Mammalian neurons are highly compartmentalized yet very large cells. To provide each compartment with its distinct properties, metabolic homeostasis and molecular composition need to be precisely coordinated in a compartment-specific manner. Despite the importance of the endoplasmic reticulum (ER) as a platform for various biochemical reactions, such as protein synthesis, protein trafficking, and intracellular calcium control, the contribution of the ER to neuronal compartment-specific functions and plasticity remains elusive. Recent advances in the development of live imaging and serial scanning electron microscopy (sSEM) analysis have revealed that the neuronal ER is a highly dynamic organelle with compartment-specific structures. sSEM studies also revealed that the ER forms contacts with other membranes, such as the mitochondria and plasma membrane, although little is known about the functions of these ER-membrane contacts. In this review, we discuss the mechanisms and physiological roles of the ER structure and ER-mitochondria contacts in synaptic transmission and plasticity, thereby highlighting a potential link between organelle ultrastructure and neuronal functions. Besides such functions, the ER also monitors normal protein folding, trafficking, and degradation. This checkpoint by the ER is well known as ER stress. Disturbance of this system leads to the accumulation of pathological protein aggregates and neurodegenerative diseases. Although neurons have multiple functionally compartmentalized domains, a cell body, dendrites, and an axon, the ER is composed of a continuous lipid bilayer throughout a cell. Similar to other cell types, the ER surrounds the nucleus and forms the nuclear envelope (NE) in the cell body of a neuron. Cisternae and tubular forms of the ER branch out from NE to form the peripheral ER, which extends throughout the neuron. Previous studies revealed that the ER contributes to the regulation of synaptic transmission and plasticity in the dendrite and the axon. Recently, the rapid development of serial scanning electron microscopy (sSEM) analyses have revealed membrane-membrane contacts between the ER and other organelles in neurons. Among such contacts, mitochondria-ER contacts (MERCs) are the most frequently observed in many cell types and are known to represent signaling hubs for various biochemical reactions. For example, mitochondria uptake Ca2+ released from the ER through a mitochondria Ca2+ uniporter (MCU) complex at this contact site. Furthermore, lipid exchange between the ER and mitochondria is required for the synthesis of glycerophospholipids, which are the major components of the plasma membrane. Additionally, MERCs have been suggested to define division sites of mitochondria, thereby regulating mitochondrial dynamics. Such functions of MERCs provide a new insight that the ER does not function as an isolated unit, but rather communicates with other organelles and forms dynamic membrane contacts in response to cellular demands in many cell types including neurons. Thus, studying the roles of these contacts in neurons holds promise for revealing new insights in the cellular mechanisms of synaptic transmission. Here, we review our current understanding of ER functions in neurons and novel modalities of ER-(other) membrane contacts in the regulation of neurotransmission.Unlike most cell types in the mammalian system, the majority of neurons in the central nervous system are produced during development and have to be maintained throughout the lifetime of an organism. Therefore, neuronal organelles and proteins are actively replaced to keep neurons functional for decades. Indeed, numerous genetic mutations in genes regulating organelles, including the endoplasmic reticulum (ER) and mitochondria, are linked to neurodegenerative diseases. The ER serves as a platform for important biochemical reactions, such as protein and lipid synthesis2 In addition, a web of anastomosed tubules made of smooth ER (SER) is present both myelinated and unmyelinated large axons of spinal ganglion neurons. Recently, advances in the automated serial electron microscopy (EM) have provided high-resolution and large-volume ultrastructural three-dimensional analyses of the axon in the brain. These studies showed that in the cortex of mice the thinnest (∼200 nm in diameter) unmyelinated axons usually contained a single narrow ER tubule, consistent with previous reports. At the branching points of SER tubules, they did not connect with each other directly but rather formed a comparably small lumen called cisternae. Notably, the structures of the ER in presynaptic varicosities take a variety of different forms. In the nucleus accumbens, the presynaptic ER forms branches and expands into small cisternae. In addition, in some presynaptic sites, ER-like tubules physically discontinuous from the ER network have been observed. On the other hand, in pyramidal neurons in layer 4/5 of the mouse neocortex, the ER bulges specifically at presynaptic sites and shows a simple sheet-like structure. Even though there are structural differences in the presynaptic ER, both reports showed that the ER forms extensive contacts with mitochondria in presynaptic varicosities, confirming studies using conventional serial EM techniques  interactions are required for the formation of elongated ER tubules. Indeed, the ER binds to MTs in the axon. Myosin Va (Myo-Va) is likewise involved in distributing axonal ER along an intermediate filament, Neurofilament-L. In contrast to the cytoskeleton that functions throughout all neuronal compartments, ER-resident proteins have been shown to regulate ER morphogenesis particularly in the axon. Genetic mutations in the ER-resident proteins Reticulon (RTN)-2, REEP1, and atlastin-1 are associated with an axonopathy, hereditary spastic paraplegia, and a mutation in the ER membrane receptor VAMP-associated protein (VAP) B is associated with another type of axonopathy, amyotrophic lateral sclerosis. In Drosophila, deletion of Reticulon-like-1, the Drosophila ortholog of RTN, and Reep proteins causes a reduction in SER in the distal portion of motor neurons and occasional discontinuities in the axonal ER. Ultrastructural analysis of the axonal ER in larvae lacking reticulon and Reep proteins showed that the diameter of the ER was increased and the number of ER tubules per section was reduced, consistent with the molecular functions of hereditary spastic paraplegia-related proteins in stabilizing ER membrane curvature. Lindhout et al. showed that VAPB and its family protein VAPA interact with cytoplasmic protein secernin-1 (SCRN1) at the ER membrane via a single FFAT-like motif, and this interaction regulates ER morphology in the axon. Notably, loss of VAP–SCRN1 interactions resulted in a decrease in synaptic vesicle cycling. Consistent with this finding, VAP or SCRN1 knockdown both resulted in a decrease in action potential (AP)-evoked presynaptic Ca2+ influx, suggesting that VAP–SCRN1-mediated ER remodeling regulates presynaptic Ca2+ homeostasis and thereby modulates AP-evoked synaptic transmission.The interaction between the ER and cytoskeleton generates the main mechanical force for membrane deformation in shaping ER networks. Deletion of ATG5, an essential protein for the autophagosome formation, leads to the accumulation of SER proteins, particularly tubular ER forming proteins, such as RTN3 and VAPB. Furthermore, ultrastructural analysis showed elevated numbers of ER tubules specifically in axons and at presynaptic sites. This indicates that neuronal autophagy constantly degrades SER in the axon. Moreover, ATG5 knockout caused an elevation of ryanodine receptor (RyRs)-mediated Ca2+ release from the ER, resulting in an increase in spontaneous release.In addition to the regulation of ER morphology, a recent study showed that the amount of ER in the axon shaft and the presynaptic bouton is regulated by ER membrane turnover via autophagy (ER-phagy). when and how such dynamic movements of the axonal ER occur remain unclear.Although live imaging of the axonal ER labeled with GFP-fused CG9186, an ER localized lipase, showed anterograde and retrograde movements of ER tubule-like structures detached from the ER networks in Drosophila neurons,3 This indicates that the axonal ER is likely to have functions other than protein synthesis. Although Ca2+ import from the extracellular space mediated by voltage-gated Ca2+ channel has been mainly focused on in terms of presynaptic Ca2+ regulation, it has been revealed that the SER functions as a Ca2+ source in the axon in several types of neurons. The increase in cytoplasmic Ca2+ can cause calcium-induced calcium release (CICR) mediated by RyRs localized in the ER membrane. CICR was shown to be required for the induction of spontaneous but not evoked neurotransmitter release by single APs with low-frequency (∼0.1 Hz) stimulation. In addition to the functions in the induction of spontaneous neurotransmitter release, RyRs were shown to be required for the enhancement of evoked GABAergic synaptic currents at the cerebellar basket cell–Purkinje cell synapse. Furthermore, although the role of CICR in the induction of short-term plasticity remains controversial, presynaptic CICR was shown to be required for inducing long-term depression (LTD) in hippocampal pyramidal neurons. Notably, CICR-mediated spontaneous firing is depotentiated by ER-phagy-mediated reduction in the amount of ER. In addition to CICR, presynaptic enrichment of inositol trisphosphate receptor (IP3R) 1 was observed in presynaptic terminals of the hippocampal excitatory Schaffer collateral synapses upon tetanic stimulation. Furthermore, IP3R-induced Ca2+ release was shown to induce GABA release downstream of kainate receptors in rat prefrontal GABAergic neurons. Consistent with these reports, impairment of ER structures by loss of ER-resident proteins such as atlastin, reticulon, and VAPs, is accompanied by a decrease in neurotransmitter release. These reports suggest that the ER functions as an accelerator of presynaptic release probability. In correlation with presynaptic Ca2+ influx, net uptake of Ca2+ was induced by neuronal activity through Sarco/ER Ca2+-ATPase. Further studies are required to resolve the complex interplay among different sources of Ca2+ at presynaptic boutons. In addition to the functions of the ER itself as a Ca2+ source or sink, store-operated calcium entry (SOCE) activated by ER Ca2+ depletion is known to increase the frequency of spontaneous neurotransmitter release in hippocampal neurons. In many cell types, Ca2+ concentration in the ER lumen is sensed by ER-resident stromal interaction molecule (STIM) family proteins. Two isoforms, STIM1 and STIM2, are known to be expressed in neurons and both proteins have weak affinity to Ca2+ via an EF-hand motif at the N-terminus. When luminal Ca2+ concentration in the ER decreases, the unoccupied EF-hand motif induces a conformational change of the STIM protein, leading to its clustering and activation. This in turn activates Orai, a plasma membrane Ca2+ channel, to induce Ca2+ influx. Of note, depleting Ca2+ from the ER increases miniature excitatory postsynaptic current (mEPSC) frequency through a mechanism requiring STIM2 but not STIM1. This suggests that STIM2-mediated SOCE potentiates spontaneous neurotransmitter release. Given that SOCE is associated with several neurological disorders such as Huntington’s, Parkinson’s, and Alzheimer’s diseases (AD), it is possible that the aberrant control of SOCE-mediated spontaneous firing leads to the onset of such neurological disorders.Mature axons exclusively exhibit smooth ER and are devoid of rough ER.44.1 Serial EM and fluorescence recovery after photobleaching (FRAP) assays showed that the ER in aspiny segments of the dendrites consists primarily of multiple tubules along the shafts with thin branches intermittently traversing the cytoplasm. At branch points and the dendritic shafts near the spines, the ER becomes