Biotherapeutics are a rapidly growing portion of the total pharmaceutical market accounting for almost one-half of recent new drug approvals. A major portion of these approvals each year are monoclonal antibodies (mAbs). During development, non-clinical pharmacology and toxicology testing of mAbs differs from that done with chemical entities since these biotherapeutics are derived from a biological source and therefore the animal models must share the same epitopes (targets) as humans to elicit a pharmacological response. Mechanisms of toxicity of mAbs are both pharmacological and non-pharmacological in nature; however, standard in silico predictive toxicological methods used in research and development of chemical entities currently do not apply to these biotherapeutics. Challenges and potential opportunities exist for new methodologies to provide a more predictive program to assess and monitor potential adverse drug reactions of mAbs for specific patients before and during clinical trials and after market approval. Biotherapeutics or biologicals are drug therapy products where the active substance is extracted or produced from a biological source ,2. TheseAs an example of the new approvals, 46 total drugs were approved by United States Food and Drug Administration (USFDA) in 2017; 24 were chemical entities and 22 were biotherapeutics. Of the 22 biotherapeutics, 10 were mAbs. These included brodalumab, dupilumab, sarilumab, guselkumab, bentalizumab, ocrelizumab, inotuzumab, avelumab, duvalumab, and emicizumab . These aTargeted mAbs for cancer therapy are typically IgG1 Fc design with immunomodulatory function . Many ofThe main objectives of the nonclinical evaluation of biotherapeutics are: (1) identification of target organs for toxicity and to determine whether the toxicity is reversible after the treatment has stopped; (2) identification of a safe starting dose for human Phase I clinical trials and subsequent dose escalation schemes, which is highly dependent on the patient population for the Phase I trial(s); and (3) provide information to monitor safety parameters in clinical trials ,18,19,20Brennan and Kiessling  provided(A) In silico review of the target biology and consequence of modulation from studies from knockout (KO) mice and humans or any available data with mAbs or other drugs against the same target or pathway. This would include pharmacovigilance studies where details are included in accessible databases ,29,30,31(B) Rational design of mAb candidates to remove any unwanted pharmacological effects such as structure of mAbs intended to suppress the immune system and/or selection of mAbs with low/no Fc effector function to avoid interaction with FcγRs on immune cells. For example, IgG4 with naturally low effector function or Fc-engineered IgG1 with low/no Fc effector function and/or use of Fab fragments would avoid receptor cross-linking ,32,33.(C) In vitro and ex vivo studies with human/animal cells and tissues to assess both on-target and off-target binding, pharmacological activity and immunogenicity potential. Tissue cross-reactivity (TCR) studies use in vitro Immunohistochemistry (IHC) techniques to characterize mAb binding to antigenic determinants in frozen human tissues and animal tissues from pharmacologically-relevant species. These techniques can be used to assist in the interpretation of any pathology-related findings toxicology animal toxicology studies as compared to the relevance of any observed binding in the human tissue panel ,37,38,39(D) In vivo studies in pharmacologically-relevant species(s). Data from studies in Points (C) and (D) would theoretically provide an understanding of expected human immune related pharmacological and potential toxicological effects of a mAb and whether this is likely to be predicted by toxicology species. Several other assays have been developed ,41,42,4310; pharmacologically active levels ; the highest safe dose (NOAEL) established in toxicology studies and/or toxicodynamic end point estimates from toxicology studies; and exposure- or concentration-related biomarker changes both in vitro and in vivo. The ED50 calculations are particularly important for FIH cancer trials [The science and regulatory aspects of FIH dose selection for mAbs have evolved into a target mechanism-based model which utilizes exposure–response relationships from both in vitro and in vivo studies . Importar trials  as a phar trials . The MABr trials  for esti(A) The nature and duration of the pharmacological effect(s) or mode of action (MOA). Is the mAb an agonist with strong immune cell activation and cytokine release potential or is it an antagonist? (B) The novelty of the molecule. Is it a standard IgG or mutivalent and/or highly engineered construct?(C) How relevant or sensitive are the nonclinical models to assess and/or predict safety of the pharmacological effect of the mAb ?human = CLanimal × b, where b is 0.85 or 0.9. This represents a single-species allometric scaling with mAbs for FIH dose estimation. Based on the target patient population, safety factors such as a 10-fold reduction in the FIH dose calculation, may be used to start a dose escalation scheme.Ideally, these parameters are measured in the in vivo and/or in vitro studies, but frequently they are predicted via modeling or allometric extrapolation. This is the standard case for estimated human doses and/or exposures . The in Antibody therapies with high functional efficiency and potential low toxicity are becoming one of the major approaches in mAb therapeutics . Based oAs another example of reducing potential on-target toxicity from mAbs, Chen and colleagues  engineerAn overall predictive toxicology program for mAbs in research and development to lessen or mitigate serious safety concerns is still considered to be in an early stage , althoug


The aim of early detection of prostate cancer (PCa) is to improve quality of life and decrease PCa-associated mortality. The use of prostate specific antigen (PSA) in a population-based screening study resulted in an increased incidence of PCa, a shift toward earlier disease stages and a reduction of metastatic disease and deaths from PCa. However, this benefit is accompanied by considerable rates of overdiagnosis which also involves potential overtreatment . In consBRCA1 dysfunction causes impairment of DNA repair mechanisms called homologous recombination (HR). This holds true also in tumors that do not harbour hereditary BRCA1 gene mutation. Moreover, this new concept called “BRCAness”  was exteth of PCa [In male, BRCA1/2 germline mutations are reported to increase the risk of PCa and correlate to its more aggressive course . Somatich of PCa . Interesh of PCa .There is some evidence, that the hierarchical model of breast cancer development might be applicable in PCa. Thereby, accumulation of so called cancer stem or progenitor cells induced by bi- or monoallelic loss of BRCA1 gene  leads toIn conclusion, BRCAness in prostate cancer has the potential to indicate PCa patients with the highest risk of metastasis . Neverth


After integrating classic and cutting-edge research, we proposed a unified model that attempts to explain the key steps of mammalian retinal neurogenesis. We proposed that the Notch signaling-induced lateral inhibition mechanism promotes oscillatory expression of Hes1. Oscillating Hes1 inhibitory activity as a result leads to oscillatory expression of Notch signaling inhibitors, activators/inhibitors of retinal neuronal phenotypes, and cell cycle-promoting genes all within a retinal progenitor cell (RPC). We provided a mechanism explaining not only how oscillatory expression prevents the progenitor-to-precursor transition, but also how this transition happens. Our proposal of the mechanism posits that the levels of the above factors not only oscillate but also rise (with the exception of Hes1) as the factors accumulate within a progenitor. Depending on which factors accumulate fastest and reach the required supra-threshold levels , the progenitor either proliferates or begins to differentiate without any further proliferation when Notch signaling ceases. Thus, oscillatory gene expression may regulate an RPC’s decision to proliferate or differentiate. Meanwhile, a post-mitotic precursor’s selection of one retinal neuronal phenotype over many others depends on the expression level of key transcription factors (activators) required for each of these retinal neuronal phenotypes. Because the events described above are stochastic due to oscillatory gene expression and gene product inheritance from a mother RPC after its division, an RPC or precursor’s decision requires the assignment of probabilities to specific outcomes in the selection process. While low and sustained (non-oscillatory) Notch signaling activity is required to promote the transition of retinal progenitors into various retinal neuronal phenotypes, we propose that the lateral inhibition mechanism, combined with high expression of the BMP signaling-induced Inhibitor of Differentiation (ID) protein family, promotes high and sustained (non-oscillatory) Hes1 and Hes5 expression. These events facilitate the transition of an RPC into the Müller glia (MG) phenotype at the late stage of retinal development. The number of people suffering from retinal diseases is projected to increase significantly in the coming decades, especially as the population of elderly patients continues to grow , which then travels to the nucleus to form a complex with the DNA-binding protein RbpJ. The NICD/Rbpj complex activates the expression of inhibitors of neuronal differentiation, such as Hes1 and Hes5, thereby keeping the trans-activated cell in an undifferentiated progenitor state or promoting MG differentiation during the late stage of retinal development  and five ligands . Notch1 and Notch2 receptors and Dll1 and Jag1 ligands play important roles in a majority of tissues during development and in adulthood, while Notch4 and Dll4 are mostly important during angiogenesis  is a process of sustained expression of a single factor” .Oscillatory gene expression is a broadly applicable phenomenon that enables us to explain myriad RPC operations and behaviors. We propose that oscillatory gene expression prevents an RPC’s transition from the proliferative progenitor state to the post-mitotic precursor state, but allows the expression of Notch signaling inhibitors, activators/inhibitors of retinal neuronal phenotypes, and cell cycle-promoting genes—since inhibitory Hes1  activity oscillates from high to low. This hypothesis can now explain the extensive heterogeneity of gene expression observed in individual RPCs  11–14 retinas contain mostly RPCs, cells collected from these retinas, in combination with bioinformatic analysis, can be used to reconstruct the oscillatory Hes1 GRN.Despite the encouraging uniformity of the results above, future studies of oscillatory gene expression in the developing retina must still be carried out and must be done very carefully in order to avoid incorrect conclusions. The simple possibility of phase shifts in expression makes it very difficult to guarantee observation of simultaneous expression of two antagonistic factors, even if a progenitor is indeed expressing both. An optimal approach to studying oscillatory gene expression in the retina will likely involve real-time/time-lapse imaging methods combined with destabilized luciferase reporters  and cone photoreceptors; and two inhibitory types: horizontal cells (HCs) and a subpopulation of early-born amacrine cells (ACs)], as well as a population of late RPCs . MeanwhiThe analysis of transcription factors regulating the development of different retinal neuronal phenotypes allows us to posit the retinal GRN (rGRN), which controls the development of all retinal neuronal phenotypes, except RGCs. On the top of this rGRN, there are