the process of morphological elaboration. This is somewhat surprising because abnormal arborization phenotypes have been described in most mouse models for ASD, including fragile X mental retardation protein (FMRP), CASPR2, PTEN, TSC1/2, MECP2 and DYRK1A -99. The SEMA5A is a class 5 member of the previously discussed semaphorin family of proteins which are involved in axonal guidance and dendritic targeting and branching. Unlike other semaphorins which mainly act as chemoattractants, SEMA5A inhibits neuronal growth cones ,101 and DSCAM is a homophilic cell-adhesion molecule which is dynamically expressed during cortical development and has been implicated in various stages of neural development . It playFinally, CNTN4 is a protein which is part of the contactin family of Ig cell adhesion molecules (IgCAMs), three of which  have been linked to ASD. CNTN4 is expressed in axons and acts as an axon guidance molecule . While iDysfunction of any of the ASD-linked genes involved in the morphological elaboration of neurons – both axonal and dendritic – could lead to wide-ranging deficits from defects in neuronal differentiation to patterns of inter-neuronal connectivity, ultimately compromising normal brain function.(4)+ channels balanced by an outward current through voltage-gated K+ channels [Cells develop intrinsic excitability as they differentiate and migrate to their target destinations by beginning to express specific combinations of voltage-gated ion channels that enable the generation of electrical potentials. The main electrical potential that permits neuronal communication, the action potential, consists of a self-regenerative influx of current through voltage-gated Nachannels . The acqOver the course of development, dynamic changes in the expression of ion transporters and channels refine the electrophysiological properties of developing neurons, including input resistance, resting membrane potential, and current kinetics . The exp2+ is an important second messenger allowing for an interface between excitability, cytoskeletal organization, and transcriptional regulation to modulate neuronal development. Voltage-gated calcium channels (VGCCs), which include L-, P-, N-, R- and T- types, are expressed in the plasma membrane and are the channels which allow the influx of Ca2+ into the cytoplasm in response to depolarization.Calcium influx in response to action potentials triggers neurotransmitter release and contributes to shaping the action potential waveform. In addition, CaOur functional categorization algorithm revealed 13 out of 100 (13%) of our ASD-linked genes to be most closely linked to the process of regulation of cell excitability.2+ channels: 3 L-type high voltage-gated channels  and 1 R-type intermediate voltage-gated channel (CACNA1H which encodes Cav3.2). Both loss and gain of function mutations in such Ca2+ channels have been associated with ASD. CACNA1H exhibits a loss-of-function mutation [Of the 13 genes, 4 encoded different types of voltage-gated Camutation , whereasmutation . Of notemutation  and enhamutation .+ channels (SCN1A which encodes Nav1.1 and SCN2A which encodes Nav1.2), and 1 encoded a voltage-gated K+ channel (KCNJ10). The Na+ channels are composed of a Na+-conductive alpha subunit and one or more regulatory beta subunits. Mutations in both Nav1.1 and Nav1.2 modify network excitability and as such have been heavily linked to epilepsy [v1.1 is most heavily expressed in GABAergic neurons, and mutations in Nav1.1 may lead to impaired firing of GABAergic inhibitory hippocampal interneurons [v1.1 may cause generalized epilepsy with febrile seizures plus (GEFS+) as a result of minor changes in Na+ channel kinetics that cause either hypo- or hyperexcitability [v1.2, on the other hand, while also broadly expressed in the CNS, is most heavily expressed in unmyelinated axons and dendrites of the cortex and hippocampus, and its mutation has been linked to seizure disorders [Of the 13 genes, 2 encoded voltage-gated Naepilepsy ,128. Brorneurons  and cererneurons . Phenotytability . Nav1.2,isorders -134. Mutisorders . In thisisorders .+ channels [+ current. In addition, on a more basic level, the complex developmental pattern of ion channel expression plays a key role in the onset of spontaneous activity and, later, enables activity-induced changes that critically instruct a number of developmental processes [In addition to ion channels, contactin associated protein-like 2 (CNTNAP2) fell into this category, likely due to interactions with Kchannels ,138. Intchannels . This marocesses .Dysregulation of any of the ASD-linked genes involved in the regulation of cell excitability at any of these levels and stages of development could thus result in cumulative dysregulation of network activity and manifestation of the autistic phenotype.(5)The elaborate morphological changes in development result in the formation of a network of synapses that will allow for the precise encoding and processing of information. Axon elongation and dendritic elaboration, allowing for proper positioning of axo-dendritic contacts, are the first steps involved in synaptogenesis. Initial synapse formation will then require adhesion to stabilize contacts, alongside the assembly of the presynaptic and postsynaptic machinery. Following this assembly, a variety of molecular players allow activity-dependent synapse modification and maturation.A series of cues, both long-range (diffusible) and short-range (surface-bound), as well as both attractive and repulsive, guide neurite outgrowth  superfamilies , cadherins, integrins, and neurexins ,151. ManSynapses eventually develop a mature structure  and matuOur functional categorization algorithm revealed 16 out of 100 (16%) of our ASD-linked genes to be most closely linked to the process of synapse formation and function. Broadly, and in light of the above synaptic development processes, most of these genes can be further subdivided into trans-synaptic adhesion molecules, postsynaptic density proteins, NMDA receptor subunits, and GABA receptor subunits.and sufficient for synaptogenesis to occur, and has greater impacts on glutamatergic than GABAergic synapse formation [Neuroligins and neurexins represent the largest sub-category of trans-synaptic adhesion molecules, with 4 out of the 16 genes (25%) having been classified in this category. Neurexins are expressed presynaptically and bind heterophilically to postsynaptic neuroligins to form a trans-synaptic complex coated on both sides by PDZ-domain containing proteins. As such, they mediate trans-synaptic adhesion as well as a variety of intracellular signaling pathways that lead to the development and maturation of the synapse. Neurexins come in six main isoforms, of which NRNX1 and NRXN3 are ASD-linked. A variety of modifications of neurexins can lead to their dysfunction and many types of synaptogenic dysregulation . Interesormation . This deormation . Similarormation ,168.An equally large proportion of our ASD-linked genes regulating synaptogenesis can be classified PSD proteins. These include Shanks  as well as synaptic Ras GTPase-activating protein 1 (SYNGAP1). Shank family proteins are multi-domain scaffolding proteins that organize and interact with an extensive protein complex (30+ proteins) at the PSD of excitatory glutamatergic synapses. This large complex performs a variety of functions, including actin-based cytoskeletal remodeling, synapse formation, AMPA receptor endocytosis, and regulation of synaptic transmission and plasticity . More spFinally, an equally large number of our synapse-linked ASD genes encode subunits of proteins that interact with glutamatergic  and GABAergic (GABRB3) receptors. Glutamate receptor interacting protein 1 (GRIP1) is a neuronal scaffolding protein that interacts directly with the C termini of glutamate receptors 2/3 (GluA2/3) through its PDZ domains. Mutations in GRIP1 induce faster recycling and increased surface distribution of GluA2 in neurons . GRIN2B Synaptogenesis clearly requires the precisely timed and spatially orchestrated assembly of hundreds of pre- and postsynaptic proteins. Major components of the synapse, including synaptic vesicles, glutamate receptors, active zone proteins, PSD scaffolding proteins, and trans-synaptic adhesion molecules must each accumulate at the right site at the right time. Disruption in the core components themselves or regulators of the trafficking and assembly of core components may disrupt synaptogenesis in different ways, one of which is by tipping the balance of excitatory to inhibitory synaptic transmission. The correct innervation of a given circuit requires the successful completion of all of these developmental steps in both pre- and postsynaptic partners, so any disruption in these processes will affect where, when and how synapses are formed.Our functional categorization of ASD genes into a