cisternae and is enriched with ribosomes.In dendrites, a large portion of the ER is SER elongated from the peripheral ER in the cortex of the cytoplasm as a continuous anastomosing network. The dendritic ER is associated with MTs via an ER integral membrane protein Climp63, which becomes enriched in the somato-dendritic compartment during neuronal maturation. Notably, knockdown of Climp63 increased branches in the dendrites as well as ER complexity. This indicates that ER complexity regulates dendritic branching. Mutual interactions between ER and MTs were also observed in Caenorhabditis elegans sensory PVD neurons. It has been shown that ER extensions into the secondary and tertiary branches of PVD neurons are dependent on MTs. ER-associated MTs were more stable than the non-ER-associated MTs. These results indicated that the ER and MTs mutually affect each other, and the ER invades the dendritic branch through MT-dependent mechanisms.As in the axon, elongated ER tubules in the shaft frequently contact MTs in dendrites. On the other hand, at the branching points where the ER forms complex structures, the ER is much more loosely associated with MTs Fig. .8) The d The source of the extrinsic cue and interplay with other signaling pathways remain to be elucidated.Regulation of ER morphology by extrinsic cues is an intriguing process to study. For example, an extrinsic signal through group I metabotropic glutamate receptors (mGluRs) phosphorylates Climp63 via activation of protein kinase C and releases the ER tubules from MTs.4.2 less than 20% of spines contained SER in rat hippocampal CA1 dendrites. A detailed three-dimensional analysis of the ER structure identified the SER and several types of non-SER compartments including clathrin-coated vesicles and pits, uncoated vesicles, tubules, and multivesicular bodies in dendritic spines of the rat hippocampal CA1 region. The SER in large spines often shows a specialized structure called ‘spine apparatus’ (SA)  of spines were visited at least once during 5 hours of imaging. These ER visits were typically short (more than 50% of visits were less than 10 minutes) and became significantly less frequent when excitatory transmission was blocked by the treatment with N-methyl-D-aspartate (NMDA) receptor inhibitor D-2-amino-5-phosphonovaleric acid (D-APⅤV) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor inhibitor NBQX. Notably, the ER preferentially visits spines enlarged by glutamate uncaging-mediated structural long-term potentiation (LTP). These reports suggest that a significant fraction of spines is visited by the ER during excitatory transmission and LTP.Studies using confocal microscopy showed that a majority of spine ER undergoes turnover in 24 hours but a small subset of spines maintain the ER for at least 4 days.5 Additionally, cargo exit sites are enriched in dendritic shafts with more SER volume or branches , is transported to the spine via recycling endosomes from the dendritic ER in a Golgi apparatus-independent manner.hes Fig. . Consideet al., performed proteomic analyses aiming for a comprehensive identification of proteins interacting with pore-forming components of AMPARs using membrane fractions from adult rat brains. Notably, among identified novel subunits of AMPARs, FRRS1l is exclusively contained in an intermediate complex (priming complex) formed by the coordinated action of several proteins on the ER. Moreover, disrupting the assembly of the intermediate complex by ablating FRRS1l significantly reduced the amount of AMPARs on the surface of the plasma membrane. This resulted in the impairment of synapse formation and the abolishment of LTP induction in hippocampal pyramidal neurons. This report suggested that the SER functions as a platform for efficient AMPARs assembly and serves as a reserve pool of AMPARs for synaptic potentiation and transmission. Considering that the SA associates with large mushroom spines and large active zones, it is possible that the large amount of membrane provided by the cisternae structure of SAs is required for promoting the assembly of the priming AMPAR complex.Besides the role as an initiation site of endosomal trafficking, the SER serves as a site for efficient assembly of AMPARs. Schwenk  Notably, using Myo-Va-deficient Purkinje neurons, Miyata et al., showed that SER insertion in the spine is required for the induction of LTD by increasing the local Ca2+ concentration. Consistent with these reports in Purkinje cells, spines with SER are shown to be preferential sites for mGluR-dependent LTD in hippocampal pyramidal neurons. These studies suggested that the insertion of SER into spines is required for proper synaptic plasticity. In line with these reports, Perez-Alvarez et al., showed that expression of a dominant negative (DN) form of Myo-Va drastically reduced the frequency of ER visits into the spines and the number of spines with ER in hippocampal pyramidal neurons. Expression of DN-Myo-Va significantly increased the spine surface expression of GluA2 and glutamate uncaging-evoked EPSCs in hippocampal CA1 pyramidal neurons. As a result, in these neurons, although low-frequency stimulation can induce LTD, pairing stimulation of both pre- and post-synapses did not induce LTP. This suggested that transient visits of the ER maintain spines at intermediate strength for providing those spines with a potential for both LTP and LTD. Although a direct link between ER dynamics in the spine and the delivery of FRRS1l-containing AMPAR to synaptic surface membranes remains unclear, both mechanisms may coordinately contribute to the induction of proper synaptic plasticity. It still remains unclear what molecules interconnect between the neuronal excitation and Myo-Va activity. The interplay of two Ca2+-binding proteins: calmodulin and caldendrin may fine-tune the activity of Myo-Va and thereby regulate the dynamics of the SER in spines in response to local Ca2+ increase.Then, the next question is how dynamic distribution of the ER is regulated in spines and what is the role of dynamic changes in the distribution of the ER during synaptic potentiation? Molecular motors moving along actin filaments play a critical role in bringing the ER network to the periphery of the neurons. Myo-Va is shown to be required for the insertion and elongation of the tubular SER from the dendrites to the spines in Purkinje neurons. This suggested that a simple tubular SER in spines is transformed to a complex SA after LTP. The SA was shown to contain the actin-binding protein synaptopodin. Synaptopodin-deficient neurons contained single ER tubules but completely lacked the SAs in the cortex, hippocampus, and striatum. This absence of SA was accompanied by a decrease in LTP in hippocampal neurons, suggesting that this structure contributes to LTP.In addition to the distribution of the ER in the spine, recent studies suggest that the ER changes its structural complexity in the spine after LTP. The ratio of spines containing SA to those containing a tubule SER was markedly increased 2 hours after theta-burst stimulation in hippocampal slice culture.6 recent advances in sSEM techniques allowed us for the first time to investigate the contact between the ER and mitochondria comprehensively in 3D. This showed that there are extensive MERCs in all compartments of neurons. However, the molecular mechanism underlying the formation of the ER-mitochondria contact in metazoans remained limited. Thus, the physiological significance of this contact had not been revealed.As mentioned above, the functions of the ER in dendrites have been rigorously studied. Although it has been suggested that part of the ER locates in close apposition to the mitochondria in dendrites from the early 1980s,et al. recently identified the ER-resident protein PDZD8 as a critical ER-mitochondria tethering protein in mammalian cells including neurons. In budding yeast, a tethering complex between the ER and mitochondria called ER-mitochondria encounter structure (ERMES) complex was identified in 2009. Three out of four ERMES complex proteins  contain a synaptotagmin-like mitochondrial lipid binding protein (SMP) domain. However, no clear functional ortholog for any ERMES protein had been identified in metazoans. Recently, the crystal structure of an SMP domain was identified, which allowed us to perform a more definitive structural homology search based on secondary and/or tertiary structures than that based on its primary amino acid sequence. This led to the identification of a candidate protein of unknown function containing a structurally-defined SMP domain, PDZD8. Indeed, a genetic complementation assay in budding yeast showed that the SMP domain of PDZD8 is functionally homologous to that of Mmm1.Hirabayashi et al. used focused ion beam-serial EM, and reconstructed mitochondria and ER structures from several hundred serial EM sections. The extent of MERCs amount quantified from the 3D reconstruction revealed that the percentage of MERC area per mitochondria surface area was greatly reduced in PDZD8 knockdown HeLa cells compared with that in wild-type cells. This suggested that PDZD8 is required for MERCs formation.Precise investigation of MERCs required EM due to the narrow (10–30 nm) distance between the ER and mitochondria at contact sites. In addition, both the ER and mitochondrial networks display highly complex topologies in 3D. Therefore, 3D rendering and quantification of MERCs areas have been very challenging in standard two-dimensional EM. To reveal the role of PDZD8 in the regulation of MERCs, Hirabayashi 2+. Therefore, whereas MERCs are proposed to serve as platforms for many important biochemical reactions, Ca2+ transfer from the ER to mitochondria is likely to be one of the main roles in neurons. The MCU, a Ca2+ channel in the mitochondrial inner membrane, is responsible for the mitochondrial uptake of Ca2+ released from the ER. To open the MCU, the concentration of Ca2+ on the mitochondrial surface must be greater than 1–5 µM depending on the components of the MCU complex. However, the concentration of Ca2+ in the cytoplasm is usually less than 100 nM. It is only in the vicinity of the ER, that the cytoplasmic Ca2+ concentration reaches levels high enough to open the MCU. Thus, contact of mitochondria with the ER is required for mitochondrial Ca2+ uptake. It has been shown that the ER releases Ca2+ upon synaptic stimulation through IP3Rs and RyRs in the dendrite. This Ca2+ release can increase the local Ca2+ concentration of cytoplasm to a level higher than the threshold value for opening the MCU. Indeed, using high-speed dual-wavelength Ca2+ imaging and genetically encoded Ca2+ sensors specifically targeted to the ER or mitochondria, it was revealed that the ER releases Ca2+ upon electric stimulation and the mitochondria subsequently buffer Ca2+ in neocortical excitatory neurons in vitro  in mature neurons. Recent reports have revealed that ER-resident proteins VAPs and TMEM24 and plasma membrane localized Kv channels are enriched in these contacts, whereas the roles of the contact in neurotransmission remain largely unaddressed. Thus, studies of ER-membrane contacts are still in their infancy and will give us new insights in the field of neurotransmission.Although organelles have been thought of as isolated units, many studies have revealed that they interact with each other. This leads to a major paradigm shift in this field. Although a lot of organelle interactions, especially ER-mitochondria contacts, have been observed in neurons, it remains unanswered how the coordinated actions among organelles contribute to neurotransmission especially in the presynaptic bouton. A clue for answering this question can be provided by studies of ER-membrane contacts in other cell types including yeast. For instance, studies in yeast and mammalian cell lines suggest that the ER-mitochondria contact is important for maintaining mitochondrial DNA that encodes genes essential for the mitochondrial ATP synthesis.