three highly expressed transcription factors in RPCs: Otx2, Foxn4, and Rorb. Otx2 and Rorb are on the top of the branch regulating the development of excitatory retinal phenotypes, while Foxn4 and Rorb are on the top of the branch regulating the development of inhibitory retinal phenotypes due to activation of Ptf1a expression  state, the more likely (higher probability) it is for the progenitor to initiate differentiation, since the required genes may have had enough time to reach the supra-threshold level, inhibiting Notch signaling according to the above-mentioned mechanism. Meanwhile, products of Y may also accumulate to reach a required supra-threshold level to activate P and initiate progenitor proliferation (Our hypothesis (based on Dr. Kageyama’s research) that Notch/Hes1-dependent oscillatory gene expression in RPCs prevents them from differentiating requires a mechanism to explain how this oscillatory expression specifically prevents the RPC transition from the proliferative progenitor to the post-mitotic precursor state. Here, we will provide this mechanism, explaining not only how oscillatory expression prevents the transition, but also how this transition happens. We previously described the oscillatory Hes1 GRN in which Hes1 acts as an engine, propelling oscillatory expression across numerous interacting genes in the GRN. We suggest that Hes1 is the only real oscillator in the GRN, its oscillatory expression resulting from the previously described mechanism and includes required negative feedback loops . Meanwhiferation . If a prferation . Both prThe mechanism proposed above affects not only the progenitor’s decision to differentiate or divide, but also affects the size of the developing retina. The progenitor-precursor transition begins when the expression of X reaches supra-threshold levels (a “switch”) in RPC. If the products of X are efficiently degraded, they will accumulate very slowly, allowing RPCs to remain in a progenitor state for a long time, thereby producing a large number of progenitor progeny that form a larger adult retina once differentiated . MeanwhiHowever, what would happen if the microRNA and 26S proteasome pathways are efficient enough to prevent any accumulation of Notch signaling-inhibiting genes at all? It seems obvious that, in this case, RPCs would not be able to stop proliferating . This isOur model of retinal neurogenesis posits that the transition of proliferative RPCs to the post-mitotic precursor state requires significantly reduced Notch signaling activity (low and sustained/non-oscillatory Hes1 levels), yet the exact mechanisms by which Notch signaling is turned off to initiate neuronal differentiation are still unclear. Here, we describe several putative mechanisms based on our own data and other published evidence.cis-inhibition of Notch signaling within a cell and trans-activation of Notch signaling in neighboring cells  and the precursor state  levels are controlled by the transcription factors Ascl1 and Neurog2 in the developing retina . They found that a slight difference in Notch receptor degradation between two progenitors preserves oscillatory expression of Neurog2, Hes1, and Dll1 in one cell, but halts oscillatory expression of these genes in the other followed by increased Neurog2 and Dll1 expression and reduced Hes1 expression. Meanwhile, if the neighboring cells lose contact with each other, the model showed that both cells halt oscillatory expression of the increasing Neurog2 and Dll1 expression and the decreasing Hes1 expression. These results are a good addition and generalization to the “clock-switch” models described above: for example, asymmetric inheritance of Ascl1/Neurog2 and Foxn4/Rorb could quite plausibly lead to different rates of Notch degradation between two daughter cells, enabling differentiation of one but not the other. The difference between our models and Barton and Fendrik’s model is their suggestion that asymmetric inheritance of Numb  leads to asymmetric Notch degradation between the two daughter cells. We call this the Numb model. The role of the Numb model in retinal development was already thoroughly investigated  might oscillate and accumulate over time in the progenitor. If the progenitor is gaining products of genes required to reduce Notch signaling activity and promote a progenitor-to-precursor transition  slowly, relative to products of cell cycle-promoting genes, then this cell will divide and produce two daughter cells . We can (1) Suppose that mother cell divides [now we know that it was in the progenitor (P) state] generating two undifferentiated cells. Although newly born undifferentiated daughter cells look alike, at the molecular level they may inherit different amounts (as we noted above) of Ascl1/Neurog2 and Dll1/Dll3 (Dll1/Dll3 model), Foxn4/Rorb and Ptf1a (Ptf1a model), miR-9 (miR-9 model), and Numb (Numb model), as well as cell cycle-promoting factors from the mother progenitor (RPC). Following what is noted above, the decision of one of these daughter cells to proliferate or become a precursor depends on the amounts of aforementioned factors inherited from the mother cell and the amounts generated in the cell over time. Again, since these events are stochastic in nature, the mother RPC exerts no direct control over its daughters’ decision to remain progenitors (P) or to differentiate D; . LikewisAt least two transcription factors are necessary to promote retinal neuronal phenotypes,” expression of Atoh7 and Onecut1/Onecut2 is very high at the early stage of retinal development and is much lower at the late stage. Vsx2 (Chx10) is a marker of RPCs at the early stage of retinal development, while Vsx2, in combination with Otx2, promotes the BC phenotype at the late stage. Thus, at the early stage of retinal development, if Atoh7 levels exceed levels of Otx2, the cell will initiate the RGC fate program ((2) As we proposed earlier, Notch signaling-mediated oscillatory expression of Hes1 and its GRN allows the expression of inhibitors  and activators  of retinal neuronal phenotypes in an RPC. If the undifferentiated cell decided to differentiate (D state), its fate depends on the respective levels of fate determination factors  stochastically inherited from the mother RPC and generated in a daughter over time . The fat program , 6B. If  program , 6B. Suf program , 6B. But program . Meanwhi program , 6B. At  program , 6B. OncAs we noted before, the stochastic model of RPC decisions described here is reminiscent of the stochastic nature of a particle’s position, momentum, energy, and higher order observables in quantum mechanics—probabilities only; no absolute, preordained numerical quantities (as Newtonian physics employed) can be used to make predictions. In this regard, much like Schrödinger’s famous cat, an undifferentiated cell in the developing retina may have all possible fates  with different probabilities  until it actually adopts one . Thus, our oscillatory expression-based model of retinal neurogenesis can perfectly explain the results of Dr. Harris lab and Gomes study  Hes1 expression is required to promote the transition of RPC into various retinal neuronal phenotypes, in this section, we will argue that high and sustained (non-oscillatory) Hes1 expression is required to promote the MG phenotype in RPC. In differentiating MG, Hes1 is expressed in a high and sustained manner that should prevent the expression of key factors promoting any neuronal phenotype  demonstrated that interaction between Notch/Hes1 signaling and the Id protein family promote high and sustained Hes1 expression followed by complete repression of pro-neuronal expression , the undifferentiated cell starts to differentiate without further proliferation, becoming a precursor (D), or it divides, concluding that it was a progenitor (P). If an undifferentiated cell divides, the factors above may become diluted in daughter cells and the process of accumulation restarts. Because of the stochastic nature of these events, probabilities are necessary to describe the process of selecting a single outcome . Thus, before a decision is made, the undifferentiated cell may be interpreted as existing in both states (P and D) simultaneously with each state assigned a probability of occurring. If the undifferentiated cell starts to differentiate (D), the selection of one neuronal phenotype out of many other possibilities depends on the level of transcription factors required for the selected phenotype. Because these factors may be inherited at different levels from the mother cell after proliferation, and because these factors may oscillate and accumulate at different levels in the post-mitotic precursor (D), the process of selection when inhibitory Notch signaling activity is ceased is purely stochastic and requires probabilistic approaches to describe the selection process . In a manner analogous to quantum mechanics, probabilities can be used to predict these cell transitions, which depend on average expression levels of phenotype-determining transcription factors in undifferentiated cells (RPCs) during retinal development. However, these probabilities may experience time-dependent fluctuations during retinal development—reduced expression of Atoh7, Onecut1, and Onecut2 in the developing retina reduces the probability of retinal progenitor differentiation into early-born neurons  during the late stage of retinal neurogenesis.While low and sustained (non-oscillatory) Notch signaling activity is required to promote the transition of retinal progenitors into various retinal neuronal phenotypes, high and sustained (non-oscillatory) Notch signaling activity synergized with high BMP signaling-induced Id protein family production is required to promote the MG phenotype in progenitors during the late stage of retinal development. This model is based on results obtained by Dr. Cepko’s and Dr. Reh’s labs. Meanwhile, if oscillatory gene expression in some undifferentiated cells/RPCs does not stop due to pathological events , these cells may begin retinoblastoma formation.in vivo, the experimental process should be predicated on building mathematical models and evaluating the models’ predictions experimentally.In this article, we have outlined a model that accounts for retinal neurogenesis from a fresh perspective. Unlike previous models that have required the existence of many intrinsically different RPCs, the model proposed here is based on Dr. Kageyama’s oscillatory gene expression paradigm and Dr. Harris’ concept on the contribution of stochasticity to the proliferation and fate choice of retinal progenitors, allowing us to explain retinal neurogenesis using only early and late RPC types. Regarding our model, observed RPC heterogeneity arises from oscillatory gene expression within these two types of RPCs. While oscillatory gene expression prevents the differentiation of progenitors into retinal phenotypes, it creates the foundation for the observed stochasticity in RPCs’ decision to either proliferate or differentiate . Thus, if our model is correct, certain core concepts in the field of ophthalmology would be fundamentally modified: in particular, any vestige of the old paradigm that depicts gene expression as a static process will be discarded, supplanted by more accurate, data-rich models built on the observation that gene expression is inherently dynamic in nature. This dynamic gene expression milieu arises from complex GRNs that contain myriad feedback loops (negative and positive). Importantly, future studies will necessitate the combination of theoretical and experimental approaches to achieve success, since oscillatory gene expression is often too complicated to analyze The datasets generated for this study are available on request to the corresponding author.DI wrote the manuscript.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.