brain development framework indicates that genetic disruptions of processes from early in the macro-architectural patterning through late-developmental synapse function may result in ASD. If this is the case, an analysis of the expression of these genes might be expected to show an array of developmental patterns. To determine this, we examined the developmental expression of our ASD gene list using developmental transcriptome data from the BrainSpan Atlas of the Developing Human Brain ,175. We In support of our functional analysis, we found a variety of expression patterns that largely support the developmental stage indicated by our functional categorization. We found an increased proportion of genes linked to cell division and survival as well as differentiation and migration expressed early in development and, conversely, an increased proportion of genes linked to synapse formation and function as well as excitability expressed late in development. There are notable exceptions such as GRIP1, CANA1H, and SPAST, respectively linked to synapse formation, excitability, and morphological elaboration, that show significantly early developmental expression patterns. Further, genes like PTEN, PON1, and PRKCB, while linked to cell division and survival, displayed relatively late expression. Such discrepancies could indicate an inadequacy in the ability of keywords to functionally categorize genes, lack of specificity in the choice of spatiotemporal timeframe fro the extraction of gene expression data, or that expression patterns may not always be directly coupled to important gene functions. However, we are able to clearly observe subsets of genes preferentially expressed early in brain development and likely most heavily involved in critical early stage brain development processes, such as proliferation, cell division and migration, as well as subsets of genes preferentially expressed late in brain development and likely most heavily involved in critical late stage brain developmental processes such as synapse formation and function. There are also genes with expression spanning the breadth of brain development which are likely important for the smooth execution or perhaps regulation of all developmental processes.Genetic sequencing from large cohorts of patients has provided us with a wealth of information to understand the pathophysiological basis of ASD. There exist a number of automated algorithms to functionally categorize sets of genes, such as DAVID  or GeneMWhile these genetic mutations appear to disrupt a slew of brain developmental processes, it is prudent to ask whether there may be a unifying principle allowing for this disparate set of genes to produce a relatively consistent complex of symptoms. The first possibility to consider is that the underlying pathophysiology of all ASD cases does not fall under one unifying theory. This does not imply that categorizing gene function is an exercise in futility since it is likely that this process will isolate relatively discrete categories of ASDs defined by similar underlying pathophysiologies. In this model, ASD types could be defined by a primary deficit in cell division, migration, differentiation, morphological elaboration, excitability, or synaptic connectivity, and we may find that the emergent impact on brain function is quite disparate and that appropriate treatments for all types of ASD need to be defined by the underlying pathophysiology. This is not to say that we will need a different treatment for each of the hundreds of potential underlying genetic deficits, but that certain related types of ASD may be more responsive to treatments tailored to that type. If ASD is truly etiologically divergent, it will be important to determine whether certain clinical features can be used to group patients into subtypes with common pathophysiology.Another possibility is that the common feature among all cases of ASD is pathological information flow through brain circuitry. While this may seem to be as futile as stating that the unifying principal of ASD is brain dysfunction, this may actually echo a more specific concept of this disorder. To develop an appropriate set of responses to our complex environment, not only must there be appropriate synaptic connectivity, but there must also be appropriate flow of activity through this circuitry. Under this model, there could essentially be two types of ASD, one in which connectivity is disrupted, and another in which the flow of information through that circuit is disrupted. Thus, mutations in genes affecting cell division, patterning and synapse formation disrupt connectivity, while mutations in genes affecting ion channel function may result in brains with relatively intact connectivity but abnormal implementation of this circuitry. However, the relationship between excitability and synaptic circuit formation could certainly mean that it is impossible for alterations in these processes to occur independently.A further unifying theory to consider, overlapping with but distinct from the above, is that all these genetic deficits result in the inability of patterned neural activity to inform appropriate synaptic connectivity during development. To develop appropriate responses to environmental cues, an initial pattern of connectivity must be established through which sensory information can flow. Cell division, differentiation, and elaboration of neurons are critical to establishing this initial circuitry. Disruption of initial patterning will thwart the ability of experience-driven plasticity to fine-tune information flow through the circuitry to generate an appropriate response to given sensory inputs. Excitability of neurons is critical to fine-tuning their morphological elaboration and ultimate synaptic connectivity, and thus, disruptions in ion channels regulating excitability could also ultimately disrupt activity-dependent plasticity. Finally, synaptic adhesion, neurotransmitter receptors, and molecules regulating neurotransmitter release are all also necessary in establishing and fine-tuning the ultimate synaptic connectivity of the brain. As such, disruption of any process from early patterning to synaptic adhesion could by this mechanism alter the ultimate synaptic connectivity of the brain.The two critical differences in the categorization of DD and ASD genes also shed light on these theories of ASD etiology. In general, DD genes did not fall as discretely into any one category. This could indicate an increased number of genes with broad function throughout development, resulting in the more generic features of developmental delay. In addition, the analysis of ASD genes yielded a significantly higher proportion linked to regulating morphological elaboration and neuronal excitability. It will be important to determine whether genes that fall into categories for cell division and differentiation also have important effects on neuronal growth and excitability as has been seen for PTEN and TCF7L2 (TCF4) ,180. ThiWhile altered synaptic connectivity and information flow could be themes that unify disparate cases of ASD, it is clear that this connectivity is not altered in the same way in all cases. Examination of excitatory/inhibitory (E/I) balance caused by certain genetic deficits clearly indicates that synaptic connectivity can be altered in different ways in different cases of ASD. We find examples of E/I increase and decrease ,181 and Whether there is a unifying dysfunction of information flow or of activity-dependent sculpting of synaptic connectivity in ASD, the underlying pathophysiology across cases of ASD is clearly divergent. This said, treatment of ASD as a single disorder is unlikely to be useful for understanding fundamental neurobiology or developing efficacious treatments. Rather, we propose that modeling divergent types of ASD based on primary genetic associations will be necessary for developing therapies for different types of ASD. Furthermore, the search for unifying principles among divergent models of ASD such as altered information flow or synaptic connectivity will shed light on the fundamental neurobiological mechanisms that give rise to complex human behavior.We find that genes associated with autism and developmental delay have functional roles in nervous system development ranging from early macroarchitectural patterning of the brain through late processes involved with sculpting synaptic networks. In comparing genes associated with developmental delay and autism, we find that autism is more often associated with genes involved in the morphological elaboration or regulation of excitability of neurons. The broad array of functions for genes associated with developmental delay or autism lays the foundation for the search for a common mechanism through which disruption in apparently disparate genes may lead to a consistent phenotypic outcome.