MLL-rearrangements. These approaches are complimentary to de novo drug discovery and have taken advantage of increased knowledge of the mechanistic basis of MLL-fusion protein complex function as well as refined drug repurposing screens. Despite the vast number of different leukemia associated MLL-rearrangements, the existence of common core oncogenic pathways holds the promise that many such therapies will be broadly applicable to MLL-rearranged leukemia as a whole.The treatment failure rates of acute leukemia with rearrangements of the Mixed Lineage Leukemia (MLL) gene highlight the need for novel therapeutic approaches. Taking into consideration the limitations of the current therapies and the advantages of novel strategies for drug discovery, drug repurposing offers valuable opportunities to identify treatments and develop therapeutic approaches quickly and effectively for acute leukemia with  De novo drug discovery and development of a drug for cancer takes approximately 10–17 years, with almost half of this time elapsing before the start of phase I clinical trials , signature matching, computational molecular docking and pathway or network mapping  gene, are a genetic hallmark and one of the major driver mutations  is only 20–50%, in comparison to over 60% for those with wild-type MLL  and the age of the patient. The standard treatment typically consists of induction chemotherapy, followed by intensive chemotherapy followed by additional therapy. Over 130 different MLL translocations with numerous different fusion partners have been described, although only a fraction of these are recurrent , a GSK3 inhibitor that also targets cyclin-dependent kinases (CDKs) and has been extensively used for the maintenance of pluripotency in human and murine embryonic stem cells (ESCs)  genes via CREB phosphorylation, leading to the induction of MEIS1-related gene expression and the subsequent maintenance of a stem cell-like phenotype in MLL-rearranged cells (MLL-rearranged leukemic stem cells (LSCs) were found to be resistant to GSK3 inhibition by lithium chloride (LiCl), an inhibitor that is approved by the Food and Drug Administration (FDA) for the treatment of epilepsy and bipolar disorder (Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase with multiple roles in several signaling pathways in physiological and pathological conditions. GSK3 is involved in mRNA transcription, cell cycle, apoptosis, cell fate determination and stem cell maintenance . As seves (ESCs) . Cell cyfibrosis , revealegression . The speed cells . In addidisorder , they codisorder .MLL-rearranged leukemia, the DNA damage response (DDR) machinery is perturbed. Some MLL-fusion proteins are suggested to predispose leukemia cells to DNA damage and ultimately cause short latency  expressing MLL-AF9 show reduced colony formation activity in comparison with Parp1+/+ counterparts. These cells were shown to be modestly sensitive to the PARP1 inhibitor olaparib. In the same study, syngeneic mice transplanted with murine leukemia cells expressing MLL-AF9 were either left untreated or were treated with doxorubicin plus cytarabine, the PARP inhibitor BMN673, or the combination of the two. The results demonstrate a synergistic effect of the PARP1 inhibitor with the cytotoxic drug combination, overcoming the resistance to olaparib  polymerase (PARP) inhibitors. Poly (ADP-ribosyl)ation of nuclear proteins supports the survival of cells with low DNA repair capacity. The role of PARP proteins has been well established in ormation . PARP inormation . Olapari cancers . The repnd RAD51 . Importand RAD51 . One stuolaparib . While iolaparib . In AML,olaparib . HOXA9 solaparib  and its olaparib . The hypnescence . Importahibition . Taking hibition , PARP inMLL-rearranged leukemia has therapeutic potential, since in mice where ATR expression is reduced, MLL-ENL AML cells growth is affected. Schoppy et al. generated a genetic system where ATR expression was conditionally reduced to 10% of normal levels in adult mice and studied the effects of this reduction in vivo comparing normal tissues and cancer. Although the suppression of ATR did not affect normal bone marrow, the authors reported inhibition of MLL-ENL-driven AML among others  is a serine/threonine protein kinase consisting of a catalytic subunit (DNA-PKcs) and a Ku heterodimer that is made up of the Ku70 and Ku80 subunits and it belongs to the phosphatidylinositol 3-kinase (PI3K)-related kinase protein family . DNA-PK MLL1, MLL2, MLL3, MLL4, SETD1A, and SETD1B)  that act as response regulators of the DDR, that strongly correlated with SETD1A and Akt expression, suggesting an anti-leukemic activity of anlotinib by targeting SETD1A- and Akt-mediated DNA damage response in MLL-rearranged leukemia cells (SET domain-containing protein 1A (SETD1A) is a histone 3 lysine 4 (H3K4) methyltransferase and is one of the six MLL family members found in mammals ( SETD1B) . H3K4 me SETD1B) . In breanescence . In leukll cycle . In a rell cycle . Anlotinll cycle . Anlotinia cells .MLL-rearrangements.These studies highlight the potential of inhibiting molecular players of the DDR pathway in acute leukemias with MLL-rearrangements is the expression of stem cell gene programs that support the leukemia-initiating activity of hematopoietic stem cells  is a critical regulator of cell-cycle progression. CDK6 and CDK4 are critical for normal hematopoiesis, as the simultaneous deletion of the two kinases leads to embryonic lethality in mice . In normem cells . The impapproach . It has proteins . MLL-AF9affected . The effaffected . The cataffected . Palbociaffected . The stude novo drug development of agents able to disrupt this interaction could be a successful strategy in MLL-rearranged leukemia. Li et al. showed that when the MLL-menin interaction is disrupted in mice, only the expression of a small number of genes is affected and importantly, these genes are not the critical downstream effectors of MLL for normal hematopoiesis or B lymphopoiesis. This indicates that menin is not required for the gene expression program regulating development of HSCs and B cells , showed synergistic effects with the MLL-menin interaction inhibitor MI-3 on cell growth and apoptosis. The combination of the two agents was also effective in in vivo experiments with a mouse xenograft model established by subcutaneous inoculation with MLL-rearranged MOLM-13 cells , as a candidate for inhibiting the growth of MLL-rearranged cell lines but not MLL wild type cells , GWAS and the Library of Integrated Network-based Cellular Signatures (LINCS) can be used among others for signature-based screening . These tMLL-rearrangements .The transcription factor c-MYB plays a central role in definitive hematopoiesis and the development of multiple hematopoietic lineages . In leukngements . Importaleukemia . Howeverleukemia . Similaria cells . Surprised cells . Mebendaed cells . This ilsubtypes   cells and compared them with the Genomic Element Associated with drug Resistance (GEAR) database -rearranged cells  SHI-1 cell transplanted mice, but not in mice transplanted with t  HL60 or t  THP-1 cells, highlighting the specificity of thioridazine against cells with the MLL-AF6 fusion. To understand the mechanism behind the MLL-AF6 specific anti-leukemia activity of thioridazine, the authors used a large-scale method combining quantitative proteomics with affinity enrichment and identified the calcium binding proteins S100A8 and S100A9, and ANXA6, a component of the annexin family, as targets of thioridazine. These three proteins are known to form a complex associated with cytoskeletal filaments  overproduction that was detrimental for the t  AML cells . This high-throughput screening approach proved very effective, as it combined the selection of a therapeutically highly relevant drug library as step one, and then the application of an all-pairs testing algorithm for identification of novel partners for combination treatments. This methodology achieves the reduction of consumables and time in the laboratory and opens up opportunities identification of novel combination therapies including the repurposing of therapeutic agents (In a study using an alternative multiplex screening approach, Lappin et al. screened a library of 80 apoptosis-inducing agents for potential combinations with compounds with therapeutic potential for pediatric AML . The scrc agents .MLL-rearrangements. The successful modelling of MLL-rearranged acute leukemia has also provided fruitful experimental strategies in the search for new therapies. In this direction, we can take advantage of the technologies used for drug repurposing and identify therapeutic agents with the potential to specifically target leukemia cells with MLL-rearrangements. Exploring drug repurposing provides a new toolkit to tackle the challenges of clinical translation. The opportunity of applying known therapeutic strategies against new targets by repurposing drugs in the clinic is developing into a core strategy in drug development.The understanding of the network of gene expression regulation driven by MLL fusion proteins in acute leukemia has given us a better insight of the molecular mechanisms that these fusion proteins hijack in order to initiate and maintain leukemogenesis. The protein complexes that cooperate with MLL fusion proteins and the subsequent gene expression changes open up numerous opportunities for pharmacological targeting specific to the leukemia cells with