Mitochondrial bioenergetics require the coordination of two different and independent genomes. Mutations in either genome will affect mitochondrial functionality and produce different sources of cellular stress. Depending on the kind of defect and stress, different tissues and organs will be affected, leading to diverse pathological conditions. There is no curative therapy for mitochondrial diseases, nevertheless, there are strategies described that fight the various stress forms caused by the malfunctioning organelles. Here, we will revise the main kinds of stress generated by mutations in mitochondrial genes and outline several ways of fighting this stress. Mitochondria play a pivotal role in eukaryotic metabolism. They catabolise redox equivalents, derived from nutrient uptake, and use them to provide the bulk of cellular energy in the form of ATP. The oxidative phosphorylation system (OXPHOS) is responsible for this energy production and it is composed of five multi-oligomeric complexes present in the inner mitochondrial membrane. Transfer of electrons through complexes I to IV reduce molecular oxygen to water. This process is coupled to proton pumping from the matrix to the intermembrane space (IMS), while the return of protons to the matrix through the F1Fo ATPase generates ATP 22+) uptake inside mitochondria is mediated by the mitochondrial calcium uniporter (MCU). Although the complex has a low affinity for Ca2+, the transport takes place due to the high concentration of Ca2+ (>10 μM) present in micro domains located in the contact sites between endoplasmic reticulum (ER) and mitochondria 2+ uptake not only shapes the cytosolic Ca2+ dynamics, which is crucial for muscle contraction, exocytosis and gene transcription, but also modulates at least three dehydrogenases of the Krebs cycle, thus regulating energy metabolism. Finally, Ca2+ overload in mitochondria regulates apoptosis due to formation of the permeable transition pore (PTP) and release of cytochrome c from the IMS In addition, mitochondria are involved in many other key cellular functions. Dissipation of the proton gradient by uncoupling proteins (UCPs) generates heat instead of energy and this plays an important role in exposure to cold or hibernation Since mitochondria take part in many different metabolic processes, mitochondrial malfunction can affect numerous aspects of the cell. As a consequence, various forms of cellular stress are generated, leading to a large variety of pathological conditions. Here, we review different forms of cellular stress caused by mitochondrial malfunction and the strategies used to fight this stress.Mitochondria have retained their own genome, the mtDNA. This small, circular, double-stranded DNA is located in the mitochondrial matrix in all cell types, and can be found with copy numbers that range from several to thousands of copies. In human, the mtDNA encodes 13 polypeptides of the respiratory chain, as well as for part of the translation machinery, required for the synthesis of these polypeptides within mitochondria: two ribosomal RNAs (mt-rRNAs) and 22 transfer RNAs (mt-tRNAs) Some features of mtDNA make it especially sensitive to oxidative damage and mutation. Firstly, mtDNA has no introns, so every single nucleotide carries information essential for protein coding; mtDNA is naked, there are no histone proteins protecting it from damage; and although DNA repair systems do exist in mitochondria, their mechanisms and extent are poorly understood Table 1, Figure 1) Pathological changes in the mtDNA can appear as point mutations in protein coding sequences, mt-tRNAs or even mt-rRNAs. In addition, major rearrangements of mtDNA, like deletions or insertions/duplications, are a cause of disease. Due to the fact that every cell contains a variable number of mtDNA molecules, mutations can be present in homoplasmy  or heteroplasmy (only a population of DNA is mutated). The level of heteroplasmy of a mutation is a critical determinant of the cellular stress of a certain tissue or organ and has a major role in the disease phenotype. Finally, a reduction of mtDNA copies (depletion syndrome) can also hamper energy production and generate cellular stress (see Table 2).Due to the diverse cellular roles that mitochondria fulfill, there are many mitochondrial processes that cause a pathology when disturbed. In the last years, massive sequencing approaches have significantly increased the number of known mutations implicated in mitochondrial diseases. Examples of this are defects in: factors involved in the biogenesis or integrity of respiratory chain complexes, those that regulate mtDNA maintenance, proteins required for transcription of mt-mRNA elements involved in translation of mtDNA encoded proteins, regulators of lipid metabolism, factors involved in cellular signalling and even enzymes of the Krebs cycle  have been associated with aminoglycoside-induced ototoxicity and mitochondrial non-syndromic hearing loss. Studies using mitochondrial cybrids derived from Hela cells and lymphoblasts have shown that these mutations affect the integrity and fidelity of the mitochondrial ribosome, therefore causing decreased mitochondrial translation, either in the presence or the absence of aminoglycosides, and resulting in a cell growth defect 67However, a study using osteosarcoma 143B derived cybrids showed no effect on mitochondrial translation after aminoglycoside treatment Table 1).This discrepancy, the phenotypic differences between asymptomatic relatives and patients all harbouring the same mutational load and the fact that only in some cases the defect arose upon antibiotic treatment, raised the search for modifying factors of aminoglycoside induced ototoxicity within the nuclear genetic background Table 1). In addition, these mutations are usually present in homoplasmy, indicating that probably other factors are involved in the development of the disorder. The molecular mechanisms underlying LHON are not yet fully understood. There have been some risks factors proposed, like specific mitochondrial haplogroups, smoking, alcohol consumption, and the use of some antibiotics. Differences in mitochondrial mass have been also postulated to play a role in the incomplete manifestation of the disease. LHON mutation carriers with no pathological phenotype have significantly higher mtDNA copy number in leukocytes than affected carriers. By comparing fibroblasts from unaffected and affected mutation carriers, along with controls, it was shown that unaffected carriers have increased mitochondrial transcripts, respiratory chain proteins and enzyme activities compared to controls and affected carriers. Therefore, increased mitochondrial mass may play a protective role in LHON and compensate for complex I dysfunction Table 1).Eye complications are also frequently found to be associated with mitochondrial dysfunction Table 1). OPA1 is a protein with eight different isoforms, processed by the mitochondrial metallochaperones YME1L and OMA1 8687Figure 1) The most common eye afflictions associated with nDNA mutations are autosomal dominant optic atrophy (ADOA), most frequently due to mutations in the Dynamin-like GTPase OPA1, and autosomal recessive optic atrophy (AROA), which has been mainly associated with mutations in the aconitate hydratase ACO2, or the uncharacterised transmembrane protein TMEM126A (OPA7) AROA presents with progressive impairment of visual capacity. The defect could either be spontaneously recovered or may lead to bilateral and progressive blindness 9394Cardiac muscle has a high energetic demand, therefore cardiac complications are frequent among mitochondrial diseases. One of the most common cardiac afflictions present in these pathologies is cardiomyopathy, which is estimated to occur in 20-40% of children with mitochondrial disease 4965Mitochondrial cardiomyopathies are characterised by abnormal myocardial structure or function that results from genetic defects that impair the mitochondrial respiratory chain 678c oxidase, caused by mutations in assembly factors and in nuclear-encoded structural subunits. Mutations in the complex IV assembly factors COX10 and COX15 have been associated with hypertrophic cardiomyopathy 16c oxidase. Mutations in COX6B1, have been associated with cardiomyopathy and encephalopathy and showed decreased levels of the mature cytochrome c oxidase complex in patient-derived tissues and cells 12106As described before (see above), genetic defects affecting the integrity of respiratory chain complexes, mitochondrial translation, maintenance of mtDNA, lipid metabolism and other metabolic pathways inside mitochondria might lead to cardiac disease. Several important perturbations have been described in subunits or factors required for the proper assembly of respiratory chain complexes. In general, mutations in these proteins cause an impairment of respiration and ATP production, increased ROS production and finally, cellular stress derived from a bioenergetics impairment. To date, pathological mutations have been found in 26 structural subunits of complex I 91112102103A center. Both enzymes have different but cooperative functions and disruptions in their function impair the maturation of cytochrome c oxidase 70717273118c oxidase assembly 741207576c oxidase, specifically affects the heart remains unclear .Although COX6B1 was thought to be a loosely interacting structural subunit of complex IV, studies have postulated Cox12 (yeast homolog of COX6B1) to be involved in the delivery of copper to Cox2, together with other metallochaperones like Sco1, Sco2 and Coa6 6162109110676811264Table 1 and Table 2).Mutations in the mitochondrial translation machinery have also been associated with cardiomyopathy. Primary defects produce a decreased synthesis of mitochondrial polypeptides, but ultimately also impair mitochondrial bioenergetics and cause cellular stress. Mutations in the 16S mt-rRNA, and the m.1555A>G mutation in the 12S mt-rRNA, have been associated with hypertrophic and restrictive cardiomyopathy 7812579127128129131132c, a pre-requisite for triggering apoptosis. Oxidized CL has been found to be involved in the opening of the mitochondrial permeability transition pore (MPTP). In addition, CL is exposed to the outer mitochondrial membrane during apoptosis, where it is used as a binding platform for pro-apoptotic factors. Therefore, CL homeostasis plays an important role in cardiomyocyte programmed death upon ischaemia or reperfusion  83Alterations of lipid metabolism inside mitochondria can also be a determinant for cardiac disease. Barth syndrome is an x-linked autosomal recessive disease, characterized by cardiomyopathy, skeletal myopathy, neutropenia, growth retardation, and 3-methylglutaconic acidurea 801341358182Mutations in another lipid related enzme, the acylglycerol kinase AGK, have been associated with hypertrophic cardiomyopathy, myopathy, cataracts, exercise intolerance and lactic acidosis (Sengers syndrome). AGK was recently described as a component of the carrier protein translocase of the inner membrane (TIM22) 84138LEU(UUR) gene, although there have been other mutations described in protein coding genes 35364080136lys gene , being the m.8344A>G the most frequent of them 49515389c oxidase  or complex III (BSC1L) 143Table 1 and Table 2, Figure 1).Similar to previously described organs and tissues and due to the high energy demands, neurological complications are commonly linked to mitochondrial disfunction. Indeed, some of the most known mitochondrial syndromes caused by abnormalities in the mtDNA present with drastic neurological symptoms: Kearns-Sayre syndrome (KSS), a multisystem disorder with progressive external ophthalmoplegia, pigmentary retinopathy, heart block and frequently other signs like ataxia, dementia or endocrine problems is associated with single deletions of mtDNA Table 2).As already mentioned, many more genes are being identified as the reason behind mitochondrial dysfunction. Defects in mtDNA maintenance may result into defective mtDNA replication and lead to quantitative loss of mtDNA (mtDNA depletion) or qualitative one (mtDNA deletion). Downstream perturbation of mitochondrial protein synthesis will final lead to a bioenergetics defect. MPV17 is a mitochondrial inner membrane protein involved in maintenance of mtDNA. It is believed