The degeneration of dopaminergic and other neurons in the aging brain is considered a process starting well beyond the infantile and juvenile period. In contrast to other dopamine-associated neuropsychiatric disorders, such as schizophrenia and drug addiction, typically diagnosed during adolescence or young adulthood and, thus, thought to be rooted in the developing brain, Parkinson’s Disease (PD) is rarely viewed as such. However, evidences have accumulated suggesting that several factors might contribute to an increased vulnerability to death of the dopaminergic neurons at an already very early  phase in life. Despite the remarkable ability of the brain to compensate such dopamine deficits, the early loss or dysfunction of these neurons might predispose an individual to suffer from PD because the critical threshold of dopamine function will be reached much earlier in life, even if the time-course and strength of naturally occurring and age-dependent dopaminergic cell death is not markedly altered in this individual. Several signaling and transcriptional pathways required for the proper embryonic development of the midbrain dopaminergic neurons, which are the most affected in PD, either continue to be active in the adult mammalian midbrain or are reactivated at the transition to adulthood and under neurotoxic conditions. The persistent activity of these pathways often has neuroprotective functions in adult midbrain dopaminergic neurons, whereas the reactivation of silenced pathways under pathological conditions can promote the survival and even regeneration of these neurons in the lesioned or aging brain. This article summarizes our current knowledge about signaling and transcription factors involved in midbrain dopaminergic neuron development, whose reduced gene dosage or signaling activity are implicated in a lower survival rate of these neurons in the postnatal or aging brain. It also discusses the evidences supporting the neuroprotection of the midbrain dopaminergic system after the external supply or ectopic expression of some of these secreted and nuclear factors in the adult and aging brain. Altogether, the timely monitoring and/or correction of these signaling and transcriptional pathways might be a promising approach to a much earlier diagnosis and/or prevention of PD. DA is ahe brain . In thisThree major cluster of DA-synthesizing neurons are typically found in the mammalian VM, collectively named midbrain dopaminergic (mDA) neurons. These comprise, in a caudal to rostral numbering, the mDA neurons located in the retrorubral field , substantia nigra pars compacta  and ventral tegmental area , as well as a dysregulated calcium (Ca2+) homeostasis are considered as the main culprit, which may be exacerbated by a local or systemic immune response  are expressed in the mammalian embryo in a ubiquitous pattern that is not restricted to the VM or mDA system, whereas PARK genes causing mostly late-onset PD symptoms  when mutated, start to be expressed only slightly later in the target areas  of the mDA neurons (PARK1/4 locus encoding mutant forms of SNCA and the PARK8 locus encoding leucine rich repeat kinase 2 (LRRK2). Abnormal accumulation and fibril formation of SNCA lead to the appearance of Lewy bodies and neurites in the PD brain as one of its histopathological hallmarks, whereas LRRK2 is a multifaceted protein with as yet ill-defined functions in the brain whose mutant variants are the second most common genetic risk factor for iPD  predisposing factor for the progression to PD in the aging individual or under additional adverse environmental and/or genetic conditions. These early mDA deficits are compensated over a significant amount of time by an enhanced DA metabolism or signaling in the remaining cells  active in the mDA domain during prenatal/embryonic development and additionally during adulthood are considered as “developmental factors.” The usability of these factors for preventive or therapeutic interventions in the PD brain is also reviewed, but the myriad of in vitro studies in this regard are not addressed here. The involvement of these signaling and/or transcriptional cascades in ongoing neurodegenerative processes associated with PD are not discussed as they were recently reviewed by others  or striatum (mDA target region)  in SNc DA neurons during late embryonic/early postnatal and adult stages  and behavioral (reduced feeding but no locomotor deficiencies) phenotypes  inducible Dat-Foxa1/2 cKO mice  and cognitive deficits in adult and especially aged mice  SNCA in the dystrophic terminals of Dat-Lmx1a/b double-mutant mDA neurons  was identified as one of the differentially expressed genes between mouse SNc and VTA, which is confined to a VTA DA neuron subset co-expressing the TF OTX2, the Ca2+-binding protein calbindin  and ALDH1A1 (Neurod6+ cells), the vesicular glutamate transporter 2  and the Ca2+-binding protein calretinin  were expressed in subsets of the Neurod6+ VTA DA neurons , and project mostly to the NAc medial shell and to a lesser extent to other parts of the NAc and lateral septum (LS) in adult mice  was less vulnerable to neurodegeneration after 6-OHDA treatment of mice, although the downstream targets of this TF conveying these neuroprotective properties remain unknown (The basic helix–loop–helix (bHLH) TF  ALDH1A1 . Other m neurons . The NEUult mice ,D. They ult mice . To detein vivo,  disrupteehaviors . The onsn subset . DeletioA subset . A caveaelopment . Most im unknown .NURR1 is so far the only mDA developmental gene with identified rare mutations in fPD and iPD cases [, displayed an enhanced age-dependent reduction of NURR1 protein levels and decreased mDA neuron numbers, increased neuroinflammation and SNCA aggregation, and L-DOPA-responsive locomotor impairments with reduced penetrance , has attracted considerable attention because of its involvement in the specification of the mDA neuron phenotype during development and its anti-inflammatory properties . NURR1 iD cases [ and refeD cases [. Homozyger birth , but hethibition , and an hibition . The indairments , 2013. TcKO mice , 2013. Ahuman PD . Interesrylation . Furtherrylation . Downregrylation . Additiorylation . Apart fptor Ret . Accordinetrance . One pos+ dorsolateral VTA (PBP) DA neurons, which also express SOX6 and high levels of the glycosylated (active) form of DAT  and eyeless (eyl) mice, or inactivation of this gene in −/−Pitx3 mice, primarily affected the vSNc DA subset, whereas the dSNc and VTA DA subpopulations were relatively spared in these mutant mice [ and DA contents as well as locomotor deficits. The selective protection from neurodegeneration of the dSNc and VTA DA subsets in the Pitx3 mutant mice appeared to be due to the mutually exclusive expression of CALB1 and PITX3 in these neurons , and heightened inflammatory responses in the adult and aging mouse VM in the absence of Pitx3. PITX3 was also shown to be upregulated downstream of retrograde (NFKB1-mediated) GDNF signaling via the RET receptor and GFRa co-receptors  and RRF, and appeared to innervate parts of the ventromedial striatum, especially NAc core and lateral shell  located in the human vSNc is the population showing the largest decline in postmortem PD brains and the highest association with PD risk genes (+ mDA neuron subset located in the dSNc that is only found in the primate (human) brain and not in rodents and other species  after forced expression of lineage-specific TFs in appropriate culture conditions  and RRF  insult  insult . AlthougApart from their endogenous roles discussed in Given their secretory nature and their potential to act over longer ranges, signaling factors are particularly amenable for external manipulation by systemic or local administration. However, their mode of action and thus effectiveness is restricted by the availability of the receiving side, i.e., the appropriate receptors must be expressed in the mDA neurons and the danger of negative side-effects or unwanted targets in other cells poses an additional challenge in this regard.in vivo also counteracted the appearance of pronounced motor symptoms in the lesioned animals, whereas the treatment with an FGFR antagonist  in the murine SNc prior to MPTP lesioning suppressed neuroinflammation in this region and rescued the degeneration of SNc DA neurons and locomotor deficits in these mice . Althoughenotype , intracehenotype . Subsequicroglia . Furthericroglia .Intranigral injection of BMP7 protein immediately before 6-OHDA lesioning had a dose-dependent (the lower dose being more effective) neuroprotective action on the ipsilateral mDA neurons and striatal projections, and reduced the motor deficits of the lesioned rats . Intracein vivo  cells to the mDA neurons  in the heterozygote +/Wnt1En1 mice .+/−En1 mice  after subchronic MPTP treatment of mice  display an adult-onset and progressive neurodegenerative process that resembles at least some of the early neuropathological features observed in human PD patients. These include a defective synaptogenesis and DA neurotransmission at their striatal terminals and dendrites in the SNr, the retrograde  degeneration of the mDA axons preceding the actual death of the mDA neurons, and an increased inflammatory response in the mutant VM. One striking observation in this context is that several of the “classical developmental TFs” for which direct target genes have been identified in the adult and aging brain, such as EN1/2, LMX1A/B and NURR1, appear to regulate the same pathways that are thought of primarily going awry in the PD brain, namely the mitochondrial energy metabolism, ALP and immune response/anti-inflammatory pathways. It is hard to imagine that these pathways and their target genes are not yet regulated in the mDA neurons in which these TFs are already active during prenatal and early postnatal development. Other “classical developmental TFs,” such as FOXA1/2, OTX2 and PITX3, appear to regulate mDA-specific target genes implicated in DA metabolism and neurotransmission, neurotrophic support and electrophysiology/Ca2+ homeostasis in the adult brain, which would have been the more expected outcome in this context. The latter targets are more related to the probably intrinsic metabolic stress load of the mDA neurons throughout their lifetime, discussed in PARK genes  also hampers current approaches to preventive or disease-modifying therapies for PD. Much more and focused research in this direction is thus urgently needed to advance this field in the near future. Remarkably, germline and especially adult cKO mouse mutants for several of the signaling and TFs discussed in RK genes , the kinRK genes . As alreRK genes , the aveainst PD .2+ homeostatic processes in the emerging mDA neurons, most likely playing a role already during the earliest stages of mDA neuron development and long before they have established a proper connectivity with their efferent and afferent targets, although their mitochondrial energy metabolism and autophagic/lysosomal clearance of misfolded proteins and defective organelles are now beginning to be unraveled in the human context in vitro , grant number PR 629/3-1 (Projektnummer 408031320) to NP.