In May 2018, the first patient was enrolled in the phase-IIb clinical trial “Safety and Preliminary Efficacy of Sequential Multiple Ascending Doses of Solnatide to Treat Pulmonary Permeability Edema in Patients with Moderate to Severe ARDS.” With the onset of the COVID-19 pandemic in early 2020, the continuation and successful execution of this clinical study was in danger. Therefore, before the Data Safety Monitoring Board (DSMB) allowed proceeding with the study and enrollment of further COVID-19 ARDS patients into it, additional assessment on possible study bias was considered mandatory.We conducted an ad hoc interim analysis of 16 patients (5 COVID-19- ARDS patients and 11 with ARDS from different causes) from the phase-IIB clinical trial. We assessed possible differences in clinical characteristics of the ARDS patients and the impact of the pandemic on study execution.COVID-19 patients seemed to be less sick at baseline, which also showed in higher survival rates over the 28-day observation period. Trial specific outcomes regarding pulmonary edema and ventilation parameters did not differ between the groups, nor did more general indicators of (pulmonary) sepsis like oxygenation ratio and required noradrenaline doses.The DSMB and the investigators did not find any evidence that patients suffering from ARDS due to SARS-CoV-2 may be at higher  risk when included in the trial, nor were there indications that those patients might influence the integrity of the study data altogether. For this reason, a continuation of the phase IIB clinical study activities can be justified. Researchers continuing clinical trials during the pandemic should always be aware that the exceptional circumstances may alter study results and therefore adaptations of the study design might be necessary. Dear Editor,In May of 2018, the first patient was enrolled in a phase-IIB clinical trial  to assess the safety and efficacy of a novel inhalative peptide  for the treatment of moderate to severe acute respiratory distress syndrome (ARDS) , 7, the Five patients were diagnosed with COVID-19 .p = 0.022, two-sample Kolmogorov-Smirnov test, ɑ = 5%), suggesting that the enrolled COVID-19 patients were less critically ill than the non-COVID-19 patients. The simplified acute physiology score 3 (SAPS3) showed similar differences, yet not significantly so . The sequential organ failure assessment (SOFA) score showed no significant differences between groups, neither at screening nor over the course of the 7-day intervention period .At the time of screening, the acute physiology and chronic health evaluation II (APACHE II) scores differed significantly between groups . Ventilation parameters  were assessed once daily during the intervention period . Oxygenation ratio (paO2/FiO2) was assessed once per day, and cumulative required doses of noradrenaline per 24 h were recorded. Both outcomes did not differ over the 7-day time span .Extravascular lung water index (EVLWI) and pulmonary vascular permeability index (PVPI) were measured at baseline and twice daily during intervention for seven consecutive days. There were no differences between the two groups of patients over time (EVLWI: Two patients died (both non-COVID), and ten made it to intensive care unit discharge  during the 28-day observation period. The distribution of ventilator-free days was widely scattered, yet not significantly different between groups . Renal replacement therapy was required in one COVID and two non-COVID patients. One COVID patient and five non-COVID patients required further escalation of the ventilator support .No major deviations from study design and procedures occurred due to the COVID-19 pandemic. Special hygienic precautions had to be used during study interventions to limit the risk of infection for the health care staff. To avoid using additional protective equipment for study interventions, thorough planning, bundling, and timing of study specific assessments and procedures  was crucial. Thankfully, our study teams did not experience any critical shortage of supply chain for personal protective equipment (PPE) arising from COVID-19. Nevertheless, a major concern was running out of PPE, putting intensive care staff and patients at risk of infection . TherefoDuring the COVID-19 pandemic, elective surgeries, especially procedures likely to require critical care support, were postponed, in order to increase capacity for patients infected with COVID-19 . As an eA main cause of direct ARDS during wintertime in the northern hemisphere is influenza pneumonia. In 2020 and 2021, the emergence of the COVID-19 pandemic had an important impact on influenza virus activity. Major reductions in influenza activity were observed globally and possible reasons include non-pharmaceutical interventions (NPIs), reduced population mixing and reduced travel, but may also include virus–virus interactions, sometimes referred to as “viral interference” .After WHO declared COVID-19 a public health emergency of international concern and characterized it as a pandemic on 11 March 2020, 30 countries began imposing measures to limit the spread of the disease. The timing of these measures directly correlated with a steep drop in influenza detections in 2020. Especially, encouragement of frequent hand-washing and basic hygiene measures and the advice on mask use for the general public had been effective in reducing influenza transmission . AnotherTaken together, emergence of COVID-19 led to less non-COVID-19 ARDS patient admissions in the ICUs than anticipated at study initiation. Therefore, patient recruitment into the study declined, especially after the Drug Safety Monitoring Board limited the quantity of SARS-CoV-2 ARDS patients allowed to be included in the “middle dose group” of the clinical study.In early 2020, the medical community was in urgent need for new therapeutic approaches to treat patients with the new COVID-19 disease. Researchers worldwide were under enormous time pressure in order to provide health care professionals with new knowledge as fast as possible.Concurrently, concerns have been raised in both the scientific and lay press with regard to the quality and integrity of data, as well as with methodology and transparency of some of this research . Twice aIn direct comparison of COVID-19-related publications to non-COVID-publications, the COVID-19-related papers were less likely to be based on randomized controlled trials (RCTs) and more likely to use case series or other observational designs. Moreover, COVID-19 papers had smaller sample sizes, shorter follow-ups, and a higher risk of bias. Furthermore, they were more likely to have a retraction or major post-publication correction . As suchWe conducted an ad hoc interim analysis of 16 patients from a phase-IIB clinical trial on the safety and preliminary efficacy of a novel inhalation agent for the treatment of moderate to severe ARDS, due to the unexpected challenges that arose with the onset of the global COVID-19 pandemic. We were provided with the data of five COVID-19 patients and eleven non-COVID-19 patients from the same solnatide dosing regime and similar treatment period, extracted from the eCRF. We found the COVID-19 patients to be a little less sick at baseline, which also showed in survivals over the 28-day observation period. Trial-specific outcomes regarding pulmonary edema and ventilation parameters did not differ between the groups, nor did more general indicators of (pulmonary) sepsis like oxygenation ratio and required noradrenaline doses.In conclusion, the DSMB and the investigators did not find any evidence to support the hypothesis that patients suffering from ARDS due to SARS-CoV-2 may be at higher  risk when included in the trial, nor were there indications that those patients might influence the integrity of the study data altogether. For this reason, a continuation of the phase IIB clinical study activities, especially against the background of the second and third wave of the pandemic, was approved by the DSMB and is well justified from a medical point of view. We recommend that researchers take into account any specific influence of a pandemic on ongoing clinical trials and hence perform adaptations in study design to successfully complete the study and avoid bias and false interpretation of the study results.In addition, clinical use of solnatide has been approved by the national medicines agencies in Austria and Italy within the scope of compassionate use programs for the treatment of moderate to severe COVID-19 patients.