to be involved in the import of deoxynucleotides into mitochondria. Pathogenic variants in MPV17 have been reported to cause hepatocerebral mtDNA depletion syndrome with liver failure, de velopment delay and other neurological manisfestations 146Table 2).Aminoacyl-tRNA synthetases (ARS), are a family of proteins encoded in the nucleus and present in either the cytosol or mitochondria that ensure the proper conjugation of an amino acid with its cognate tRNA molecules. All mt-ARS are synthesized in the cytosol, imported to mitochondria due to an N-terminal targeting sequence (presequence) which is cleaved upon translocation to the matrix. Pathogenic variants of mt-ARS will affect mitochondrial translation and have been implicated in human neurological disorders of the brain, spinal cord and motor neurons in addition to other symptoms. Some of the most typical presentations are leukoencephalopathy with involvement of the brainstem and spinal cord and high lactate due to mt-aspartyl-tRNA synthetase (DARS2) mutations Figure 1) Nowadays there is no actual treatment for mitochondrial diseases. Nevertheless, in the last years a number of therapeutic strategies have been proposed, mainly in animal models. They can be classified into those acting on common pathways, and therefore applicable to different diseases, and those which aim to ameliorate a particular disorder  is the master regulator of mitochondrial biogenesis. It increases the activity of several transcription factors, like the nuclear respiratory factors (NR1 and NR2), thereby controlling the expression of OXPHOS related genes. In addition, PGC1alpha interacts with the peroxisomal proliferator activator receptors (PPARs), which regulate the expression of fatty acid oxidation genes Saccharomyces cerevisiae NADH reductase (Ndi1), which transfers electrons from NADH to coenzyme Q (CoQ), has been used to bypass CI defects CoQ to molecular oxygen in different organisms, has been used to bypass CIII and IV defects in cell culture 159in vivo in mammals.Another possible strategy is to bypass the block in the respiratory chain from specific complex defects. In such a way, electrons would flow again and reduce ROS production. Concomitantly, unaffected complexes would pump protons across the inner membrane and increase ATP production. The yeast in vivo from many insults, such as ischemia/reperfusion, denervation/induced muscle atrophy, and OXPHOS deficiency 89162As previously described (see above), the dynamin-like GTPAse OPA1 is required for proper mitochondrial shaping. Regulating fission and fusion helps fight mitochondrial malfunction. Increasing the expression of long isoforms of OPA1 improves respiration efficiency by enhancing supercomplex assembly and protects 10 , directly transfer electrons to the respiratory chain and bypass defective complexes. Others, like EPI-743 and RP103, enhance the biogenesis of glutathione, an important cellular antioxidant. KH176, can reduce altered cellular ROS levels and protect OXPHOS deficient cells against redox stress by targeting the Thioredoxin/Peroxiredoxin system In order to cope with increased oxidative damage generated in damaged mitochondria, different small molecules with antioxidant properties have been tested. Some examples, like Idebenone, lipoic acid, or Coenzyme QFinally, genetic approaches can be used to correct mutations at a genomic level. Mitochondrially targeted restriction endonucleases have been used to shift heteroplasmy levels in cell lines with mutations in mtDNA and in heteroplasmic mice. Introduction of TALE and zinc finger nucleases (TALEN and ZFN) enabled the addition of specificity to the nucleases so that mutant DNA molecules could be selected for by directing unspecific restriction enzyme (FokI) to appropriate specific sequence assembling ZFN or TALE modules 166Mitochondrial diseases show very complex and various clinical presentations. Because of the dual genetic origin of mitochondrial proteins, the number of genes susceptible of causing a mitochondrial pathology is large. Thus, the diagnostic process of mitochondrial diseases is usually complicated and very long, and in many cases although there is a clear suspicion of a mitochondrial defect, the final defect behind the phenotype remains undercover de novo serine biogenesis. This metabolic switch was proposed to be a specific and rapid response to cellular stress/mtDNA damage in skeletal muscle and heart Despite the heterogeneity of the diseases and the genetic defects the final kind of cellular stresses are similar. In the most cases, there is a bioenergetic defect or an increased production of ROS. It remains unclear why although many genetic defects are present in the whole body, only certain tissues are affected. There may be different mechanisms to cope with mitochondrial stress, which may be tissue specific There have been several approaches proposed to treat mitochondrial diseases, however their application to the clinics is still a challenge


Plants have evolved a remarkable ability for reprogramming cell identity to facilitate tissue repair and developmental plasticity. During cell fate transition, an unorganized pluripotent cell mass, called a callus, is formed. Two major types of calli are observed depending on the inductive external stimuli: mechanical wounding alone or wounding followed by incubation on high auxin-containing callus-inducing medium (CIM) -PHYTOCHROME A SIGNAL TRANSDUCTION 1 (PAT1) complex to replenish collapsed cells through active reentry into the cell cycle. The ERF115-PAT1 complex possibly promotes LBD16 and LBD29 promoters and activates expression through deposition of the active H3K36me3 mark, stimulating proliferation of pericycle competent cells and conferring root primordium characteristics in the callus   gene to facilitate pluripotency establishment in calli , SCARECROW (SCR), PLETHORA 1 (PLT1), and PLT2 are expressed in callus cells  and SHOOT MERISTEMLESS (STM), are slowly induced, rather than being rapidly activated, in a confined domain in response to high cytokinin (Gordon et al., Consistent with the fact that callus tissues have similarity to root primordium, A remaining question would be what characteristics of the root primordium-like tissues are advantageous to calli for pluripotency. Accumulating evidence has suggested that the root primordium has an efficient cell proliferation system. Callus cells originate from the pericycle cells, which have low endoreduplication activity and thereby active cell division (Blakely and Evans, de novo organogenesis, which is required for the in vitro tissue culture process. This type of pluripotency, which resembles the root primordium, possesses a genuine competence for tissue regeneration with a cost-effective cell division process.Overall, unlike wounding alone, high auxin plus wounding provides limited flexibility of cell fate transition and thereby ensures stepwise transition of cell identity. This facilitates planned All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Understanding how variation in gene expression contributes to morphological diversity is a major goal in evolutionary biology. Cichlid fishes from the East African Great lakes exhibit striking diversity in trophic adaptations predicated on the functional modularity of their two sets of jaws . However, the transcriptional basis of this modularity is not so well understood, as no studies thus far have directly compared the expression of genes in the oral and pharyngeal jaws. Nor is it well understood how gene expression may have contributed to the parallel evolution of trophic morphologies across the replicate cichlid adaptive radiations in Lake Tanganyika, Malawi and Victoria.prdm1a, a skeletogenic gene with modular anterior-posterior expression, displayed higher pharyngeal jaw expression and modular expression pattern only in carnivorous species. Furthermore, we found the expression of genes in cichlids jaws from the youngest Lake Victoria to exhibit low modularity compared to the older lakes.We set out to investigate the role of gene expression divergence in cichlid fishes from these three lakes adapted to herbivorous and carnivorous trophic niches. We focused on the development stage prior to the onset of exogenous feeding that is critical for understanding patterns of gene expression after oral and pharyngeal jaw skeletogenesis, anticipating environmental cues. This framework permitted us for the first time to test for signatures of gene expression underlying jaw modularity in convergent eco-morphologies across three independent adaptive radiations. We validated a set of reference genes, with stable expression between the two jaw types and across species, which can be important for future studies of gene expression in cichlid jaws. Next we found evidence of modular and non-modular gene expression between the two jaws, across different trophic niches and lakes. For instance, Overall, our results provide cross-species transcriptional comparisons of modularly-regulated skeletogenic genes in the two jaw types, implicating expression differences which might contribute to the formation of divergent trophic morphologies at the stage of larval independence prior to foraging.The online version of this article (10.1186/s12862-019-1483-3) contains supplementary material, which is available to authorized users. The evolution of jaws in ancestral vertebrates was pivotal in their subsequent colonisation of trophic niches . Jaws arCichlids are one of the most striking examples of trophic diversity as they have evolved highly specialized pharyngeal jaws in addition to oral jaws . The funOne ecologically important key stage of cichlid development is the stage 26 defined in . This stIn this study, we investigate the expression of a set of modularly regulated genes in the oral and pharyngeal jaws of 12 haplochromine cichlid fish species at stage 26, the end of larval development and prior to the onset of exogenous feeding. The selected candidate genes are known to be involved in the development and morphogenesis of jaw skeletal elements in teleost fishes and also have modular effects along the anterior-posterior axis during viscerocranial skeletogenesis  among the candidate reference genes in both jaws  for relative gene expression analyses of our target genes.To measure the expression of our selected target genes in oral and pharyngeal jaws, it was essential to first validate reference gene(s) with least expression variation among the jaw samples across different species . The 12 foxq1b with highest expression to fgf8a with lowest expression in both jaws  in the oral and pharyngeal jaws, but their expressions were quite variable, from foxq1b, satb2 and lbh genes in LM and LV. The only gene with clear herbivore-carnivore expression differentiation was prdm1a in LT indicating that none of the selected target genes can act as differentiating factors in herbivore versus carnivore skeletal morphogenesis in the oral jaw at the end of larval development  in the algae-browser species of LV but such tendency was not observed in the two other lakes  [fos) is also implicated in adaptive phenotypic plasticity of pharyngeal jaw in response to mechanical strains in cichlid [lbh affects jaw skeletal morphogenesis in a similar manner as plastic mechanical responses exert effects on jaw morphogenesis through transcriptional regulation of Ap-1 complex. The reason for selecting lbh as a target gene in our study was based on the fact that it is the only identified gene with a polymorphism associated with continuous morphological variation in the cichlid jaw and its function during jaw morphogenesis is characterized [lbh along anterior-posterior arches at early craniofacial patterning seems to be a determinant of jaw morphological variations in later stages of development in cichlids [lbh expression in pharyngeal jaw than oral jaw at the end of larval development, and the algae-grazer species in each lake appeared to have different jaw-specific lbh expression compared to the other trophic niches. Also, lbh displayed fairly similar differential expression pattern between the contrasting trophic niches in both jaws of LM and LV species. This suggests potential role of lbh in divergent jaw morphogenesis long after early developmental patterning of craniofacial skeleton. Interestingly, lbh