Citrobacter rodentium, the model for enteropathogenic Escherichia coli, Vibrio cholerae, Shigella species, Salmonella enterica species, and Clostridioides difficile. A comparison of what is known about the humoral immune responses to these pathogens reveals considerable variance in specific features of humoral immunity including establishment of high affinity, IgG class-switched memory B cell and long-lived plasma cell compartments. This article suggests that such variance could be contributory to persistent and recurrent disease.Bacterial enteric pathogens individually and collectively represent a serious global health burden. Humoral immune responses following natural or experimentally-induced infections are broadly appreciated to contribute to pathogen clearance and prevention of disease recurrence. Herein, we have compared observations on humoral immune mechanisms following infection with  Enteric pathogens rapidly activate host innate and adaptive defense mechanisms upon infection. These mechanisms include activation of innate immune cells, their production of cytokines and chemokines, and antigen presentation necessary for the recruitment of inflammatory cells , and iniVibrio cholerae and Clostridioides difficile secrete enterotoxins that mediate the pathogenesis and the inflammatory responses which often leads to tissue injury and loss of intestinal barrier integrity. Other bacteria such as Salmonella and Shigella overcome the intestinal barrier through invasion via the microfold cells (M cells) which are specialized epithelial cells that overlie Peyer's Patches of the intestine . C. rodentium infection is a well characterized murine model of infectious colitis  similarly to EPEC and EHEC  is necessary for limiting bacterial colonization and promoting clearance. MyD88-dependent Toll-like receptor 2 and 4 (TLR2 and TLR4) signaling mediates production of pro-inflammatory cytokines such as Tumor necrosis factors (TNF), and recruitment of neutrophils, macrophage and innate lymphoid cells (ILCs) dependent on Interleukin 6 and 23 (IL-6 and IL-23)  –28. At td IL-23) –31. ThisC. rodentium is mediated by CD4+ T cell and B cell responses  cells. Following C. rodentium infection, Tfh cells expand in the mesenteric lymph nodes and spleen, and secrete IL-21 and IL-4  has been shown to mediate transport of IgG from the blood to the intestinal lumen to mediate defense against trophils . The FcRtrophils –41. Thisand IL-4 . Other Cesponses –46. The odentium , 48. CD4ffectors .Vibrio cholerae, a highly motile Gram-negative facultative bacteria  which consists of two different subunits (A and B). Cholera toxin B binds to intestinal brush border cells leading to the endocytosis of subunit A which targets adenylyl cyclase and increases cyclic adenosine monophosphate (cAMP) levels. The increase in cAMP causes excessive secretion of chloride ions which results in the accumulation of water in the gut  and efflux pumps, and by inhibiting their binding . These memory B cells induce a robust and rapid recall response upon reinfection and play a significant role in a longer-lasting protection against cholera  serovars, such as S. Typhimurium, S Enteritidis, and S. Dublin cause a gastroenteritis which can manifest with acute diarrhea, abdominal pain, fever and vomiting. In some cases, largely in infants, older individuals and the immunocompromised, NTS can cause an invasive infection including bacteremia meningitis and septic arthritis  of the small intestinal epithelium that overlie the Peyer's patches -encoded virulence factors including two distinct TTSSs. The TTSSs transport their effectors in the host cells where they disrupt the actin and microtubule cytoskeletons and the cell membrane to form a membrane ruffle that facilitates engulfment of the bacteria. In the cytoplasm, the bacteria replicate in a vacuole, termed the Salmonella containing vacuole (SCV), that transcytoses to the basolateral membrane and allows invasion of the submucosa  secretes various effectors encoded in the Shigella virulence plasmid  antigens, the invasion plasmid antigens (Ipa), shiga toxins, and other TTSS effectors. These responses are Shigella serotype-specific and there is no cross-protection against infection from different strains  in hospitalized patients resulting in 12,800 deaths and costing the healthcare system more than one billion dollars  ranges from mild diarrhea to pseudomembranous colitis, toxic megacolon, or sepsis. CDI is further complicated by a high and increasing frequency of recurrence; up to 35% of patients will relapse with often more severe disease , flagella, adhesins such the surface layer proteins (SLPs), and hydrolytic enzymes , the surface layer proteins (SLPs), and the adhesins (Cwp66 and Cwp84) have also been shown to induce antibody responses in some patients. However, whether they play a role in the defense against recurrent disease has not been examined  mediates this IgG transport in C. rodentium (Infections with odentium , but litAs regards humoral immunity to enteric bacterial pathogens, future directions may include:More complete investigation of the Th/Tfh, Bmem cell, and plasma cell responses to infection in both animal models and human patients.Determining the mechanisms of antigen presentation in the gut, and how this influences activation and programming of B cell and Th/Tfh responses.Determining whether secreted toxins subvert humoral immunity and the mechanisms by which this is achieved.A systematic genomic approach to obtain in-depth immune profiles of the humoral response to infection and immunization.Single cell BCR sequencing to determine the plasma cell and memory B cell repertoires.C. difficile and Salmonella that are characterized by persistent and/or recurrent disease. This may lead to a better understanding of the mechanisms by which memory B cell and plasma cell responses are limited, and could contribute to determining causes of persistent or recurrent disease.The application of newer technologies to the humoral response to enteric pathogens may be particularly important for infections with SA and ML wrote and edited the article. SA devised and produced the illustrations. All authors contributed to the article and approved the submitted version.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.