Metabolic reprogramming and cellular plasticity drive tumorigenesis. However, how these cellular events collectively contribute to the oncogenic process is poorly understood. Epithelial-mesenchymal transition (EMT), a fundamental mechanism of cellular plasticity, is governed by the EMT transcription repressors such as Snail. In the present study, through establishment and characterization of inducible overexpression of Snail in A549 lung cancer cells, we aim to define the metabolic reprogramming in response to Snail in the EMT of lung cancer cells.Our metabolomic analysis suggests that forced expression of Snail accompanied reduced diversion of glycolytic metabolites to the serine/glycine metabolic shunt, a critical metabolic branch that distributes glucose catabolic intermediates to the major anabolic pathways. Our gene expression profiling and molecular characterization revealed that Snail actively suppressed the expression of glycine decarboxylase (GLDC), a key enzyme on the serine/glycine metabolic shunt, through binding to an evolutionarily conserved E-box motif and thereby inhibiting the promoter of the GLDC gene. Besides, knockdown of GLDC led to a cellular function shift from proliferation to migration.This study has revealed a novel molecular link that integrates the serine/glycine metabolism with the Snail-mediated EMT program in cancer cells. For example, the elevation of Snail has been shown to augment differential responses in the glycolysis, the primary catabolic pathway of cancer cells, with the upregulated and downregulated glycolysis seen in the breast and lung cancer, respectively. Therefore, it is imperative to elucidate the molecular relationship between the EMT transcription factor and the metabolic target for a better understanding of the metabolic basis for EMT.Interestingly, while gaining migratory capacity during EMT, the cells often slow down proliferation, implying that significant rearrangement of cellular states including cellular metabolism would occur in the EMT processes.,14 Synthesized from the serine that is derived from the glycolytic catabolites, the glycine is broken down to form the folate-mediated one-carbon unit, supplying key anabolic intermediates for supporting cell proliferation.,14 Therefore, high turnover of glycine is a unique metabolic feature associated with rapidly proliferating cancer and embryonic stem cells. Consistently, the key enzymes of this high-flux serine/glycine metabolic shunt including phosphoglycerate dehydrogenase, serine hydroxymethyltransferase, and glycine decarboxylase (GLDC) which are responsible for the synthesis and catabolism of serine and glycine, respectively, are all found to be significantly elevated to drive cancer proliferation.–19 However, given a clear functional shift from proliferation to migration during EMT, it is not known whether the serine/glycine metabolic shunt is reprogrammed at the molecular level in this important cellular process.The small amino acid glycine is a key metabolic intermediate that connects the major catabolic and anabolic pathways in cancer cells.GLDC gene and thereby suppresses the GLDC promoter. Thus, these observations have revealed a novel molecular link that integrates the serine/glycine metabolic rearrangement with the Snail-induced EMT program.In the current study, through characterization of an EMT tissue culture model, we identified that GLDC, the glycine catabolic enzyme is a novel direct target of Snail. We found that Snail binds to an evolutionarily conserved proximal E-box motif in the Human lung cancer A549 and H1299 (ATCC) and human embryonic kidney 293FT (Invitrogen) cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U of penicillin/ml, and 0.1 ng of streptomycin/ml. and pcDNA3.1 (Invitrogen) to yield pInducer20-Snail-HA and pcDNA3.1-Snail-HA for doxycycline-inducible and constitutive overexpression of wild-type Snail, respectively. For construction of the expression vector for the transcriptionally inactive Snail mutant that lacks SNAG domain, the sequence encoding the amino acids at the positions of 2–8 at the N-terminal of Snail protein, was deleted in pcDNA3.1-Snail-HA to yield pcDNA3.1-SnailΔSNAG-HA using a site-directed mutagenesis kit (Stratagene). For construction of the luciferase reporter for CDH1 promoter, a ~400 bp sequence spanning the nucleotide positions-365 to +48 of CDH1 gene was amplified from human genomic DNA and cloned into pGL3-basic (Promega). For construction of the luciferase reporters for GLDC promoter, a ~1 kb sequence spanning the nucleotide positions-962 to +55 of GLDC gene was amplified from human genomic DNA and cloned into pGL3-basic (Promega) to yield the wild-type reporter GLDC-luc. The mutated reporter constructs with targeted deletion of either the proximal or the distal E-box motif were generated using site-directed mutagenesis and designated as E1 mut-luc and E2 mut-luc, respectively.For construction of the Snail expression vectors, the full-length human Snail cDNA was fused with a 3’ in-frame HA epitope tag sequence and was cloned into pInducer20For establishment of A549-Snail cell line, the lentiviral particles were produced by cotransfection of pInducer20-Snail-HA with pMD2.G and psPAX2 packaging vectors into 293FT cells, and were then infected into A549 cells. The infectants were selected in the media containing 500 μg/ml of G418, and the drug-resistant cell clones were screened for doxycycline-inducible overexpression of Snail-HA protein using Western blotting.For knockdown of GLDC, the siRNA oligos (Origene) was transfected into A549 cells using Lipofectamine RNAiMAX reagent according to the vendor’s specifications.Cells were washed twice in phosphate-buffered saline (PBS) and lysed in the hypotonic lysis buffer . Protein concentrations were determined with the Bradford reagent (Bio-Rad). Cell lysates (40 μg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred onto nitrocellulose filters. The blots were saturated with 5% nonfat milk and probed with antibodies against GLDC , AMT , DLD , GAPDH , E-Cadherin , N-Cadherin , Snail , or HA . Following a wash with PBST (PBS containing 0.1% Tween 20), the blots were incubated with peroxidase-coupled goat anti-rabbit immunoglobulin G . The immunolabeled protein bands were detected by enhanced chemiluminescence method (Perkin Elmer).Cells grown in the chamber slides were fixated with 4% paraformaldehyde, and subsequently permeabilized with 0.2% Triton X-100. After saturation with 3% Donkey serum, cells were incubated with the primary antibody against HA  or E-Cadherin , and then incubated with the TRITC-conjugated Donkey anti-rabbit  or FITC-conjugated Donkey anti-mouse  secondary antibody. The immunofluorescence was visualized in the cells using confocal microscopy (Olympus).,23 To measure cell proliferation, equal number of cells were seeded on 6-cm plates, and were subjected the indicated treatment. The number of cells was then counted using a hemocytometer at the indicated time. To measure cellular capacity for migration, the Boyden chamber matrigel migration assay was performed. In brief, cells were seeded in the serum-free media in 8 μm Boyden chamber (Corning) coated with Matrigel (BD Bioscience), and the chamber was placed inside a bottom plate containing complete growth media as migratory attractants. Following 24 h incubation, invasive cells retained on the filter were fixed with 1% paraformaldehyde and stained with 1% crystal violet, and quantified under a microscope.Cell proliferation and migration were examined as previously described. Data are presented as the relative abundance of each individual metabolite in reference to the level of the control cells without Snail induction.Cell metabolism was quenched by flash freezing of the cells in liquid nitrogen. Cellular metabolites were extracted in 80% aqueous methanol, and then quantitated using the LC-MS/MS methods as previously described.Total cellular RNA was isolated using the Trizol reagent (Invitrogen). To profile the differential expression of mRNAs in the A549-Snail cells with or without Snail induction, cDNA libraries compatible for Illumina sequencing were prepared by using the QuantSeq 3’ mRNA-seq Reverse (REV) Library Prep Kit (Lexogen) according to the manufacturer’s instruction. The resultant cDNA libraries were assessed using a TapeStation (Agilent), and subjected to 100 bp single-end sequencing using the Illumina HiSeq 2500 system at the Wayne State University Applied Genomics Technology Center. The sequencing reads were mapped to the human reference genome, and the differential gene expression was determined.T), and normalized to PPIA mRNA level of PPIA or β-actin housekeeping gene. The results were expressed as the fold changes relative to the untreated control, and the standard deviation (SD) bar graphs were plotted. The PCR primers used are listed in To determine the mRNA expression using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), cDNA libraries were constructed by random priming using the SuperScript III First-Strand Synthesis System (Invitrogen). The synthesized cDNA was used as a template for qRT-PCR with SYBR Green qPCR Master Mixes (Thermo Scientific) on an Mx3000P cycler (Stratagene). All mRNA levels were determined as the delta-delta threshold cycle (ΔΔCRenilla reniformis luciferase control vector Prl-CMV (Promega) into 293FT or A549-Snail cells using Lipofectamine 2000 (Invitrogen). The levels of Snail proteins were increased either by cotransfection of the 293FT cells with pcDNA3.1-Snail-HA or pcDNA3.1-SnailDN7-HA vector, or treatment of A549-Snail cells with doxycycline. Dual luciferase assays were performed according to the manufacturer’s instructions (Promega).Firefly luciferase reporter plasmids (2 μg each) were cotransfected with 0.04 mg of A549-Snail cells were treated with doxycycline to induce the expression of Snail-HA protein for 2 days, and were then cross-linked in formaldehyde. The nuclei were isolated and sonicated using conditions resulting in prominent DNA fragments between 500 and 750 bp. Chromatin samples were immunoprecipitated with anti-HA antibody (Santa Cruz) and Protein G sepharose . DNA samples purified from the precipitated chromatin were used as PCR template. Primers for PCR reactions were: GLDC promoter: 5’-GCTGGGAAGAGGGTAGGAAG-3’ (forward) and 5’-GCGCTCAACCAAGACACTC-3’ (reverse); CDH1 promoter: 5’-GTGAACCCTCAGCCAATCAG-3’ (forward) and 5’-TCACAGGTGCTTTGCAGTTC-3’ (reverse); and GAPDH promoter: 5’-CGGCTACTAGCGGTTTTACG-3’ (forward) and 5’-GCTGCGGGCTCAATTTATAG-3’.t-test.Statistical analyses were performed with GraphPad Prism 6 software . Data are presented as means ± SD statistical significance between two groups was determined by unpaired in vitro model for EMT. As shown in To recapitulate the EMT process in tissue culture, we engineered the lentiviral-mediated, doxycycline (Dox)-inducible overexpression of Snail in the A549 cells, a lung carcinoma cell line that has been widely used as  We observed that Snail induction had negligible effects on the levels of the metabolites of the tricarboxylic acid (TCA) cycle [Wondering if Snail induction triggers metabolic alteration, we measured the levels of numerous metabolites in the A549-Snail cells using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis. We observed that Snail induction reduced the levels of the nucleotide biosynthetic intermediates . Warburg As GLDC is a metabolic oncogene previously reported in lung cancer, and it locates on the serine/glycine metabolic shunt that diverts the glycolytic metabolites to the major anabolic pathways, we characterized how Snail regulates the expression of GLDC in detail. We first examined the effects of Snail overexpression on the levels of the transcripts of each GCS components in two clones