was the only gene among our candidates showing expression correlation between the two jaws in different comparisons (i.e. lake- and trophic-based comparisons) suggesting its non-modular transcriptional regulation along anterior-posterior feeding skeletal structures. It is already known that lbh can be itself a downstream target of Wnt signaling [One of the candidate target genes of paramount importance in our study was tebrates . The funelopment , 49, 50,elopment , as wellelopment . It has rodimer) . Moreove cichlid , 19. Thicterized . The difcichlids . In our ignaling , a key signaling . Differeignaling , 54, 55.prdm1a encoding a transcriptional activator and repressor regulating neural crest development in zebrafish embryos [prdm1a is a direct downstream effector of retinoic acid (RA) signalling pathways [prdm1a to have consistently higher expression in the pharyngeal jaw than the oral jaw in carnivore species of the three lakes at the end of larval phase indicating its potential role in adaptive divergence of posterior trophic skeleton in haplochromine cichlids at later stages of development. Interestingly, a correlation in prdm1a expression was observed between the two jaws only in herbivore species which could implicate a transcriptional regulatory decoupling of prdm1a between the two jaws in carnivore species. It is worthy to further investigate whether receiving different diets during juvenile phase can influence the distinct herbivore-carnivore expression pattern of prdm1a in cichlid jaws. In particular, prdm1a might be a mediator of the effects of activated RA through different nutritional conditions on trophic skeletogenesis in later stages of development such as the early feeding period [Perhaps the most striking finding of our study was the expression pattern of  embryos , and sel embryos . prdm1a pathways , which ipathways , 38. We g period .bapx1, foxq1b and wnt9b with a specific role in the formation of oral jaw skeletal elements. The first gene, bapx1, encodes a member of the NK family of homeobox-containing proteins and plays an important role in positional specification of the oral jaw joint and its articulation in gnathostomes [bapx1 is required for morphogenesis of the retroarticular process and mandible and its expression is controlled by activity of FGF, BMP and Endothelin signaling pathways [bapx1 would be of paramount importance for investigations of the molecular basis underlying divergent jaw morphogenesis [bapx1 in the oral jaw than pharyngeal jaw of most species, but such difference was either not observed or not pronounced in the invertebrate-picker species. Furthermore, among LM species, the herbivore species had markedly higher oral jaw expression of bapx1 compared to the carnivores. The oral and pharyngeal expression of bapx1 was found to be correlated in herbivore species across the three lakes whereas such correlation was not observed in carnivores. These observations suggest a potential role of bapx1 in divergent morphogenesis of oral jaw skeleton in later stages of development in cichlids.We tested expression of three target genes, hostomes –30. The pathways , 30. Theogenesis , 32. In foxq1b, encodes a member of Forkhead-Box transcription factor family that is known to have a specific developmental expression pattern confined to the oral jaw in zebrafish [foxq1b plays a role in the formation of the lower jaw, particularly Meckel’s cartilage and associated structures, and it is a major mediator of the effects of Aryl hydrocarbon receptor (AHR) pathway in early patterning and later larval stages of jaw development in zebrafish [foxq1b is shown to be associated with morphological divergence of jaw skeleton during larval development in Arctic charr, a salmonid species [foxq1b in all species at the end of larval development suggesting its conserved role in oral jaw formation in teleost fish. We also found lower expression of foxq1b in the pharyngeal jaw of algae-grazer species compared to other trophic niches indicating its potential role in morphological divergence of the pharyngeal jaw in haplochromine cichlids. The foxq1b expression also showed a correlation between the pharyngeal and oral jaws in both herbivorous and carnivorous species, which proposes shared transcriptional regulatory mechanism at a late stage of larval development.The second gene, ebrafish , 34. foxebrafish . The AHRebrafish , which pebrafish , 54, 55.ebrafish  and diff species . In our wnt9b, is a member of the WNT gene family encoding a secreted signaling protein required for dorso-ventral patterning of oral jaw and outgrowth of upper jaw in zebrafish [wnt9b in all species except the herbivorous species from LT, also, more similar expression patterns for wnt9b were found in the jaws of species from LM and LV. These observations could reflect more diverged wnt9b transcription in LT haplochromine cichlids compared to the two other lakes.The third gene, ebrafish , 56. Expfgf8a, a member of the fibroblast growth factor (FGF) family, and satb2, a DNA binding protein which binds to nuclear matrix acting as a multifunctional determinant of craniofacial patterning and osteoblast differentiation during development [fgf8a expression in pharyngeal pouches is required for migrating pouch-forming cells towards mesodermal guideposts, a crucial mechanism in viscerocranial skeletogenesis [fgf8 (together with other FGF family members) is a determinant of skeletal size in posterior pharyngeal arches [fgf8a at the late larval stage 26. However, this pattern was not consistent across the three lakes and even an opposite expression pattern concerning trophic niche was observed between LM and LT. Importantly, fgf8a was the only gene showing no expression correlation between the two jaws in the different comparisons, indicating that its regulatory decoupling along the anterior-posterior axis might be associated with the emergence of the pharyngeal jaw in cichlids. Finally, satb2, was the only gene showing expression correlation between the two jaws specifically in carnivores at the late larval stage, indicating its potential role in distinct and modular morphogenesis of the two jaw types in the herbivore cichlids. satb2 gene has been shown to have high degree of conservation in gnathostomes [In addition to the abovementioned candidates, we tested the expression of two other genes, elopment . Activatelopment , 58. Morogenesis . In latel arches , 61. In hostomes  and its hostomes .The morphology of cichlid jaws has evolved repeatedly, along similar morphological trajectories in Lake Tanganyika, Malawi, and Victoria. The cichlid radiations in these lakes essentially represent the same process of evolutionary diversification, but at different stages linked to their age , 65. TheThis is the first attempt to study modularity in gene expression between cichlid oral and pharyngeal jaws simultaneously, across diverse trophic niches in parallel adaptive radiations. Our results provide evidence of distinct modular expression of key genes involved in jaw morphogenesis in relation to trophic niche specialization prior to the onset of independent feeding in cichlid larvae. We also show that the gene expressions of the jaws in cichlids from the younger lake are less modular, consistent with previous studies on their morphological integration. Our findings shed light on the molecular and transcriptional organization of the oral and pharyngeal jaws at the end of postembryonic development, in anticipation of environmental stimuli. The expression divergence prior to plastic response to feeding for modularly-regulated genes and the differences between the two jaw types might contribute to pre-feeding trophic canalization in cichlids, and therefore, requires further investigations. This initial exploration can be expanded upon by adding more genes or even whole transcriptomes to unravel transcriptional basis of cichlid jaw modularity and parallelism. Another interesting example for a future study could be the genes involved in bone-remodeling, as it has been already observed in other teleost fish that their differential expression during early and late developmental stages (prior to foraging) could differentiate between contrasting trophic jaw morphologies , 67.Twelve haplochromine cichlid species from Lakes Victoria, Malawi, and Tanganyika, which are adapted to two major trophic niches via convergent evolution, were selected for this study  together with one 1.4 mm ceramic sphere to crush the jaws. The jaws were homogenized using a FastPrep-24 Instrument  and RNA was isolated based on the instructions by the manufacturer adjusted for small amounts of fibrous tissue. In brief, the instruction includes the mixing of the lysis buffer and homogenized tissue with isopropanol and passing it through a column provided by the kit, several RNA washing steps and an in-column DNase treatment. The quantity of RNA was checked using a Nanophotometer  and the quality was assessed with RNA ScreenTapes on an Agilent 2200 TapeStation (Agilent Technologies). The RNA samples with a RNA integrity number above seven were subjected to first strand cDNA synthesis using 700 ng of RNA and High Capacity cDNA Reverse Transcription kit (Applied Biosystems). The resulting cDNA of each sample was diluted 1:10 times in nuclease-free water to be used for qPCR steps.bapx1, foxq1b, wnt9b, fgf8a, lbh, prdm1a and satb2, involved in modular morphogenesis of trophic skeletal system along the anterior-posterior axis in teleost fishes (described in the discussion section). The primers were designed at conserved sequence regions using the available transcriptomes of five East African haplochromine species  and two more distantly related cichlid species (Oreochromis niloticus and Neolamprologus brichardi) [Oreochromis niloticus annotated genome in the Ensembl database (http://www.ensembl.org) [We selected seven candidate reference genes with the highest expression levels in transcriptome data of the jaws from LT Haplochromine cichlids which also had shown no significant expression differences between the two jaw types and across species at stage 26 . Furtherichardi) , 73. Thembl.org) . The primbl.org) . Primer http://LinRegPCR.nl) [The instruction suggested by Maxima SYBR Green/ROX qPCR Master Mix (2X)  was used to produce qPCR reactions. The amplification steps were performed in 96 well-PCR plates through ABI 7500 real-time PCR System (Applied Biosystems). For each biological replicate two technical replicates were assigned and we followed an approved experimental set-up known as sample maximization method to obtain to optimal qPCR conditions . The qPCgPCR.nl)  (Additioreference), and then ΔCq of each target gene was calculated (ΔCq target = Cq target – Cq reference). In expression comparisons within the jaw types for each target gene a pharyngeal jaw replicate of an algae-grazer species in each lake was used as a calibrator sample and rest of the samples were normalized to its ΔCq value to calculate ΔΔCq values . In expression comparisons between the jaw types for each target gene a pharyngeal jaw replicate for each species was used as a calibrator sample. Relative expression quantities were calculated for the normalized values using E−ΔΔCq [http://www.r-project.org).Three different methods were utilized to validate the most stable reference genes including BestKeeper , NormFing E−ΔΔCq  and theng E−ΔΔCq . The sigAdditional file 1:Information about qPCR primers used in this study. (XLSX 12 kb)Additional file 2:Statistical results and raw gene expression data. (XLSX 31 kb)