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction: Outcome reporting bias in vaccine studies is a widespread problem among all researchers who have a tendency to report selective results and conclusions that support their beliefs and values or those of sponsoring agencies. Especially during the COVID-19 pandemic, this bias surfaced through the unprecedented proliferation of conflicting vaccine studies. Many researchers strongly recommend and report on the safety and effectiveness of the COVID-19 vaccine. Those researchers who embrace the COVID-19 vaccine and vaccines, in general, are often dismissive of other researchers who present views that differ from medical orthodoxy and oppose medical consensus.Methods: The aim of this analysis is to critically evaluate seven vaccine studies using qualitative and/or quantitative approaches to identify outcome reporting bias and assess its potential impact on the stated conclusions that align with medical consensus. Four studies claim to have found no association between autism and (a) blood levels of mercury, (b) measles, mumps, and rubella (MMR) vaccine, and (c) thimerosal-containing vaccines. Three other studies claim no association exists between infant mortality rate and the number of vaccine doses, universal varicella vaccination and herpes zoster, and pandemic influenza vaccines and fetal losses.Results: The presence of outcome reporting bias and independent reanalysis demonstrated an impact on both the direction and magnitude of the observed effect - raising questions concerning the robustness of the original study design and conclusions and challenging the current medical consensus. Medical consensus has exonerated vaccines as having any causal relationship to autism spectrum disorders (ASDs), yet no other reasonable cause has been proposed. Medical consensus attributes significant ASD increases to better case ascertainment and broadened clinical diagnosis. According to 2018 data, an estimated 1 in 44 eight-year-olds has been identified with ASD. From 1990 to 2019, there have been an estimated two million new cases of ASD in the US, with lifetime social costs exceeding $7 trillion (in 2019 dollars). Can perpetuating medical consensus impede the advancement of public health? Or has it already done so?Conclusions: Conflicts of interest  that abound between health regulatory agencies and the pharmaceutical industry impact what is ultimately reckoned as medical consensus. Outcome reporting bias that is inherent to all researchers to some degree, obscures medical and scientific truth. Advancement of public health requires that researchers have integrity and an openness and willingness to collaborate to resolve contradictory findings. In fact, it is usually through meticulous, rigorous, scientific investigation of contradictory findings that medical science has advanced and contributed to improvements in public health - since medical consensus and orthodoxy can be incorrect. Outcome reporting bias in vaccine studies is a widespread problem among all researchers  who haveEspecially during the COVID-19 pandemic has outcome reporting bias surfaced through unprecedented proliferation of conflicting vaccine studies. Many researchers strongly recommend and report on the safety and effectiveness of the COVID-19 vaccine. Those researchers who embrace the COVID-19 vaccine and vaccines in general are often dismissive of other researchers who present views that differ from medical orthodoxy and oppose medical consensus. Those presenting factual data and analyses critical of medical consensus are blamed (censored) for precipitating vaccine hesitancy through "misinformation." Marcia Angell (who previously served as an Executive Editor and later as the Editor-in-Chief) of The New England Journal of Medicine and Editor-in-Chief of The Lancet, Richard Horton, both caution that a proliferation of vaccine studies published in prestigious peer-reviewed medical journals possess such an overwhelming degree of outcome reporting bias, that they serve, in large part, as a marketing arm of the pharmaceutical industry ,4. OutcoData in the various studies referenced are available from the corresponding study authors or journal editors. The appendices provide referenced email communications obtained through Freedom of Information Act (FOIA) requests, a form letter from a journal editor declining publication, and pertinent extracts from CDC websites.The methods of analyses vary from study to study, and specific methods are discussed in detail in each referenced study. Where applicable, statistical significance was taken at p < 0.05. Empirical analysis of outcome reporting bias has in some cases involved the methods summarized as follows: Study 1 uses a two-tailed t-statistic to investigate whether there is a statistically significant difference between the blood mercury levels of (a) children with autism spectrum disorder (ASD) and (b) normally developing children. Study 2 computes a rate ratio of children diagnosed with ASD following measles, mumps, and rubella (MMR) vaccination to those children not administered MMR vaccination. A rate ratio greater than 1 indicates an increase in ASD among those vaccinated. Study 3 assesses the discrepancy between statements communicated among authors via email and statements reported concerning study data trends and conclusions. Study 4 notes the impact of a biased dataset and the use of heterogeneous and homogeneous data on linear regression analyses of infant mortality rates (IMRs) of reported nations as a function of the corresponding number of vaccine doses specified in the nation's infant immunization schedule. Study 5 illustrates the importance of analyzing a full dataset instead of a selected sample that is not characteristic of the population. Study 6 qualitatively considers HZ incidence reported over 60 years starting in 1945 and investigates a two-segment, piecewise linear model and the year of its breakpoint to determine if universal varicella vaccination had an impact on increasing HZ incidence rates. Study 7 investigates the number of fetal losses reported annually for each influenza season from 1990/1991 through 2002/2003 and presents unadjusted rates of fetal loss per 1 million pregnant women vaccinated as well as an ascertainment-corrected rate derived through the use of capture-recapture statistics. A comparison was made of fetal-loss reports in influenza seasons both before and after a large spike in fetal loss reports that occurred during the influenza pandemic of 2009/2010.The aim of this analysis is to critically evaluate seven vaccine studies using qualitative and/or quantitative approaches to identify outcome reporting bias and assess its potential impact on the stated conclusions that align with medical consensus. These studies claim to have found no association between (1) blood levels of mercury and autism, (2) MMR vaccine and ASDs, (3) thimerosal-containing vaccines and autism incidence rates, (4) number of vaccine doses and IMRs, (5) MMR vaccine and pervasive development disorders (PDDs), (6) universal varicella vaccination and herpes zoster (HZ), and (7) pandemic influenza vaccines and fetal losses.After each study was critically evaluated by one or more independent researchers, reanalysis yielded results that challenged medical consensus and orthodoxy, thus confirming Nickerson’s maxim, "Many beliefs may be held with a strength or degree of certainty that exceeds what the evidence justifies." Study 1: Association between blood levels of mercury and autismp = .15) difference between the blood mercury levels in 82 children with ASD and 55 normally developing children  increase before vs. after the introduction of the varicella vaccination program" . HoweverFigure Study 7: Unusual pattern in fetal losses following pandemic influenza vaccinesUtilizing data from the Vaccine Adverse Event Reporting System (VAERS), Moro et al.  establisOutcome reporting bias might explain why the CDC-sponsored study by Moro et al. chose to consider data through 2008/2009  and stopin utero mercury exposure to the developing fetus that could potentially exceed the FDA safety level  [In 2009, the CDC’s ACIP recommended pregnant women "get the seasonal flu shot and the 2009 H1N1 flu shot" on the basis that (1) the seasonal influenza vaccine was associated with low fetal-loss reports to VAERS occurring during the prior 19 single-dose seasons and (2) the pandemic A-H1N1 vaccine "is made in the same way and in the same places as the seasonal flu shot" (Appendix IV). This qualitative assessment of safety was subject to outcome reporting bias since it overlooked the fact that during the first 14 of the prior 19 seasons, a more precautionary vaccination approach prevailed during which only those pregnant with special circumstances or those beyond their first trimester were vaccinated. Moreover, despite the majority of seasonal and pandemic influenza vaccines each containing 25 mcg of mercury, no warning or concern was issued by the CDC regarding pregnant women receiving up to 50 mcg of mercury - resulting in an Later, in another study, Moro et al. concluded , "the reTwo different CDC-sponsored studies by Moro et al. investigating VAERS reports concluded there were "no unusual pattern"  and no "Vaccines are not associated with autism: an evidence-based meta-analysis of case-control and cohort studies [The presence of outcome reporting bias and independent