of the A549-Snail cells. We observed that overexpression of Snail significantly reduced the level of the GLDC transcript without affecting those of the other GCS components including AMT, GCSH, and DLD indicating that Snail selectively suppresses GLDC, the rate-limiting component of GCS [GLDC gene in lung cancer cells.We then investigated the molecular basis for which Snail induces metabolic changes. As Snail is a transcription repressor, we performed gene expression profiling to identify the metabolic enzyme transcripts that decreased in the Snail-overexpressing cells. We found that the transcript encoding GLDC was among the top hits that decreased in response to Snail induction in the A549-Snail cells. GLDC is the rate-limiting component of the glycine cleavage system (GCS), a mitochondrial multimeric enzyme complex that breaks down glycine and concomitantly synthesizes a one-carbon unit, which is a crucial anabolic intermediate required for cell proliferation.t of GCS . Consistt of GCS . Thus, o We, therefore, tested whether an inverse relationship between Snail and GLDC expression exists in the cells exposed to TGF-β. As expected, treating the cells with increasing amounts of TGF-β led to concomitant upregulation of Snail and downregulation of GLDC at both the transcript and protein levels in dose-dependent manners [Transforming Growth Factor (TGF)-β is a cytokine that actively induces Snail and thus promotes EMT in A549 cells. manners . As cytoGLDC. As Snail belongs to a family of sequence-specific transcription repressor that recognizes the E-box motif with a consensus of 5’-CAGGTG-3’,,6 we searched for the possible Snail binding site(s) in the sequence of human GLDC gene. We found two perfectly matched E-box motifs in the promoter of GLDC that located at nucleotide positions −23 to −18 and −303 to −298, respectively, relative to the transcription start site. Multi-sequence alignment demonstrated that the proximal motif (positioned at −23 to −18) but not the distal one (positioned at −303 to −298) conserved in two rodent species [in vivo, we induced the expression of the Snail-HA protein in A549-Snail cells and performed the chromatin immunoprecipitation (ChIP) assay using anti-HA antibody. The ChIP analyses demonstrated that Snail interacted with the promoter of GLDC as well as the promoter of CDH1, a bona fide Snail target, and not with the promoter of GAPDH, an irrelevant negative control gene [GLDC E-box motifs only has 278 nucleotides that were not separable in the ChIP assay, we conclude that Snail interacts with at least one of the E-box motifs in the promoter of GLDC in A549 cells.We then investigated the molecular mechanism of how Snail represses the expression of  species . To testrol gene . As the GLDC promoter, we generated a set of luciferase reporters that carry either the wild-type or the mutated GLDC promoter [ΔSNAG, inhibited the promoter of GLDC as well as the promoter of CDH1 in a dose-dependent manner [GLDC promoter to Snail [GLDC.To examine whether and how Snail represses the activity of promoter . The luct manner . In addito Snail . TogetheGLDC reduced the expression of GLDC protein by ~ 80%, and significantly decreased the proliferation and increased the migration of A549 cells [To examine how reduced expression of GLDC influences the cell behavior, we knocked down GLDC in A549 cells using small interference RNA (siRNA). The data demonstrated that transfection of the siRNA oligos specific for 49 cells . Thus, r–9 it is interesting to address whether these events are functionally coordinated. Concerning to tumor biology, it is mechanistically insightful and therapeutically useful to elucidate how metabolic reprogramming couples with the EMT program, which could help for developing novel therapeutic approaches against metastasis and chemoresistance through disrupting the metabolic basis for tumor plasticity.,29 In this study, we examined how gain of Snail, a prototypic EMT transcription factor and an oncogenic protein, affects the fluctuation of the backbone metabolic pathways in lung cancer cells, and further identified that GLDC is a novel metabolic component integral to the Snail-induced EMT program.As metabolic reprogramming and cellular plasticity are common features for the adaption of cells to the environment, We predict that the sharp divergence of the alterations of the glycolytic metabolites under the condition of Snail induction may reflect a significant rearrangement of a metabolic branch that connects with the glycolytic pathway at the interface of GAP and 3PG. Indeed, the serine/glycine metabolic shunt, a vital connector between glycolysis and key anabolic pathways, is right started using 3PG as metabolic substrate. Thus, our metabolite analysis predicted that Snail significantly rearranges the serine/glycine metabolism in the EMT commitment of the lung cancer cells.To robustly examine the metabolic fluctuation in the process of EMT, we established an inducible EMT tissue culture model through ectopic expression of Snail under the control of doxycycline-inducible promoter in the lung cancer A549 cells. We demonstrated that induction of Snail robustly activated EMT, as manifested by the appearance of the critical molecular and functional signatures of EMT, including concomitant reduction of E-cadherin and elevation of N-cadherin, as well as increase of migration and decrease of proliferation in the Snail-expressing cells. Using targeted metabolomics analysis, we probed the influence of Snail overexpression on the metabolite fluctuation of the backbone metabolic pathways, those including nucleotide synthesis, glycolysis, and TCA cycle. We observed that the levels of nucleotide biosynthetic intermediates were uniformly decreased in the Snail-expressing cells, suggesting the metabolic demand for DNA synthesis possibly decreased when the cells commit to EMT and slow down proliferation. In contrast, the levels of TCA cycle metabolites remained unchanged in the Snail-expressing cells, indicating that mitochondrial oxidation is likely bioenergetically necessary for supporting the EMT-committed cells. Interestingly, a biphasic response in the levels of the glycolytic metabolites to the Snail induction was observed. After Snail induction, dividing at the interface between GAP and 3PG, the levels of the upstream glycolytic metabolites were decreased, yet the levels of the downstream ones remained unchanged. At present, we do not fully understand the logic behind this clear split in the alterations of the glycolytic metabolites in response to Snail induction. However, a clue may lie in the fact that in addition to feeding the TCA cycle, glycolysis diverts its metabolic flux to specific key branching pathways including the pentose phosphate pathway and the serine/glycine metabolic shunt.GLDC gene mediates the binding and thereby the inhibition of Snail to the GLDC promoter. Also, we demonstrated that treatment of cells with TGFβ, a canonical EMT-inducing cytokine, led to concomitant induction of Snail and suppression of GLDC in dose-dependent manners. Thus, we demonstrated that this newly identified Snail/GLDC axis is an integral component of the EMT program. GLDC is the rate-limiting enzyme of GCS that catabolizes glycine for the synthesis of critical anabolic substrate – the folate-mediated one-carbon unit. GLDC is a metabolic oncogene sufficient to drive the proliferation of cancer cells. For metabolic networking, GLDC, as a bottleneck enzyme, is essential for cancer cells to sustain the metabolic flux through the serine/glycine shunt. In this sense, it is plausible that GLDC functions as a regulatory checkpoint that influences the activity of the serine/glycine branch and in turn, the distribution of glycolytic flux in response to Snail induction. Indeed, the functional significance of this newly identified Snail/GLDC axis is further supported by the observation that reduction of GLDC is sufficient to induce a switch of cellular phenotype from proliferation to migration in both the present [ studies.Interestingly, our gene expression profiling identified that the expression of GLDC, a key enzyme of the serine/glycine metabolic branch was significantly reduced in response to Snail induction. Our molecular characterization further defined that an evolutionarily conserved proximal E-box motif in  present  and prevIn summary, the present study has identified that GLDC is a novel metabolic target directly controlled by Snail. It further suggests that the Snail/GLDC regulatory axis, as an integral part of the EMT program, poses a significant impact on the diversion of glycolytic flux to biosynthetic pathways, and thereby the arrangement of global anabolic/catabolic activities in the process of EMT. However, the functional importance of this novel regulatory link in the context of tumor biology needs further investigation.1


Retinal vein occlusions (RVOs), including central retinal vein occlusions (CRVOs) and branch retinal vein occlusions (BRVOs), are a common cause of morbidity in elderly patients. We present the case of a healthy 74-year-old female patient who initially presented with blurry vision in her left eye in the setting of a symptomatic COVID-19 infection. She was diagnosed with a branch retinal vein occlusion that did not immediately require treatment. Three months later, she again presented with worsening vision and was found to have cystoid macular edema (CME) secondary to the vein occlusion, thus was treated with an intravitreal dexamethasone implant. This case serves to highlight the growing evidence of increased thromboembolic risk associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the possible correlation of COVID-19 infections with ocular pathology, including retinal vein occlusions. Despite the mounting collection of scientific research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, the pathogenesis of this severe acute respiratory syndrome (SARS) virus continues to be poorly understood. Among those treating COVID-19 patients, the SARS-CoV-2-induced cytokine storm is a recognized cause of morbidity and mortality and a growing collection of data supports COVID-19 predisposing patients to thromboembolic events with a multitude of systemic adverse effects, including pulmonary emboli and deep vein thromboses [In the realm of ocular disease, retinal findings such as cotton wool spots and retinal hemorrhages have been found in otherwise healthy COVID-19 positive patients , leadingRetinal vein occlusions (RVOs) are a widespread cause of morbidity in elderly patients and are known to be associated with thromboembolic events. We report a 74-year-old individual diagnosed with a branch retinal vein occlusion (BRVO) while symptomatic with COVID-19.A 74-year-old, Caucasian female with a past medical history of well-controlled hyperlipidemia presented to the emergency department with worsening vision of the left eye. Approximately three months prior, the patient had been diagnosed with SARS-CoV-2. While symptomatic from COVID-19, she developed blurry vision in the left eye and was diagnosed with a branch retinal vein occlusion (BRVO) that did not require treatment at that time. The exact time of COVID-19 onset to retinal vascular event is not known, however, our patient was symptomatic with COVID-19 when she became symptomatic from a change in her vision in her left eye.On this examination three months later, the vision in the right eye was 20/25, while the vision in the left eye was best corrected to 20/50 at distance. Intraocular pressures were normal at 11 and 12 millimeters of mercury (mmHg) in the right and left eyes, respectively, measured with a TonoPen tonometer. Anterior examination was within normal limits for age, with nuclear sclerosis present in both eyes. Dilated