Three-dimensional neural models or cerebral organoids possess an innate advantage over two-dimensional neural cultures as they can recapitulate tissue organization and cell type diversity that resemble the developing brain. Brain organoids also provide the exciting opportunity to model the integration of different brain regions in vitro. Furthermore, recent advances in the differentiation of non-neuronal tissue from stem cells provides the opportunity to study the interaction between the developing nervous system and other non-neuronal systems that impact neuronal function. In this review, we discuss the potential and limitations of the organoid system to study in vitro neurological diseases that arise in the neuroendocrine and the enteric nervous system or from interactions with the immune system.The complex development of the human nervous system has been traditionally studied using a combination of animal models, human post-mortem brain tissue, and human genetics studies. However, there has been a lack of experimental human cellular models that would allow for a more precise elucidation of the intricate dynamics of early human brain development. The development of stem cell technologies, both embryonic and induced pluripotent stem cells (iPSCs), has given neuroscientists access to the previously inaccessible early stages of human brain development. In particular, the recent development of three-dimensional culturing methodologies provides a platform to study the differentiation of stem cells in both normal development and disease states in a more  The formation of the complex nervous system is a fascinating and crucial series of events that occurs during human development. From the formation of the neural plate in the third week of gestation to the migration of neural crest cells throughout the periphery of the embryo, the nascent nervous system undergoes a series of dramatic changes that produce an intricate network of neural cells that make up the central and peripheral nervous systems (CNS and PNS). This is achieved by the careful coordination of multiple cellular events that include: neuronal progenitor cell (NPC) proliferation, neuronal or glial cell differentiation, neuronal morphogenesis, maturation, and migration. After the formation of the different brain regions, neurons establish connections with specific target cells that are critical to their pre-determined functions.Most of our current understanding of the intricacies of human nervous system development have come from human genetic studies , post-moin vitro cultures. 2D stem cell derived neural models provide a human platform to conduct molecular, cellular, or physiological studies and are amenable to high throughput drug screening . The BBB is a specialized endothelial cell membrane  infections and AD pathogenesis. We also discuss models of the BBB and its role in mediating neuro-immune responses and potentially modeling the neuro-vascular axis.The relevance of neuro-immune interactions in brain health is further highlighted by studies on maternal immune activation in the etiology of neuropsychiatric disorders like autism and schizophrenia . In thisin vivo organization of the cerebral cortex is closely mirrored in self-directed and forebrain patterned organoids . These results suggest that the neurogenic and migratory pathways associated with in vivo cortical development are also replicated in vitro , the subventricular zone (SVZ), and the outer subventricular zone (OSVZ) . Remarkarganoids , which din vitro .ZIKV infection of the fetal brain , showing a primarily reduced cerebral cortex  plaques within extracellular spaces and neurofibrillary tangles, formed by deposition of hyperphosphorylated TAU within neurons. The Aβ hypothesis of AD pathology posits that the accumulation of Aβ plaques leads to neurofibrillary tangles and induces a state of chronic neuroinflammation, which in turn worsens AD pathology . In AD, ar space . The Aβ4symptoms . Furthersymptoms . HyperphDpp1-1APP  and Dpp2-3APP ) and PSEN1 mutation (A264EPSEN1) , which results in diminished APP processing . These sN1A264E) . HoweverN1A264E) , emphasiAPOE gene has three polymorphic alleles: APOE2, APOE3, and APOE4, each with different binding affinities for Aβ plaques  forebrain cortical organoids. APOE4 did not affect the migration or incorporation of microglia into forebrain organoids. However, it induced morphological changes in the organoid neurons, such as elongated processes with increased Aβ fragments. These results suggest that APOE4 microglia might be defective in clearing the Aβ peptides  excitatory motor neurons eliciting contraction of the intestinal smooth muscle proximal to the site of IPAN activation by acetylcholine (Ach) release. Descending interneurons synapse with inhibitory motor neurons and induce smooth muscle relaxation distal to the site of IPAN stimulation by release of multiple neurotransmitters including: nitric oxide and vasoactive intestinal peptide (VIP). The combination of contraction and relaxation of the intestinal smooth muscles is the basis for the peristaltic reflex in the GI tract. Glial cells are also present in the ENS, and like the astrocytes in the CNS, protect the endothelial barriers in the GI while also maintaining optimal conditions for neuronal function. However, the cellular complexity of the ENS makes it challenging to model in vitro.Upon closure of the neural tube, neural crest cells migrate into the primitive gut to form the future ENS. The ENS is part of the autonomic nervous system and modulates peristaltic reflexes, fluid secretion into the lumen of the GI tract, and vasodilation of GI tract blood vessels . The ENSin vivo in the GI tract. However, they did not contain all the enteric neural cell types as only IPANs, ChAT+ excitatory neurons, dopaminergic neurons, and some types of inhibitory neurons were observed. Notably HIOs + ENS lacked glia as well as enteric inhibitory neurons, which actively express nitric oxide synthase (NOS) and function in smooth muscle relaxation. The functionality of in vitro derived organoids in terms of their capacity to mature and integrate in vivo has been tested in “chimeric” models transplanting human stem cell derived tissue into live animals. HIOs + ENS engrafted into live mouse kidneys had increased NOS-expressing inhibitory neurons, reduced ChAT+ neurons, and diminished numbers of enteric neuronal cell bodies compared to actual human intestine. These results suggest that aggregation of NCCs with HIOs do not contain all of the intrinsic cues that are essential for the timely appearance of ENS excitatory and inhibitory neurons. Alternatively, the kidney environment might not have been as supportive for ENS development as would have been engraftment into the GI. Despite the alterations in neuronal cell type composition, neurons within the HIOs + ENS were functional as shown by Ca2+ efflux measurement. HIOs + ENS neurons mediated sustained intestinal smooth muscle contraction and relaxation independent of ICCs. An important discovery using the HIOs + ENS model is that the ENS promotes cellular proliferation and differentiation of intestinal cells. HIOs + ENS, but not HIOs alone, lead to increased cell proliferation in the intestinal crypt, which correlated with increased expression of genes regulating proliferation and intestinal cell development.Waardenburg syndrome and Hirschsprung’s disease are severe enteric neuropathies that are often fatal; thus, highlighting the essential role of ENS in regulating intestinal tissue activity . Unfortu“in vivo” like ENS containing organoids enriched in multiple enteric neuronal and glial cells. ENS neurons were derived from iPSCs cultured in RA, inhibitors of BMP and TGF-β signaling, and activators of GSK signaling. A key difference with the HIOS + ENS method, was that NCCs and HIOs were seeded onto polyglycolic acid scaffolds, and were implanted into the omentum of irradiated mice for in vivo maturation. The omentum is a fold of the peritoneum connecting the stomach with other abdominal tissues. Therefore, this could provide a more permissive environment to the development of more in vivo like ENS. This organoid model referred to as ENCC-HIO-TESI  contained epithelial and mesenchymal cells associated with mature human intestinal tissue. ENCC-HIO-TESI also contained excitatory ChAT+ neurons, serotonin+ IPANs, ascending and descending interneurons, inhibitory NOS+ neurons and enteric glial cells. Enteric glia (SOX10/GFAP+ and SOX10/S100β+) was also found throughout the ENCC-HIO-TESI mesenchyme. This was in stark contrast to the HIOs + ENS , a complex multisystem genetic disorder, is characterized by short stature, muscle hypotonia, hyperphagia, obesity, hypogonadism, behavioral problems, and developmental delay . Childreryoid body-like aggregates with quick re-aggregation (SFEBq).” This culture method utilizes aggregates of ES cells in culturing conditions suitable for differentiation into ectodermal derivatives. Under these conditions, the ES cells also spontaneously form highly ordered structures and display self-organization (Since 3D organoids can be used to model embryonic development, they are ideal for studying the development of the pituitary gland and hypothalamic tissue. nization . Using Snization . Removinnization .In addition to establishing hypothalamic tissue from mouse ES cells using the SFEBq cultures, researchers were also able to generate pituitary hormone cell types. By increasing the size of the ES cell aggregate in vitro in human ES cells using the SFEBq method. They found that the anterior pituitary self-formed after these large aggregates generated both hypothalamic and oral ectoderm. Interestingly, a single aggregate could contain multiple LHX3+ pouches, while only one pouch normally develops in the embryo. Unfortunately, there did not seem to be a high success rate for the development of these pouches in vitro, with only 4.0 ± 0.4 vesicles per eight aggregates, suggesting that the organoid culture conditions can be improved to more efficiently generate pituitary progenitors. The most common cells types produced in the hESCs pituitary organoids are corticotrophs and somatotrophs (totrophs . GonadotThe key interaction in the large aggregates in SFEBq culture is likely the interaction between neural ectoderm and surface ectoderm, which can generate a placode, similar to the adenohypophyseal placode that generates Rathke’s pouch. in vivo tangential migration (Formation of forebrain, retinal tissue, choroid plexus and hindbrain were original observed by igration  and the igration .in vitro, which were only shown to mature into blood vessels after transplantation into a live animal. iPSC-derived cerebral organoids were co-cultured for up to 3 to 5 weeks in vitro with endothelial cells from the same donor and transplanted at day 54 and analyzed after 2 weeks (in vitro. Future directions might incorporate bioengineering approaches like 3D printing of blood vessels or “organ in a chip” type of technology (in vitro system.As organoids grow in size and complexity, another major challenge is to provide oxygenation, nutrients, and structural integration of the vasculature to the organoids. Current methodologies have allowed researchers to grow brain organoids up to 510 days , 2018 in 2 weeks . The forchnology  to overcin vitro. Alternatively, the impact of different immune signaling molecules could also be assessed in co-culture systems of microglia and brain specific organoids, in self directed cerebral organoid systems that contain microglia or in “assembloyds.”With respect to the neuro-immune axis one major challenge will be tomodel how neuroinflammation might affect fetal development. Growing evidence suggests a role for maternal inflammation during fetal development as a contributing factor to the complex etiology of neuropsychiatric disorders like autism and schizophrenia . Currentin vivo using transsynaptic spread of a GFP labeled rabies virus followed by electrophysiological measurements in response to glucose uptake. This circuit was recreated in vitro using a co-culture system of enteroendocrine cells and nodose vagal neurons (With respect to the Gut-brain axis the recent key discovery that a subset of ENS enteroendocrine cells can directly synapse with cranial nerve axon terminals  opens up neurons . Future Finally, with respect to the neuroendocrine axis, the assembly of multiple components this system could interrogate mechanisms underlying neural interactions with the hypothalamic-pituitary-adrenal gland axis, which has been previously associated to individuals at high risk of developing psychosis. While at the moment there are organoid models of adrenal glands, one could envision that being able to develop the hypothalamic-pituitary-adrenal gland axis in a dish could provide mechanistic insight on how corticosteroids could modulate neural circuits in the hypothalamus.SBL, EC, and AO conceived and developed the manuscript. SBL, EC, AO, and SD wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dendritic cell (DC) activation is characterized by an acute increase in glucose