reanalysis of each study demonstrated an impact on both the direction and magnitude of the observed effect - raising questions concerning the robustness of the original study design and conclusions and challenging the current medical consensus. Each of the seven study outcomes experienced a reversal, yielding statistically significant positive correlations between (a) MMR vaccine and ASD (and PDD), (b) thimerosal-containing vaccines and autism, (c) the number of vaccine doses and IMRs, (d) universal varicella vaccination and HZ incidence, and (e) pandemic influenza vaccines and VAERS fetal-loss reports. Once "overwhelming" evidence has accumulated in a given area of research (reinforced by publication bias that serves to suppress studies with undesirable results), the medical consensus is further sustained by the publication of a synthesis of the results of all conducted studies via meta-analyses, such as that exemplified by Taylor et al., titled  studies .Complicating factors underlying outcome reporting biasin utero mercury exposure to the fetus) [In the US, the American Academy of Pediatrics recommended that thimerosal be removed from routine childhood vaccines in 1999; however, the existing, unexpired doses of thimerosal-containing vaccines continued in use throughout 2002. In 2002, the CDC recommended two new thimerosal-containing influenza vaccines for children aged 6-23 months. Moreover, in 2004, CDC recommended that women, in any trimester of pregnancy, be administered a seasonal influenza vaccine (resulting in an e fetus) , served Especially during the COVID-19 pandemic, the media has played a key role in stimulating outcome reporting bias concerning vaccine efficacy. This was alluded to by Peter Daszak, President of EcoHealth Alliance, three years prior to the start of the COVID-19 pandemic in February 2016:Daszak reiterated that until an infectious disease crisis is very real, present, and at an emergency threshold, it is often largely ignored. To sustain the funding base beyond the crisis, he said, we need to increase public understanding of the need for MCMs  such as a pan-influenza or pan-coronavirus vaccine. The media is a key driver, and the economy follows the hype. We need to use that hype to our advantage to get to the real issues. Investors will respond if they see a profit at the end of the process, Daszak stated .A more elaborate form of outcome reporting bias in vaccine studies  occurs when input data are manipulated until various statistical computer programs output selective results that appear correct, demonstrate statistical significance, and support a predetermined conclusion. Under such a scenario, the COVID-19 vaccine may be purported to be "safe and effective" based on selective data showing a lower incidence of hospitalization and death due to COVID disease among COVID-vaccinated vs. unvaccinated patients. However, a comprehensive risk-benefit assessment may yield public health concerns when data with wide disparity have been properly stratified, or additional data are available for all-cause morbidity and mortality - especially if ascertainment-corrected vaccine-induced events  exceed population background rates for those same events. Consider the nonsteroidal anti-inflammatory drug (NSAID) Vioxx, licensed by the FDA in 1999 for patients experiencing arthritic pain. While Vioxx was initially believed to reduce the risk of serious gastrointestinal complications by 50% relative to the NSAID pain-reliever naproxen, by 2004, Vioxx was withdrawn from the market after studies reported a four-to five-fold increased risk of cardiovascular events .Outcome reporting bias prolongs the vaccine/autism debateBetween 2001 and 2007, more than 5,000 families filed autism claims with the federal Vaccine Injury Compensation Program (VICP). In these cases, many parents describe their children as exhibiting late-onset autism-like features shortly following vaccine administration, i.e., children who could perform tasks and follow directions, as well as speak and maintain eye contact, suddenly regress, losing these acquired skills and abilities. Poling et al. document such a case in a 19-month-old female with a pre-existing, previously undiagnosed, mitochondrial disorder that predisposed her vulnerability to the receipt of five vaccines administered in a single physician’s visit against nine vaccine-preventable diseases  . Years lVaccine and Autism: A Tale of Shifting Hypotheses conclude:Medical consensus has exonerated vaccines from having any causal relationship to ASD. Gerber and Offit in Twenty epidemiologic studies have shown that neither thimerosal nor MMR vaccine causes autism. These studies have been performed in several countries by many different investigators who have employed a multitude of epidemiologic and statistical methods. The large size of the studied populations has afforded a level of statistical power sufficient to detect even rare associations. These studies, in concert with the biological implausibility that vaccines overwhelm a child’s immune system, have effectively dismissed the notion that vaccines cause autism. Further studies on the cause or causes of autism should focus on more promising leads .All epidemiologic studies have methodological limitations. Any study  whose data are unavailable for independent reanalysis  should be retracted since reported results and conclusions are unverifiable. Despite accolades ascribed to epidemiologic studies (including those "unverified") that endorse medical consensus, we must ask: Do they present the truth so convincingly that researchers should "focus on more promising leads" given the thousands of parents who have reported their children regressing following vaccine administration? Do health regulatory authorities believe that every single one of these late-onset autism cases is merely coincidental and that no evidence of a dose-response effect or plausible biological mechanism exists? Can medical consensus be trusted when it attributes significant ASD increases to better case ascertainment and changes in clinical diagnosis? What are the chances that at least some children have undiagnosed mitochondrial disorders similar to the Poling child discussed earlier and experienced similar vaccine injury?Studies that rely on epidemiologic methods may not be able to successfully control for confounding and bias due to their inability to distinguish congenital cases of autism  from cases of late-onset autism . Without access to specific childhood immunization records, epidemiologic studies have limited ability to ascertain the potential synergistic effects of various combinations of vaccines. Additional fields of neurology, toxicology, virology, pharmacology, and pharmacokinetics may help medical professionals to better elucidate the pathogenesis of adverse vaccine effects due to exposures to aluminum, thimerosal (ethyl-mercury), and other vaccine components.Of the many experts who are certain that the MMR vaccine and thimerosal-containing vaccines have no causal relationship to ASD, not one has yet to propose another reasonable cause. Instead, on February 8, 2007, CDC director Dr. Julie Gerberding admitted, "… we can't yet tell if there is a true increase in ASDs or if the dangers are the result of our better studies" . Dr. GerSegal and Shoenfeld have proposed a plausible mechanism that is suspected in adverse vaccine reactions : "MolecuAccording to 2018 data, an estimated 1 in 44 eight-year-olds has been identified with ASD, according to the Autism and Developmental Disabilities Monitoring (ADDM) Network. In other terms, Cakir et al. report that from 1990 to 2019, there have been an estimated two million new cases of ASD in the U.S. with lifetime social costs exceeding $7 trillion (in 2019 dollars) . Can perLimitationsThe main limitation of this analysis was that outcome reporting bias was investigated in seven vaccine studies, yet it impacts thousands of vaccine studies and is present to some degree in all research.This study examined several examples of outcome-reporting biases that are found in many vaccine studies. Conflicts of interest  that abound between the FDA, CDC (or foreign entities such as the Danish Staten Serum Institut), and the pharmaceutical industry impact what is ultimately reckoned as medical orthodoxy or scientific consensus. Moreover, regulatory agencies seemingly attempt to control the narrative that vaccines are "safe and effective" through their funding or sponsorship of selective studies that often lack data transparency and, in some cases, cross bioethical boundaries. Where vaccine study data are available, independent researchers experience an astonishing level of censorship by medical journal editors who deny publication when outcome reporting bias in the original published studies is exposed and reanalysis leads to conclusions contrary to the medical consensus. This conduct, as well as the outcome reporting bias that is inherent to all researchers to some degree, obscures medical and scientific truth.Ideally, if conflicts of interest could be eliminated among vaccine study sponsors, regulatory agencies, and the pharmaceutical industry, the fact that various researchers might obtain contradictory results and conclusions would not be worrisome. It may be the case that two contradictory presentations - the one supporting the medical consensus and the one that is antagonistic - may both be flawed! Advancement of public health requires that researchers have integrity and an openness and willingness to collaborate to resolve contradictory or mixed results. In fact, it is usually through meticulous, rigorous, scientific investigation of contradictory findings that medical science has advanced and contributed to improvements in public health - since medical consensus and orthodoxy can be incorrect. The narrative that COVID-19 vaccines are "safe and effective" is not immune to outcome reporting bias. Those in support of medical consensus lose credibility and ultimately undermine public confidence in vaccine programs by censoring researchers reporting deleterious effects, especially when the numbers of adverse reactions become so prevalent that the medical community can no longer claim they are coincidental or when a given vaccine's risk-benefit ratio is shown to be no longer favorable.