fundus examination showed a choroidal nevus peripherally in the right eye. In the left eye, tortuous vessels with intraretinal hemorrhages and microaneurysms were present in the inferior-temporal quadrant of the retina with macular edema centrally, in addition to a peripheral choroidal nevus with overlying drusen and without surrounding subretinal fluid. Imaging was obtained for further evaluation and a diagnosis of BRVO with cystoid macular edema (CME) of the left eye was confirmed . Alternatively, intravitreal anti-vascular endothelial growth factor (Anti-VEGF) therapy could have been implemented. She continued to follow closely with her primary care physician to ensure that her hyperlipidemia continued to be well controlled, as well as be monitored for blood pressure and glucose control. A further hypercoagulability work-up was not done. Due to the BRVO being symptomatic three months after its initial presentation and the patient no longer being symptomatic from the SARS-CoV-2 virus, systemic treatment, such as an anticoagulant, was not initiated.Retinal vein occlusions (RVOs) are a common cause of morbidity in elderly patients. RVOs include central retinal vein occlusions (CRVOs) and branch retinal vein occlusions (BRVOs) and may cause blurry vision or severe loss of vision, depending on the location and severity of obstruction. Causes of visual decline in BRVOs include macular edema, ischemia, neovascularization, and hemorrhage. A multitude of risk factors have been indicated in RVOs including atherosclerotic disease, hypertension, diabetes mellitus, open angle glaucoma, connective tissue diseases, thrombophilia, and other causes of hypercoagulability. In our patient, her development of a BRVO of the left eye coincided with her diagnosis and symptoms of COVID-19.Coronaviruses (CoVs) affecting ocular tissue is not a novel discovery. Seven of the CoVs currently infect humans and, while most cause respiratory tract infections, three of the seven have also been detected in ocular tissue or shown to cause ocular symptoms, including the current SARS-CoV-2 virus . HCoV-NLWhile reasonable to consider a retinal vasculitis, such as that induced by the feline CoV, causing the retinal findings in our patient, rather than a thromboembolic event that we propose, other findings of a retinal vasculitis were not evident at three months after the initial BRAO on the fluorescein angiography. However, a retinal vasculitis rather than a thrombotic event should still be considered on a differential for a COVID-19 patient with ocular changes.Ribonucleic acid (RNA) from the SARS-CoV-2 virus has been detected in many diverse ocular specimens, including in the tears and conjunctival secretions of patients with coronavirus conjunctivitis . FurtherRecent publications have recognized retinal hemorrhages and cotton wool spots in COVID-19 positive patients without other retinal disease . SeveralThis is the first reported case, to our knowledge, of a branch retinal vein occlusion coinciding with a diagnosis of COVID-19.In this case report, we present a branch retinal vein occlusion occurring in association with the diagnosis of COVID-19. Admittedly, the relationship between COVID-19 and RVOs has yet to be completely elucidated. It is fully possible that due to the high numbers of COVID-19 cases worldwide at this time, the simultaneous diagnoses of COVID-19 and retinal vein occlusions is coincidental, however, with the association of COVID-19 and thromboembolic phenomenon, there is also reason to suspect that this is more than a coincidence. Further studies should be performed to better understand the correlation between COVID-19 and retinal pathology, including retinal vein occlusions.


The warning by the Italian Medicines Agency on the high shortage of azithromycin in the country in January 2022 represents a paradigmatic lesson learnt from improper use of antibiotics during COVID-19 pandemic. On 13 January 2022, the Italian Medicines Agency  warned about a high shortage of azithromycin in the country, mainly due to its improper use against COVID-19. The difficulty in finding the molecule seems to be related to an excessive prescribing in the last two months, coinciding with the rapid increase in cumulative incidence of SARS-CoV-2 cases sustained by the B.1.1.529 variant (Omicron Variant of Concern) [The COVID-19 pandemic has led to a race towards treatments against the disease. Among the possible medications that have been proposed for improving its prognosis, there is no recommendation for treating COVID-19 with antibiotics. In particular, extensive evidence has demonstrated that azithromycin is not associated with a protective effect against the infection and COVID-19 outcomes, despite being initially suggested as one such possible treatment by virtue of potential interference with SARS-CoV-2 infectivity [infodemic, with people constantly updated with fake news [In Italy, patients can obtain antibiotics in community or hospital pharmacies under prescription by a doctor. It can be thus inferred that azithromycin hoarding represents a symptom of excessive prescribing by healthcare providers, virtually attributable to convincing solicitation by COVID-19 patients or those who are afraid of being infected with SARS-CoV-2. Indeed, COVID-19 is a perfect storm for ake news ,4.The principal outcome of the inappropriate prescribing is the delayed care of other infections, such as bacterial community-acquired pneumonia, acute bacterial exacerbations of chronic obstructive pulmonary disease, non-gonococcal or gonococcal urethritis and cervicitis, and other bacterial infections . For aziIn the case of antibiotics, organisms’ drug resistance is a key additional concern, one of the biggest challenges to public health and healthcare. In Italy, according to the AIFA National Report on Medicines Use for 2020, antibiotics are among the most commonly prescribed drugs, with more than 20 daily defined doses consumed per 1000 persons per day; around 4 in 10 people have received at least one antibiotic prescription ,6. As a The rapid spread of the COVID-19 pandemic caught off guard healthcare systems worldwide, with enormous impact and disruption of healthcare systems and practices . Indeed,Taken together, risks associated with indiscriminate and improper antibiotic use—exacerbated by the COVID-19 pandemic—call for informative intervention targeting providers, including primary health care physicians and pharmacists, as well as the general public.Specifically, Health and Public Health Authorities should promote specific initiatives tending to improve awareness and knowledge towards antimicrobial resistance and appropriate antibiotic use. These may include the introduction of mandatory programs in medical degree and trainings; evidence-based initiatives tending to reduce the level of inappropriate prescriptions; and the setting up of data flows to adequately inform stakeholder on antimicrobial agent use and resistance through digital health facilities ,12.syndemic of COVID-19 and antimicrobial resistance.In a nutshell, while we will continue to struggle with COVID-19 pandemic and its consequences, it is necessary to learn the lesson from the case of azithromycin in Italy and promote urgent global public efforts to tackle an irredeemable


RNAs are a class of molecules and the majority in eukaryotes are arbitrarily termed non- coding transcripts which are broadly classified as short and long non-coding RNAs. Recently, knowledge of the identification and functions of long non-coding RNAs have continued to accumulate and they are being recognized as important molecules that regulate parasite-host interface, parasite differentiation, host responses, and disease progression. Herein, we present and integrate the functions of host and parasite long non-coding RNAs during infections within the context of epigenetic re-programming and molecular crosstalk in the course of host-parasite interactions. Also, the modular range of parasite and host long non-coding RNAs in coordinated parasite developmental changes and host immune dynamic landscapes are discussed. We equally canvass the prospects of long non-coding RNAs in disease diagnosis and prognosis. Hindsight and suggestions are offered with the aim that it will bolster our understanding for future works on host and parasite long non-coding RNAs. Genomic sequencing has continued to reveal an increasing number of transcripts termed non-coding RNAs (ncRNAs) due to the hypothesis that ncRNAs have no protein-coding potential. Meanwhile, advances in research are regularly giving evidence to show that some ncRNAs have protein-coding potentials , and theMoreover, lncRNAs may be functional during the development of organisms, cell proliferation, motility, inflammation, and gene regulation during host-pathogen interactions . These fPlasmodium falciparum, Cryptosporidium, Eimeria necatrix, and Toxoplasma gondii), Kinetoplastida , Parabasalia , and Helminth . This review seeks to expand and consolidate on the concept of RNAs in parasitism  by interfering with transcription, stability, or translation of gdv1 mRNA  suggests lncRNAs might play crucial roles in the rapid transition and adaptation of adult S. mansoni to a parasitic mode of life in mammalian host  trimethylation mark has been proposed as the basis for n silent . The genormation . This prPlasmodium parasite expressing episomal antisense lncRNAs than un-transfected or mock-plasmid transfected parasites  knock-out enhanced the expression of antigenic proteins by histone H3 lysine 36 trimethylation (H3K36me3) of var genes. Jiang et al. further revealed that var gene in wild type P. falciparum had low levels of H3K36me3 and that silent var genes displayed high H3K36me3 methylation at the same exonic region to indicate a positive correlation between PfSETvs-dependent methylation and var lncRNA silencing  when infected with T. gondi high- and low-virulent strains  as communication channels that vehiculate the transfer of ncRNAs during host-parasite interactions. There has been demonstration of inter-communication between y ncRNAs . It is ey ncRNAs   infection in myDD8 (wild type) and myDD8-/- (knock out) mice macrophages, siva1–205 and nfkb1–210 exhibited greater expression in myDD8 than Myd88-/- macrophages when infected with T. gondii RH  traits. LncRNAs are activators/suppressors of host immune regulatory cascades and could be important tools for diagnosing parasitic diseases. Although there are existing gaps in our understanding of lncRNA functional threshold in parasitic infections, especially in helminths, it is in no doubt that these RNA molecules are paving the way for better understanding of parasite development and parasite-host crosstalk JC proposed the theme and provided guidance. JO organized the paper frame and drafted the manuscript. BO read the manuscript. All authors contributed to the article and approved the submitted version.Key Technologies Research and Development, R&D program, 2017YFD0500403JC.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