metabolic flux that is required to fuel the high anabolic rates associated with DC activation. Inhibition of glycolysis significantly attenuates most aspects of DC immune effector function including antigen presentation, inflammatory cytokine production, and T cell stimulatory capacity. The cellular nutrient sensor mammalian/mechanistic Target of Rapamycin (mTOR) is an important upstream regulator of glycolytic metabolism and plays a central role in coordinating DC metabolic changes and immune responses. Because mTOR signaling can be activated by a variety of immunological stimuli, including signaling through the Toll-like Receptor (TLR) family of receptors, mTOR is involved in orchestrating many aspects of the DC metabolic response to microbial stimuli. It has become increasingly clear that mTOR's role in promoting or attenuating inflammatory processes in DCs is highly context-dependent and varies according to specific cellular subsets and the immunological conditions being studied. This review will address key aspects of the complex role of mTOR in regulating DC metabolism and effector function. As the quintessential professional antigen presenting cells of the immune system, dendritic cells (DCs) play a central role in coordinating both innate and adaptive immune responses through efficient recognition and uptake of extracellular material and the potent ability to provide both presented antigen and costimulatory signals required for proper T lymphocyte activation . DC actiWhile historically the field of immunology has focused on immune cell regulation at the transcriptional and translational levels, the recent emergence of the field of “immunometabolism” has provided the scientific community with a new framework for thinking about immune cell activation; specifically, how nutrient availability and usage controls the cellular effector functions important for immunological protection. One of the cornerstone findings of recent advances in the field is the observation that immune cell activation, in both the lymphoid and myeloid lineages, is broadly characterized by an increased flux of glucose metabolism, often termed in the literature as “aerobic glycolysis” as both a historical nod to the analogous “Warburg metabolism” described in cancer cells [reviewed in Potter et al. ] and to The hierarchical regulation of cellular metabolism and energy homeostasis can be functionally partitioned into two opposing “programs,” anabolism and catabolism, each governed by a distinct central upstream regulator. Energetic anabolism, generically characterized by reduced metabolic activity coupled to energy conservation and production, is controlled by AMP-activated protein kinase (AMPK) in response to low cellular ATP levels or nutrient starvation [reviewed in ]. Catabode novo synthesis of lipids, proteins, and amino acids, serving as an important checkpoint in converting increased cellular fuel consumption into processes such as cell division and protein production that have obvious implications for broad physiological responses, including those carried out by immune cells  and mTOR complex 2 (mTORC2). While mTORC1 is primarily responsible for cellular energy expenditure and protein translation, mTORC2 serves an important role as a positive regulator of mTORC1. For the purposes of this review, “mTOR activity” will refer to mTORC1 functions unless otherwise noted. It is notable that while mTOR promotes sustained catabolism of carbohydrates, it concurrently supports the ne cells . As a done cells .Activation of DCs via TLRs promotes significant upregulation of aerobic glycolysis, which regulates the immune function of both human and mouse DCs , 30, 31.Rapid induction of glycolysis in DCs, occurring within minutes of TLR stimulation, is controlled by a PI3K/TBK1/IKKε/Akt signaling axis that promotes the rapid translocation of Hexokinase 2 (HK2) to the mitochondria which supports the rapid flux of glucose catabolism associated with DC maturation . GlucoseWhile a number of studies have concluded that that mTOR, and one of its downstream transcription factors HIF1α, are required for DC glycolytic reprogramming , 33–35, Metabolite tracing studies have shown that the rapid catabolism of glucose in TLR-stimulated DCs is closely linked with a number of biosynthetic pathways including the preferential generation of citrate through the TCA cycle , 20. CitBecause pharmacological inhibitors of mTOR function attenuate lymphocyte proliferation, mTOR signaling has classically been considered to play a broadly pro-inflammatory role in the immune system and has been used extensively for its systemic tolerogenic properties in the clinic. Recent studies at the cellular level have revealed that mTOR can exert both inflammatory and anti-inflammatory effects depending on the physiological context and cellular subsets in question. A summary of these findings for mTOR's documented impact on various aspects of DC effector function are delineated below.3 was shown to induce an mTOR-dependent tolerogenic phenotype in monocyte-derived human DCs (moDCs) that was characterized by decreased surface expression of CD80, HLA-DR, and CD86, and increased production of IL-10  mRNA expression  are potent NO producers and are required to control a number of different types of both bacterial and viral infections (in vivo including blood circulating CD1a+ DCs (It noteworthy to consider that only specific subsets of DC populations express iNOS in both mice and humans. GM-CSF -differentiated mouse BMDCs classically induce iNOS expression when stimulated by LPS and IFN-γ . From anfections –77. Howefections , 78, 79,fections , 80. DesD1a+ DCs  and DCs D1a+ DCs –84. GiveD1a+ DCs .While mTOR -dependent NO production has been strongly implicated in promoting DC cell death in iNOS-expressing DC subsets , 15, theThe process of autophagy, whereby cytoplasmic components are ingested, degraded, and their molecular components recycled, plays important roles in DC antigen presentation [recently reviewed in ]. It hasGiven the importance of metabolic changes in supporting the immune activation of DCs, it is not surprising that the central metabolic regulator mTOR plays a critical role in coordinating activation-associated changes in DC metabolism and function Figure . While mAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sirtuins (SIRT) are ubiquitous histone and protein deacetylases and a member of this family, SIRT1, is the best-studied one. Its functions in endothelial cells encompass branching angiogenesis, activation of endothelial nitric oxide synthase, regulation of proapoptotic and proinflammatory pathways, among others. Defective SIRT1 activity has been described in various cardiovascular, renal diseases and in aging-associated conditions. Therefore, understanding of SIRT1-deficient, endothelial dysfunctional phenotype has much to offer clinically. Here, we summarize recent studies by several investigative teams of the characteristics of models of global endothelial SIRT1 deficiency, the causes of facilitative development of fibrosis in these conditions, dissect the protein composition of the aberrant secretome of SIRT1-deficient endothelial cells and present several components of this aberrant secretome that are involved in fibrogenesis via activation of fibroblasts to myofibroblasts. These include ligands of Wnt and Notch pathways, as well as proteolytic fragments of glycocalyx core protein, syndecan-4. The latter finding is crucial for understanding the degradation of glycocalyx that accompanies SIRT1 deficiency. This spectrum of abnormalities associated with SIRT1 deficiency in endothelial cells is essential for understanding the origins and features of endothelial dysfunction in a host of cardiovascular and renal diseases. The broad spectrum of deacetylation targets and dense network of metabolic connections of SIRT1  have conThe sirtuin (SIRT) family consists of seven mammalian NAD-dependent histone deacetylases. SIRT 1 and 2 are expressed in the nucleus and the cytosol, SIRT 3–5 have been identified as mitochondrial proteins and SIRT 6 and 7 are solely expressed in the nucleus . It has SIRT1-null mice exhibit high rates of perinatal mortality and those that survive show developmental deficits, such as growth retardation, sterility, impaired DNA damage response, genome instability, impaired lipid metabolism and liver steatosis, defective immune responses, impaired cognitive functions, and stem cell differentiation, among other abnormalities, which collectively hamper their use . TherefoOne of the dire characteristics of SIRT1-deficient endothelial cells is the reduced endothelium-dependent vasorelaxation. Functional SIRT1 deacetylates the endothelial nitric oxide synthase (eNOS) on lysines 496 and 506 in the calmodulin-binding domain of eNOS. With the deacetylation of eNOS its activity is increased leading to elevated levels of bioavailable endothelial nitric oxide . Endotheendo-/-) and have demonstrated that those mice not only develop vasculopathy and premature senescence, but also tubulointerstitial fibrosis at an early age. One of the mechanistic links presented is the observation that endothelial SIRT1-deficient mice have lower expression of the master matrix metalloproteinase, MMP-14. Lower MMP-14 leads to impaired degradation of deposited matrix proteins, and as a result of reduced matrilytic activity, endothelial cells lose their ability to navigate within the interstitial matrix and recover metabolism and regeneration of the affected organs  are protected against tubulointerstitial fibrosis  and chronic phase of folic acid nephropathy), have a better preservation of renal microvasculature, show a better renal blood flow, and have a less profound tissue hypoxia than TGFβRIIendo+/+ control mice. TGFβRIIendo+/- mice also show suppressed endothelial-to-mesenchymal transition in these fibrogenic models , we obtained and analyzed conditioned media of high-purity populations of RMVEC from control wild-type, SIRT148 hours . 332 nonendo-/- mice upon stimulation with TGFβ. Both pathways previously have been implicated in the development of fibrosis, are mechanistically interconnected , Notch signaling is SIRT1-dependent  and theradation . Indeed,radation . The facradation .Regarding the Wnt pathway in the development of kidney fibrosis, 16 out of 19 Wnt proteins and 8 out of 10 Frizzled receptors have been found to be elevated in renal tubular cells in kidney fibrosis due to UUO . Zhou etAmong the DKK protein family, the role of DKK3 and its interactions with the Wnt pathway is controversial. Dickkopf1 (DKK1) is a well-studied Wnt antagonist and The Notch pathway has been established to be of significant importance during kidney development . For insRegarding the impact of Notch signaling in the development of kidney fibrosis it is crucial to mention that the transcript and protein levels of members of the Notch pathway are highly elevated in patients with CKD, such as diabetic nephropathy, lupus nephritis and focal segmental glomerulosclerosis. It has been demonstrated also that Notch1 expression in renal tubules is associated with tubulointerstitial fibrosis and decreased kidney function in patients with CKD . Supportcis- or trans-position. Among the ligands for the Notch pathway we found Jagged1, which was exclusively secreted by SIRT1-deficient endothelial cells after TGFβ stimulation. 