Cataract and presbyopia are the leading cause of vision loss and impaired vision, respectively, worldwide. Changes in lens biochemistry and physiology with age are responsible for vision impairment, yet the specific molecular changes that underpin such changes are not entirely understood. In order to preserve transparency over decades of life, the lens establishes and maintains a microcirculation system (MCS) that, through spatially localized ion pumps, induces circulation of water and nutrients into (influx) and metabolites out of (outflow and efflux) the lens. Aquaporins (AQPs) are predicted to play important roles in the establishment and maintenance of local and global water flow throughout the lens. This review discusses the structure and function of lens AQPs and, importantly, their spatial localization that is likely key to proper water flow through the MCS. Moreover, age-related changes are detailed and their predicted effects on the MCS are discussed leading to an updated MCS model. Lastly, the potential therapeutic targeting of AQPs for prevention or treatment of cataract and presbyopia is discussed. The majority of cases of age-related vision loss in the world today are associated with the lens pathologies presbyopia and cataract . Presbyovia the MCS. Further, we hypothesize that age-related changes in AQP structure and function lead to cataract formation. Finally, we will discuss therapeutic strategies being developed in other tissues that are targeting AQPs with the view to inform efforts to develop novel therapies on the lens.It has been shown in animal lenses that movement of water through the lens generates a substantial and highly regulated pressure gradient , deliver+ ions, which primarily enters the lens at the poles and travels into the lens via the extracellular spaces between fiber cells (+ ions cross fiber cell membranes and return towards the surface via an intercellular outflow pathway mediated by gap junction (GJ) channels (Cx46 & Cx50)  , Outflow equator , Efflux. equator . This fl equator . Mapping equator  and conf equator  has reve equator . It has  sutures .+ pump  in mammals and act to move water bidirectionally across biological membranes through osmotically driven passive diffusion . ClearlyThe water permeability rate varies between aquaporin isoforms; for instance, AQP0 water permeability is approximately 20-fold lower than AQP1 and AQP5 . In addiH2O), is enhanced by the presence of AQPs in cell membranes, we can expect that differences in AQP expression, subcellular distribution, lipid and protein interactions, and function will all contribute to the directed movement of water into and out of the lens. In addition, other AQP properties, such as cell adhesion, could also be important in establishing the lens MCS and will be discussed below. There are few reports on lenticular AQP7 and AQP8 expression and both appear to be expressed exclusively in lens epithelial cells  with each protein displaying unique localization and abundance patterns, discussed in detail below . Since tal cells . Althougal cells , the spein vitro in high glucose. Recently, it has been reported  in the rat lens . SubsequAs fiber cells differentiate from lens epithelial cells at the lens equator, we expect normal differentiation-related processes, such as protein synthesis/degradation, protein trafficking and post-translational modification, to govern protein expression and function. However, fully mature lens fiber cells are organelle-free and therefore lack the ability to synthesize new protein . Thus, rThe following mechanisms have been shown to alter AQP function and their potential roles in lens AQP regulation are discussed:Phosphorylation: Reversible protein phosphorylation is one of the most common posttranslational modifications and provides dynamic posttranslational control of protein function  binding and the interaction of CaM through binding to the short amphiphilic helix region (Leu227-Gly237) of AQP0 is reduced upon phosphorylation . The classic example is the phosphorylation of the AQP2 C-terminal residue S256 that promotes the trafficking of AQP2 to the apical membrane of epithelial cells that line the cortical collecting duct of the kidney (In AQP5, two consensus PKA sites are found: S156 in ICL2 in fficking  and altee kidney . In thise kidney  and indee kidney  of phospMembrane Trafficking: As mentioned above, phosphorylation-dependent trafficking to and from the membrane may be a way to modulate PH2O of fiber cell membranes, however it needs to be remembered that fiber cells are essentially elongated epithelial cells that retain their distinct apical and basal membrane domains but have dramatically elongated lateral membranes (embranes . To achiembranes  (Zampighembranes . The apiembranes  to form embranes . Howeverembranes .In contrast, AQP5, a close homologue of AQP2, appears to traffic by a different mechanism from that of AQP0 . AQP5 diProtein Interactions: Multiple proteins have been reported to interact with AQP0 and such interactions occur most often through the AQP0 C-terminus (H2O in those specific regions of the lens.terminus . As mentterminus  and positerminus . In addiAQP0 associates with gap junction plaques in a narrow zone of the lens bow region and this interaction may facilitate the assembly of Cx50 into nascent gap junction plaques during the early stages of embryonic lens development . The AQPStudies of proteins that interact with AQP5 in the lens are limited, however, in other tissues, AQP5 function can be regulated by protein-protein interactions . For exaLipid Interactions: Intrinsic membrane proteins are embedded in a biological membrane where lipids dynamically interact with them and can affect their function as well as structural properties such as folding, packing and stability (tability . Similartability . Others tability  and permtability . Althougtability , how difNumerous studies of age-related modifications to lens proteins reveal extensive age-related modifications to AQPs including truncation , deamidaTruncation: Non-enzymatic truncation is a prevalent age-related modification observed for most of the abundantly expressed lens proteins, including AQP0 (ing AQP0  and AQP5AQP0 undergoes extensive C-terminal truncation with two major truncation sites at Asn 246 and Asn 259 . TruncatConsistent with western blot results, AQP5 undergoes only minor C-terminal truncation in the lens core . Using hDeamidation: One of the most abundant age-related modifications in human lenses is deamidation (midation  and AQP0midation  even in midation . The funmidation , this momidation ; therefoCrosslinking: It has been widely recognized that lens protein crosslinking and loss of solubility contribute to the development of age-related lens opacity ( opacity . Recentl opacity  and AQP0 opacity . AQP0 is opacity . The fun opacity . Thus, cH2O that facilitate the outflow and efflux of water from the lens driven by the local osmotic gradients generated by the ion fluxes that drive the MCS (via an intracellular pathway mediated by gap junctions where at the lens surface AQP channels mediate the efflux of water from the lens. In this section, we now present an updated model of regional differences in AQP functionality which includes recently acquired data (Experimental confirmation of the existence of water fluxes  along wi the MCS . However the MCS  (Zampigh the MCS . Thus, Ared data  that sugH2O is dependent on both AQP0 and AQP5, which undergo distinctly different differentiation-dependent changes in their subcellular location and post-translational modification which by altering local PH2O contribute to the overall magnitude and directionality of water fluxes that circulate through the lens. Based on our current understanding of these changes to AQP0 and AQP5 expression we propose that full length AQP0 provides a basal level of water permeability in the outer lens cortex and this permeability can be altered by AQP0 phosphorylation/calmodulin binding (H2O, can be modulated by either mechanically or pharmacologically altering zonular tension via a process that is mediated by TRP channels (H2O of the plasma membrane relative to the PH2O of the gap junctions would tend to facilitate the cell-to-cell removal of water via the intracellular outflow pathway mediated by gap junctions. In this regard it has been shown that AQP0 becomes associated with the periphery of gap junction plaques in this region (H2O permeability by phosphorylation/calmodulin binding and interaction with binding partners, while the altered lipid environment in the lens is predicted to reduce the PH2O of AQP0 based on in vitro studies (via full length AQP5 in the core rather than via truncated AQP0 water channels.In this updated model , AQP1 is binding . In thischannels . In the channels , that machannels . Recent channels . A majorchannels . Furthers region . In conts region . Whethers region  remains  studies . Since i studies , we propvia AQP0 water channels (via AQP0 and/or AQP5 channels located in the lateral membranes of fiber cells. In the inner cortex the restriction of extracellular space would reduce this lateral extracellular movement of water away from the sutural influx zone and direct water flow towards the lens core. In this inner cortical region movement of water from the suture into the fiber cells via AQP5 can be dynamically modulated by changes in zonular tension in the anterior (H2O in this region of the lens.Since the diffusion of water through water channels is driven by the local osmotic gradient established by ion transport we will now propose how these observed spatial differences in AQP0 and AQP5 location and functionality combine to drive water transport through the lens . In paraanterior . We specvia an intracellular outflow pathway that delivers water to an equatorial efflux zone where water leaves the lens. In this outflow pathway (H2O of fiber cells in the efflux zone can be regulated in parallel to the ion fluxes that drive the transport of water throughout the lens. Taken together, it is clear that the expression level, the subcellular localization, the extent of modification, the local lipid environment, and the extent of protein-protein interactions all play roles in the regulation lens water transport which has been shown to be so critical for the maintenance of the transparent and refractive properties of the lens (Once delivered to the central lens, water moves towards the surface of the lens  pathway , PH2O ofthe lens .Based on our knowledge that AQP mutations or AQP deletion leads to cataract formation  and thatHeliotropium indicum extract was found to alleviate selenite-induced cataract and also increase AQP0 levels (In other tissues where AQP dysfunction has been linked to numerous diseases, AQPs have been promoted as therapeutic drug targets . For exa0 levels . Conside0 levels , the acc0 levels , therapeThere are many features of our working model for lens water transport that require further investigation. Given the known effects of post-translational modifications, lipid environment and expected effects on local water permeability, spatial definition of these lens properties is necessary to define the molecular basis of lens water transport. It is important to recognize that many previous studies have used either homogenized whole lenses or separation methods with low spatial resolution  resultin