COVID-19 altered research in Clinical and Translational Science Award (CTSA) hubs in an unprecedented manner, leading to adjustments for COVID-19 research.CTSA members volunteered to conduct a review on the impact of CTSA network on COVID-19 pandemic with the assistance from NIH survey team in October 2020. The survey questions included the involvement of CTSAs in decision-making concerning the prioritization of COVID-19 studies. Descriptive and statistical analyses were conducted to analyze the survey data.P < 0.01). Likewise, institutions with internal funding were 3.88 times more likely to establish COVID-19 dedicated research . CTSAs were instrumental in securing funds and facilitating establishment of laboratory/clinical spaces for COVID-19 research. Workflow was modified to support contracting and IRB review at most institutions with CTSAs. To mitigate chaos generated by competing clinical trials, central feasibility committees were often formed for orderly review/prioritization.60 of the 64 CTSAs completed the survey. Most CTSAs lacked preparedness but promptly responded to the pandemic. Early disruption of research triggered, enhanced CTSA engagement, creation of dedicated research areas and triage for prioritization of COVID-19 studies. CTSAs involvement in decision-making were 16.75 times more likely to create dedicated diagnostic laboratories (95% confidence interval [CI] = 2.17–129.39; The lessons learned from the COVID-19 pandemic emphasize the pivotal role of CTSAs in prioritizing studies and establishing the necessary research infrastructure, and the importance of prompt and flexible research leadership with decision-making capacity to manage future pandemics. La Peste (the Plague). Taking place in North Africa in the 14th century. Camus describes disease that swept across Europe and North Africa, killing almost 50% of the population. Camus wrote, “They fancied themselves free, and no one will ever be free so long as there are pestilence” .P < 0.05). Likewise, early disruption from January to March 2020 was 8.1 times more likely to being associated with the creation of a dedicated COVID-19 research area  as compared to the CTSAs not affected until after March 2020. Among all variables selected for the multivariate analyses, institutions with CTSA’s involvement in the institutional decision-making were 16.8 times more likely to create a COVID-19 diagnostic laboratory than those without CTSA’s involvement in the decision-making process. Among all funding sources, institutional funding was associated with the creation of a COVID-19 research area . Federal funding was significantly associated with the creation of a new diagnostic laboratory . When CTSAs were involved, 52.9% institutions hired new staff while no hires were reported in 47.1% when CTSAs were not directly involved in the COVID-19 response. Involvement of CTSA leaders in institutional decision-making was significantly associated with hiring new personnel for COVID-19-related studies . Fifty-two (86.7%) of the institutions reported establishment of a feasibility committee to prioritize review and performance of COVID-19-related studies. Active involvement of CTSA leaders in institutional decision-making was significantly associated with the formulation of such committees .A total of 64 CTSA hubs were invited to participate in the study. Of these, 60 (90% response rate) participated in the study. The response rate to the survey was very high, suggesting that the hubs did find this topic of great value. Our analysis indicates that early disruption of research  and institutional funding for COVID-19 were associated with the creation of a dedicated COVID-19 research area  prioritization of COVID-19-related research; 2) rapid startup; 3) whether previously approved human subjects research projects should continue; (4) facilitating information sharing and removing barriers; (5) securing funding; and (6) collaborating with other institutions and provide CTSA resources. Responses from 60 CTSAs focused on how they were involved in the decision-making process are presented in Table  rapid st whether n = 26) reported having Biosafety Level (BSL)-3 facilities ready for use; but for others, such facilities had fallen into disuse. BSL-2 facilities could be rapidly enhanced to BSL-2+ facilities but were still insufficient for handling respiratory tract samples. Many institutions re-equipped their BSL-2+ facilities with the equipment to process and store samples for SARS-CoV-2 biorepository specimens but did not attempt to modify them to BSL-3 facilities. Discrepancies in requirements for similar procedures based on whether they were labeled research (NIH required) versus clinical (institution and CAP influenced) was a source of confusion. The CTSA hubs also participated in other key activities, such as feasibility assessments and drafting policies and standard operating procedures to conduct research.A few CTSA sites (12%) had begun planning even before the first US case had been identified. Most hubs (86%) were engaged in the decision-making process at a leadership level for research. Some institutions (Our data showed that human resources (HR) in many institutions allowed all interviewing and hiring to be performed with virtual technology. Some institutions used agencies and clinical research organizations to assist with staffing solutions, such as travel nurses, to fill time-sensitive research positions. In most institutions, COVID-19-related hires were fast-tracked with expedited on-boarding processes, including rapid hospital credentialing. Research staff from non-COVID-19 studies were often quickly redeployed to COVID-19 studies. Within divisions, staff was quickly shifted between studies, including redeployment from outpatient studies to inpatient trials, and then to outpatient vaccine studies. In some institutions, HR initiated a matching program between COVID-19 trial personnel needs and individuals who had been furloughed based on lack of work, allowing those at risk of termination to migrate to COVID-19 studies. Others pooled the coordinators, although sharing was not always easy due to competition for subjects among investigators conducting research that overlapped in inclusion criteria.Despite efforts to rapidly hire staff for studies, some institutional Human Resources departments had difficulty navigating conflicting institutional policies regarding hiring and furloughs that differed for university-wide compared to hospital/medical research personnel. During the first 4 months of the pandemic, delays in dissemination of campus-wide policies regarding pay, leave of absence, and benefits slowed hiring. One institution suggested that during a pandemic, the School of Medicine should have been exempted from campus-level reviews to allow for timelier hiring for critical positions. Although some institutions awarded premium pay or compensatory time for evening and weekend work, many felt that compensation strategies failed to match the COVID-19 research needs.n = 24), whereas others reviewed all COVID-19-related studies (n = 12) that included databases and survey studies. These served as triage to limit duplication, optimize resources, and align with institutional priorities, patient availability, and staff capacity.In this study, 52 (86.7%) of the institutions reported establishment of a feasibility committee to review COVID-19-related studies to facilitate best practices. Involvement of the CTSA in the decision-making at a leadership level was significantly associated with the formulation of such committees. The scope of these committees also varied in that some reviewed therapeutic studies only  and realized a keen interest among investigators to conduct COVID-19 research.It is our view that recruitment of patients with COVID-19 who were eligible for a trial came with challenges. For outpatient clinical trials, often no clear relationship existed between the subject and the institution, and as such, an adequate method to contact these patients had to be developed. For inpatient trials, obtaining approval of the treating health care team became logistically challenging as the treating physician changed frequently in pandemic situations. Clinicians, patients, and families were not aware of the clinical trials; as such, communication was key, and an information sharing process needed to be developed so that all patients had the opportunity to participate if they desired. Some sites reported that it was highly recommended that clinicians advise patients of trial opportunities or at least be aware of patients’ interest in participating in clinical trials in general and guide them . Howevera priori superior to the other and any potential benefit of a studied therapy might only benefit future patients [Given the abundance of clinical trials for COVID-19, there was potential for competing trials  . This rapatients , 21 or aAlthough IRBs are charged with protecting the rights of the persons between the arms of a trial, some COVID-19 committees did not believe that all open studies at a single institution could be equipoised. Changes were made to the regulatory process of IRB review in some institutions with a goal of reducing administrative barriers without compromising human subjects protections while facilitating urgent research (see the article on changes in IRB during the COVID-19 pandemic in this issue) . The choCreation of dedicated areas for clinical COVID-19 research: Many institutions (50%) created centrally located spaces for clinical COVID-19 research  and assent of the treating clinician. COVID-19 offered several challenges to the conduct of research. Since some trials excluded some patients treated with therapies that had become standard of care , there was little time to identify the subjects. Where competing trials existed, potential subjects needed to be educated on the opportunities available while maintaining equipoise in recruiting trials. Clinicians providing care for patients ill with COVID-19 may not have participated in the selection of trials, and therefore, may not agree with investigators on which, if any, trial might be of greatest interest or benefit to their patients. Programs generally engaged a multidisciplinary panel composed of infectious diseases and pulmonary specialists, intensivists, and ad hoc trialists to assist in defining treatment strategies and allocating patients into enrolling studies. In order to optimize patient’s allocation into trials, the mission of such a patient treatment panel should be to (1) guarantee that standard of care is applied to every patient as per institution’s policy to prevent heterogeneity between groups, (2) explore research opportunities using a hierarchy , (3) adapt strategies based on daily literature reviewed through PubMed and national scientific societies, and (4) limit the number of trials to assure feasibility and equipoise with emphasis on coordination and communication between the multiple stakeholders.COVID-19 is not a short-term event and is likely not the last pandemic that we will encounter. What has become clear is that there was a need to have a state of readiness to meet the challenge. The CTSA network provides the ideal infrastructure to serve that purpose and should be fully integrated into university and hospital leadership decision-making. Institutions created various forms of a centralized review process to identify approaches to research that are best suited for their community. Although the optimal method and structure of an oversight committee may not be well established, a coordinated review process is required to make the best use of constraint resources. Constant review of evolving knowledge must be used to improve and refine processes.As traditional methods to prevent the spread of infection  were not been fully adopted, it appears that therapeutic interventions will be needed to tame the pandemic. Clinical trials designed and implemented with scientific rigor must be conducted without distraction from political background noise. We must have the ability to identify eligible subjects and offer them the opportunities available with the premise that we will adhere to Good Clinical Practice .The future should focus on preparedness and on not repeating the errors. The CTSA network is well positioned to play a pivotal role in this goal, as it provides the infrastructure to rapidly mobilize in response to such emergencies and should be supported and utilized to protect the health of the nation.