2-D culture studies also showed higher endothelial-mesenchymal transition after exposure to Jagged1 or Jagged2, especially in combination with TGFβ1 (unpublished data). Additional experiments confirmed the activation of the Notch pathway by demonstrating NICD (intracellular domain of the Notch protein) and αSMA-positive RMVEC (unpublished data). In order to investigate the combined effects of Jagged1 on renal fibroblasts and RMVEC we used a co-culture system utilizing microfluidic devices based on previously published protocols . Once thFigure 2) fibroblasts. Endothelial cells deficient in SIRT1 secrete DKK3 and Jagged1 and induce the conversion of fibroblasts into myofibroblasts and contribute to the dysfunctional endothelium-dependent facilitation of tissue fibrosis, which we termed, the “third pathway” of fibrogenesis (igure 2) .Having demonstrated the effects of DKK3 and Jagged1 secreted from dysfunctional endothelial cells exerting paracrine effects on renal fibroblasts, it was only plausible to expect their autocrine effects. In experiments on DKK3 application to cultured RMVEC we found that DKK3 significantly increased the amount of RMVEC undergoing endothelial-mesenchymal transition . We alsoThe endothelial glycocalyx consists of hyaluronic acid cords, heparan sulfate chains and a mixture of dermatan, keratan, and chondroitin sulfates, which give the endothelial glycocalyx a net negative charge . The scaThe rich metabolic network of SIRT1 is at the core of pathologic manifestations of its deficiency in endothelial cells. It not only disrupts the homeostatic processes in these cells, but also affects their functions leading to development of endothelial dysfunction. In addition, endothelial SIRT1 deficiency distorts the normal angiocrine profile of secreted molecules maintaining the structural integrity of organs, thus acquiring secretomic components, which are capable of activating fibroblasts and promoting tissue fibrosis. Furthermore, studies of the secretome of SIRT1-deficient cells revealed that a key component of endothelial glycocalyx is shed, its fragment is pro-fibrogenic, whereas the aberrant composition of endothelial glycocalyx could further exacerbate endothelial dysfunction. These findings provide added support to the therapeutic interventions to correct SIRT1 expression and activity.ML designed the studies, performed the experiments, analyzed the data, and wrote and approved the manuscript. HD designed the studies, performed the experiments, analyzed the data, and approved the manuscript. GM designed the studies and approved the manuscript. MG designed the studies and wrote and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although diagnoses of human immunodeficiency virus (HIV) infection among persons who inject drugs in the United States are declining, an HIV outbreak among such persons in rural Indiana demonstrated that population’s vulnerability to HIV infection (Cluster cases met one or more of the following criteria: 1) HIV infection diagnosis in a woman or MSW in 2018, with partner services data indicating sex or sharing injection-drug equipment with a person in a previously identified cluster case; 2) HIV infection diagnosis in 2018 in a woman or MSW living homeless in the outbreak area; 3) molecular analysis indicating HIV infection with a strain related to those identified among persons meeting either of the first two criteria (HIV-TRACE genetic distance ≤1.5%)  at an emergency department (ED) after being evaluated with fever (patient 6) . He did All 14 cases diagnosed in 2018 occurred in persons living homeless in an area of approximately 3 square-miles; 11 were in women who identified as cisgender, nine of whom reported exchanging sex for money or nonmonetary items, and 12 were in persons who inject drugs, 10 of whom used both heroin and methamphetamine.Analysis of all newly reported HIV infections during January 1–November 30, 2018, identified 27 cases of HIV infection among women and MSW who inject drugs in King County. This represents a 286% increase over the seven cases diagnosed in 2017. On August 3, 2018, a PHSKC disease intervention specialist identified epidemiologic links among patients 5, 6, and 7. Four days later, the health department issued an alert to medical and social service providers concerning the cluster and the increase in HIV diagnoses among persons who inject drugs and who are living homeless. The HIV/Sexually Transmitted Diseases program also contacted several local EDs and the hospital closest to where the patients lived. These EDs have asked providers to increase screening of persons who inject drugs and persons who are living homeless, and at least three are developing more systematic, risk-based opt-out HIV screening programs. To date, ED screening has identified one case of HIV infection (patient 11). On August 20, the King County Jail expanded HIV testing, including opt-out testing at health assessments at 10–14 days and, when resources permit, at time of jail booking. This effort has identified one new case of HIV infection, which has not been linked to the cluster. PHSKC also initiated an expanded program of outreach testing, condom distribution, and syringe services among persons living homeless in north Seattle. As of November 15, 2018, that initiative had tested 534 persons and identified four related cases of HIV infection .PHSKC is increasing access to HIV testing and preexposure prophylaxis (PrEP) among persons who inject drugs through its sexually transmitted disease clinic and SSPs and via a collaboration with a mobile clinic serving north Seattle women who exchange sex or are living homeless. PHSKC is also conducting a rapid assessment to define the medical and social service needs and preferences of persons who inject drugs or who are living homeless in north Seattle with the goal of expanding services, including medication-assisted treatment. Investigations of this cluster and efforts to link infected persons to care are ongoing.This report describes an outbreak of HIV infection in a population of women and MSW who inject drugs and the sex partners of these persons. The outbreak was part of a cluster of 23 persons, nine of whom received a diagnosis of HIV during 2008‒2017. The data suggest that HIV transmission from persons with these earlier diagnoses, some of whom were not virally suppressed, or from their sex partners without a diagnosis, led to a rapid expansion of transmission during 2017–2018, with 14 related infections diagnosed in 2018 in a small geographic area.4). The rate of new diagnoses of HIV infection in King County declined 51% from 2008 to 2017 . PHSKC SSPs provided >7 million syringes to persons who inject drugs in 2017; 79% of persons who inject drugs report using SSPs, and syringe sharing among persons who inject drugs has declined over time  were diagnosed from December 2016 to August 2018, when the cluster was first identified. Had the molecular data been available and analyzed more quickly, it might have been possible to respond earlier, possibly averting some cases. CDC recently initiated a national effort to expand the use of molecular HIV analyses to identify growing clusters of cases  infection among persons who inject drugs in the United States are declining, an HIV outbreak among such persons in rural Indiana demonstrated that population’s vulnerability to HIV infection.In 2018, disease investigation and molecular HIV surveillance in Seattle, Washington, identified 14 related HIV diagnoses among heterosexuals who were living homeless, most of whom injected drugs. From 2017 to mid-November 2018, the number of HIV diagnoses among heterosexuals in King County, Washington, who inject drugs increased 286%.Persons who inject drugs, particularly those living homeless, remain vulnerable to outbreaks of HIV infection, even in cities with large HIV prevention programs and shrinking HIV epidemics.


It is well established that a family of dual-specificity MAP kinase phosphatases (MKPs) play key roles in the regulated dephosphorylation and inactivation of MAP kinase isoforms in mammalian cells and tissues. MKPs provide a mechanism of spatiotemporal feedback control of these key signalling pathways, but can also mediate crosstalk between distinct MAP kinase cascades and facilitate interactions between MAP kinase pathways and other key signalling modules. As our knowledge of the regulation, substrate specificity and catalytic mechanisms of MKPs has matured, more recent work using genetic models has revealed key physiological functions for MKPs and also uncovered potentially important roles in regulating the pathophysiological outcome of signalling with relevance to human diseases. These include cancer, diabetes, inflammatory and neurodegenerative disorders. It is hoped that this understanding will reveal novel therapeutic targets and biomarkers for disease, thus contributing to more effective diagnosis and treatment for these debilitating and often fatal conditions. •A comprehensive review of the dual-specificity MAP kinase Phosphatases (MKPs)•Focus is on MKPs in the regulation of MAPK signalling in health and disease.•Covers roles of MKPs in inflammation, obesity/diabetes, cancer and neurodegeneration The regulation, structure, catalytic mechanism and substrate selectivity of the MKPs have been extensively reviewed . Mo. MoDUSP110/MKP-5 . Despite2.2DUSP2  was first identified as a mitogen-inducible gene in human T-cells and is most closely related to DUSP1/MKP-1 and DUSP4/MKP-2, sharing 71% and 68% amino acid identity, respectively . I. Iet al DUSP16/MKP-7 and revealed an essential role for this MKP in brain development. Embryos lacking DUSP16/MKP-7 exhibited congenital obstructive hydrocephalus together with brain overgrowth. This was secondary to blockage of the midbrain aqueduct by the expansion of neural progenitor cells, eventually preventing the outflow of cerebrospinal fluid. Interestingly only an increase in cells staining positively for phospho-p38 was observed in the affected regions of the CNS indicating that this MAPK, rather than ERK or JNK could be the relevant target [Finally, a recent study has explored the reason for the embryonic/prenatal lethality in mice lacking t target .DUSP16−/− mice recapitulates aspects of different human neurodevelopmental disorders suggests that either DUSP16/MKP-7 or the pathways it regulates may be implicated. They also reveal specific defects in innate and adaptive immunity in mice lacking DUSP16/MKP-7. In particular the specific role of DUSP16/MKP-7 in promoting autoimmunity make it a potential therapeutic target in a range of human disorders such as inflammatory bowel disease, rheumatoid arthritis and lupus.Taken together these studies reveal the first essential role for a member of the MKP family of enzymes in brain development and as the phenotype of 5The past decade has seen an acceleration in the use of GEM models to probe the physiological and pathophysiological roles of the MKPs and these have provided a wealth of information for certain members of the family, particularly in relation to the functional regulation of the immune system, but also in metabolic disease and cancer. As is the case for other classes of protein phosphatases, it is abundantly clear that MKPs are not merely passive “erasers” of protein phosphorylation, but instead form a complex network of activities in cells and tissues that act to regulate the spatiotemporal activity of the different MAP kinase pathways and play essential roles in regulating key physiological outcomes.DUSP1/MKP-1 in metabolic control [DUSP1/MKP-1 for inactivation of p38 MAPK in macrophages and dendritic cells [in vivo. This must be addressed in future work as must the validity of putative non-MAPK substrates for certain MKPs invoked in disease models. The latter include the possibility that STAT3 is directly targeted by DUSP2 in innate immunity [DUSP6/MKP-3 in metabolic regulation [DUSP7/MKP-X in oocyte meiotic progression [Several themes have emerged, one of which is the importance of compartmentalised regulation of MAPK signalling by activities in the nucleus and cytoplasm as exemplified by the regulation of nuclear JNK activity by  control  and nucl control . It is aic cells ,35 whereic cells . Thus faimmunity , the potgulation  and endogulation  respectigression .DUSP1/MKP-1 in combatting obesity and depression [DUSP2 as a potential anti-inflammatory drug target [DUSP1/MKP-1 could also result in more severe inflammatory responses and many existing anti-inflammatory agents, such as glucocorticoids, actually enhance the expression and activity of this MKP as part of their mode of action. Clearly specificity of action towards individual MKPs would also be crucial in any strategy to target these enzymes and the avoidance of undesirable side effects. In this regard and due to the involvement of a redox active cysteine residue in catalysis, the PTPase superfamily has long been regarded as “undruggable”. However, recent progress in the development of allosteric PTPase inhibitors [Finally, there are numerous examples where knockout phenotypes indicate that certain MKPs may be therapeutic targets in human disease. These include inhibition of pression ,73 and Dg target . Howeverhibitors ,201 giveTransparency document.Image 1