The risk of bias was assessed using the Newcastle Ottawa Scale and 20 studies were eligible for narrative analysis. Limitations included the heterogeneity in detection assays used, the small and uneven sample size, a lack of vitreous data in earlier disease stages, and not accounting for patients with other systemic co-morbidities. Analysis showed that inflammasome biomarkers IL-1β and IL-18 increased significantly from non-proliferative DR to proliferative DR in both vitreous and serum, suggesting the inflammasome pathway is activated as DR progresses and that serum inflammasome levels could be explored as potential biomarkers for DR progression.Activation of the NOD-like receptor protein 3 (NLRP3) inflammasome pathway has been implicated in Diabetic retinopathy (DR) pathogenesis, but its impact on DR development and progression remains unclear. Therefore, the primary aim of this systematic literature review was to determine the role of the inflammasome in DR development. Furthermore, the secondary aim was to determine whether systemic inflammasome activity can be used to predict DR progression. Studies measuring vitreous and/or serum inflammasome biomarkers in DR patients with Type 2 Diabetes Mellitus (T2DM) were searched systematically using online databases EMBASE, PubMed and Web of Science with the last search conducted on 29The online version contains supplementary material available at 10.1186/s12886-022-02439-2. Diabetic retinopathy (DR) is the leading cause of preventable blindness in the working-age population worldwide . In 2010DR is characterized by the loss of retinal vascular integrity, which results in the development of vascular lesions including microaneurysms, dot and blot hemorrhages and hard exudates –5. Proli., 2021 [., 2019 [Several treatment options are available for DR and DME, including pan-retinal photocoagulation, intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections and intravitreal corticosteroids . However., 2021  reported[., 2019  also sho[., 2019 . Other c[., 2019 –15. ManaThe pathogenesis correlating chronic systemic hyperglycemia with the development of vascular lesions in DR is not fully elucidated. In contrast to previously proposed mechanisms which reflect DR as a single hyperglycemia-induced process, including formation of advanced glycation end products as well as the polyol, protein kinase C and hexosamine pathways, recent studies have demonstrated that diabetic complications, including DR, are underpinned by a complex interplay between metabolic and inflammatory changes , 17. ThiWhilst these studies highlight the importance of the inflammasome pathway in DR, the direct or indirect impact of the inflammasome on DR onset and progression has not been investigated extensively. Therefore, the primary aim of this systematic literature review is to determine the role of the inflammasome in DR development and progression by correlating serum and vitreous inflammasome levels with DR progression. The secondary aim is to determine whether systemic inflammasome activity can be used to predict DR progression by testing whether higher inflammasome activity is associated with more rapid DR progression. This systematic literature review suggests a close association between the development and progression of DR and vitreous inflammasome biomarkers. Increased levels of inflammasome biomarkers were also found in the serum of DR patients with more advanced DR compared to those at early stages of the disease, suggesting that blood tests could potentially be used clinically to predict the development and progression of DR. This study provides further insight into the role of the inflammasome in DR pathogenesis, and opens up opportunities for the development of new drugs which specifically target the NLRP3 inflammasome pathway.Using pre-specified keywords in the search strategy Table S, 305 stuQuality and risk of bias were assessed using the predefined criteria and scoring system in the modified Newcastle Ottawa Scale (NOS) Table S, which i., 2018 [., 2018 [., 2015 [., 2018 [., 2020 [The clinical profiles of participants in the included studies are summarized in Table S., 2018 , which h[., 2018  [., 2018 . IL-18 [., 2009  showed s[., 2005  showed a[., 2018  also shoThere was a surprising lack of literature investigating the role of the inflammasome in the development and progression of DR as there are already a number of NLRP3 inflammasome inhibitors in research which have been proposed as potential DR therapeutics. This includes MCC950 which directly disrupts the oligomerization of the NLRP3 inflammasome complex , and Pep., 2014 [Several other significant gaps in literature were revealed in this review. Firstly, most studies only provided vitreous biomarker levels in PDR and control but not in earlier stages of the disease, making it difficult to determine whether the inflammasome is activated in all T2DM patients or only in patients with manifested DR, and whether there is a significant difference between NPDR and PDR. It would be valuable to have the data of vitreous biomarker levels from NDR and NDPR groups; however, this is generally unethical, given the highly invasive nature of vitrectomy. For the control group, studies have retrieved vitreous from patients requiring vitrectomy due to non-diabetic retinal diseases such as macular hole or epiretinal membranes, which may imply the presence of other systemic conditions and may thus not be true controls. However, it is generally very difficult to collect vitreous samples from patients with both early DR and these other retinal diseases. This further emphasizes the need for serum-based DR screening tests that use systemic inflammasome biomarkers to predict DR progression. Secondly, there is a significant lack of longitudinal study that monitors inflammasome biomarker levels over time as DR develops and progresses. Preciado-Puga et al., 2014  was the There are a few limitations in this systematic literature review. Firstly, data were analyzed qualitatively because a meta-analysis could not be performed due to the heterogeneity in DR grading scales, the detection assays used, the small and uneven sample size as well as the small number of studies included. The differences between detection assays are important to take into account as Luminex and CBA, which quantify results based on fluorescence intensity, are more sensitive compared to an ELISA, a colorimetric assay. Secondly, other types of diabetes besides T2DM were not included as the study was designed with minimal differences between study populations in order to make valid comparisons. Lastly, this systematic review did not account for diabetic patients with systemic co-morbidities other than DR as these conditions may potentially cause an exponential increase in pro-inflammatory biomarker levels, masking the difference between different DR severity levels. The reality is that T2DM patients with severe DR are often diagnosed with other microvascular and macrovascular co-morbidities associated with systemic inflammation. As such, it is likely that systemic inflammation drives the development and progression of DR, further supporting the need to measure serum inflammasome biomarkers to predict DR development and progression.A comprehensive search strategy, including pre-specified keywords used to identify articles that have measured biomarker levels in the vitreous and serum of patients with T2DM with and without DR, is provided in Table SStudies that fulfilled the prespecified inclusion criteria Table  were deeth of September 2021 to identify all pertinent studies using online electronic databases EMBASE, PubMed and Web of Science. The references of the included studies were also searched to identify further relevant studies. EndNote X9 was used to manage the identified studies and record the eligibility status.A systematic search was conducted on the 29Two authors (CYJK and OOM) independently searched the online databases, screened the studies by titles and abstracts and subsequently identified eligible studies as outlined above. Any discrepancies were resolved through discussion and consultation with the third, independent author (IDR).Two independent authors (CYJK and OOM) extracted the following information from each study: name of author(s), year of publication, study type, biomarker location, DR grading scale and measurement assay Table S. CYJK exA modified version of the NOS was used to assess the quality and risk of bias quantitatively and qualitatively. The specific criteria and scoring system were predefined prior to the study Table S. The thrChanges in the levels of vitreous and serum pro-inflammatory, anti-inflammatory, chemotactic and cell growth biomarkers in DR patients compared to controls were summarized Table . Levels Additional file 1: Table S1. Search strategy using Ovid EMBASE. This search strategy was used to identify literature relevant to this systematic literature review.Additional file 2: Table S2. Modified Newcastle Ottawa Scale (NOS) for quality and risk of bias assessment. The quality and risk of bias of each study was assessed using 8 questions in the domains of selection, comparability, and exposure/outcome.Additional file 3: Table S3. Clinical profile of subjects included in the selected studies. Age, gender, duration of diabetes and HbA1c results from each of the included studies were extracted for comparison.Additional file 4: Table S4. Study characteristics. Table summary of the author, year, study type, biomarker location, DR grading scale and measurement in each study.Additional file 5: Table S5. PRISMA Checklist. A 27-item checklist addressing the completeness of the introduction, methods, results, and discussion sections of a systematic literature review.


Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice.DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5′-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast  DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. DSB repair failure can cause loss of chromosome fragments and cell death, while their inaccurate repair can result in chromosome rearrangements, genome instability, and ultimately cancer .DNA DSBs can be repaired by multiple pathways with different levels of fidelity. The main mechanisms are non-homologous end joining (NHEJ), which results in a direct ligation of the broken DNA ends , and homThe choice between DSB repair pathways with different outcomes and mutagenic potential is decisive for the maintenance of genomic integrity and it is subjected to complex regulatory mechanisms. The origin and the structural features of the DSBs, as well as the phase of the cell cycle in which they occur, are important determinants of DSB repair pathway decision. DNA end resection commits DSB repair to HR by creating 3′ single-stranded DNA (ssDNA) overhangs necessary for HR, while destroying the substrate for NHEJ repair ,7. DurinThe ratio between NHEJ and HR varies across phylogenetic groups. HR is generally prevalent in organisms with a small genome and with low abundance of repetitive sequences, such as budding yeast. In mammals and plants, NHEJ is the preferred pathway. On average, 75% of the DSBs that occur in mammalian cells are repaired by NHEJ, while HR repairs the remaining 25%. However, HR is always used as the preferred mechanism to repair DSBs that occur during S phase ,13.The response to DSBs should be considered within the context of chromatin, a highly organized structure in which the DNA is wrapped around octamers of four core histones H2A, H2B, H3, and H4 forming the nucleosomes . NucleosChromatin structure around a DSB can be modified by removing entire nucleosomes, thanks to the action of chromatin remodeling factors, and/or by modifying histones, thus increasing chromatin flexibility and accessibility of repair enzymes to the DSB ,14. HistSaccharomyces cerevisiae and in mammalian cells. Considering the evolutionarily distance between these two organisms and the extensive variations of histone marks during the cell cycle and in different cell types in mammals, one would expect that these chromatin modifications could be extremely different in the two species. In contrast, many histone PTMs and histone-modifying enzymes are well conserved, although more intricate and generally redundant pathways have evolved in metazoans compared to yeast. Taking these differences into account, we believe that the acquired knowledge of chromatin landscape and its effects on DSB repair pathway choice in yeast was and still is pilot for identifying proteins and pathways sensitive to specific histone PTMs also in mammals.An extensive work has been carried out to define the role of histones dynamics in DSB response in both the model organism In this review, we will focus on histone PTMs and discuss recent insights into their functions in DSB repair pathway choice, mainly referring to the latest discoveries in yeast. We and others recently reviewed the findings obtained in mammals and the regulatory models which have been proposed ,19,20,21S. cerevisiae), which recognize aberrant DNA structures and phosphorylate a multitude of targets involved in both repair and checkpoint pathways, thus promoting DSB repair and ensuring its completion before the cells progress through the cell cycle ,43,4]. AHR is initiated by resection of both DSB ends to create 3′-ended ssDNA tails in a two-step process that requires the concerted action of helicases and nucleases. The first resection step (short-range resection) requires MRX/MRN and Sae2/CtIP, whose action is particularly important to process DNA ends with protein adducts or “dirty” ends, while it is dispensable for resection of “clean” DSB ends, as those induced by endonucleases . Mre11 pAfter DSB end resection, the most common HR mechanisms require strand pairing and strand invasion, which strictly depend on the RecA/Rad51/Dmc1 family of recombinases that form a nucleoprotein filament on ssDNA. Rad52 together with a set of Rad51 paralogs, replaces RPA with Rad51 in yeast, while in mammals the main loader of RAD51 is BRCA2, along with several RAD51 paralogs ,4. Once For a long time, scientists interested in HR mechanisms have wondered about this “search problem”, thus showing that repair of a DSB is influenced by the proximity between the damaged and the template sequences. This homology search could also be facilitated by DNA damage-stimulated changes in chromosome movement .After the D-loop formation, HR can proceed via the canonical pathway that involves the formation of a double Holliday junction, whose resolution can generate either crossover or non-crossover products b, centerA specialized homology-dependent DSB repair pathway which does not require strand invasion and D-loop formation is the SSA. This mechanism relies on the annealing between two homologous sequences flanking a DSB and leads to the deletion of one of the repeats and of the intervening region c 5]..5].S. cerevisiae) . F. F131]. Histone H1 mediates higher-order chromatin folding in metazoans and it has a well-established role in chromatin organization during DNA repair . RNF20/4HHO1 inactivation was found to increase the resistance to DNA damage of mutants with defective NHEJ, but not of recombination mutants, suggesting that Hho1 may specifically inhibit HR ..ESA1 genNuA4 is recruited to the DSB ends by the MRX complex. Through the acetylation of both histones and non-histone proteins, NuA4 promotes resection and HR, while it inhibits NHEJ . AcetylaThe SAGA complex is another HAT that contributes to NuA4 recruitment to the DSBs and participates in resection stimulation . SAGA acIn mammalian cells, TIP60 depletion causes defects in DSB repair ,153,154.S. cerevisiae, defects in the acetylation of histones that are part of nucleosomes reconstituted during replication are associated with sensitivity to genotoxic agents [In c agents ,157. Celc agents ,163,164.c agents . This cac agents ,166. Sinc agents , its DNAc agents ,169.H3 K56 acetylation promotes cell survival after transient exposure to genotoxic agents that cause DNA damage during replication . When H3In the mammalian system, both reduction and increase of H3 K56 acetylation have been observed after DNA damage. Deacetylation by Sirt6 and Sirt3 HDACs promotes NHEJ by recruiting the chromatin remodeling enzyme SNF2H and 53BP1 to the DSB sites ,171.Chromatin around a DSB undergoes extensive changes that are regulated at multiple levels to allow DSB repair. Histone PTMs play a pivotal role in the modification of chromatin structure and in the subsequent DSB repair, as highlighted by the hypersensitivity to DSB-inducing agents of cells lacking histone modifier enzymes. In mammalian cells, the regulatory network of histone PTMs involved in DSB response is very complex due to redundancy of the modification machinery and to the variability of the modifications in different cell types. From this point of view, the yeast system has the advantage of a simplified histone PTM machinery. Many components and effectors of this machinery, as well as the biological processes controlled by histone PTMs, are conserved between yeast and vertebrates. Therefore, deciphering the chromatin landscape around a DSB and the effects of histone PTMs on DSB repair pathway choice in yeast should help unveil how these systems operate in mammals.Although inadequate DSB repair is one of the main causes of genomic instability and tumorigenesis, it also offers therapeutic opportunities to selectively kill cancer cells. In this context, targeting histone modifications required for DSB repair is expected to increase the hypersensitivity of cancer cells with defects in other key factors involved in the DSB response. A comprehensive view of histone PTMs in the surroundings of DSBs and their effects on DSB repair pathway choice is needed both to elucidate the contribution of histone modifications in controlling DNA metabolism and to predict the possible effects of targeting histone modifiers in therapy.