The sense of smell essentially contributes to social communication, guides nutrition behaviour and elicits avoidance towards environmental hazards. Olfactory smell impairment may hence entail severe consequences for affected individuals. Compared with sensory loss in other modalities, reduced olfactory function is often unnoticed by those affected and diagnosed late. Those patients seeking help frequently suffer from long-term impairments resulting in reduced well-being and quality of life. The current review provides an overview of aetiology, prevalence and specifics of diagnostics in acquired and congenital olfactory loss and focusses on short- and long-term consequences. Compensation strategies are elaborated, and treatment options are mentioned. Individual characteristics associated with the development of serious mental health impairment are discussed in order to help practitioners identifying populations at risk. The smell of freshly baked bread, salty sea air or strawberries in summer are all sensory impressions enriching our lives. Yet, an intact sense of smell is often not worth mentioning until symptoms of olfactory loss are experienced. Although mainly unconscious for the perceiver, olfaction guides human behaviour in various life domains—nutrition, avoidance of harm, sexual interaction or social communication  ranges from of 3.6 to 5.8% within the general population  conductive dysfunction, (2) sensorineural dysfunction, and (3) central dysfunction . A majoThe lack of ability to subjectively assess olfactory performance may hinder an early diagnosis. As a consequence, the unnoticed impairment may cause exacerbation of side effects. Although a majority of anosmic individuals does not appear to be bothered in daily life, a considerable number of patients declare negative effects of their sensory loss, often manifested in reduced life quality or depressive symptoms  or disease might lead to infection or staying in situations at risk, and indeed, ICA patients reported more frequent accidents in household compared with healthy controls , retronasal qualities of olfaction should not be neglected  is a continuous, dynamic process involving adaptive coping strategies, such as active coping, namely focusing on positive aspects, as well as “realistic emotional acceptance” of the situation  leading to acceptance without judgement (Kabat‐Zinn Although a vast majority of patients with olfactory dysfunction seems to deal well with the olfactory loss, some suffer from concomitant impairment on several life domains. This can result in reduced well-being, the development of depressive symptoms and overall reduced quality of life. Delayed diagnostic procedure, e.g., due to poor sensitivity of self-report, may hinder prompt adequate treatment. When identifying populations at risk, particular attention should be paid to patients exhibiting aggravating tendencies as well as to young women as those report a high individual significance of olfaction in their daily life. Patients with olfactory dysfunction dispose a wide array of compensation strategies which comprise emotion- and problem-focused adjustment to the disorder. In addition, promising treatment options are available aiming at partial restorage of the olfactory function. At the same time, strategies that convey acceptance of the disorder should be promoted, as this reflects a key feature of adaptive coping and is linked to improved mental health outcomes.


Chromosomal instability (CIN) is associated with many human diseases, including neurodevelopmental or neurodegenerative conditions, age-related disorders and cancer, and is a key driver for disease initiation and progression. A major source of structural chromosome instability (s-CIN) leading to structural chromosome aberrations is “replication stress”, a condition in which stalled or slowly progressing replication forks interfere with timely and error-free completion of the S phase. On the other hand, mitotic errors that result in chromosome mis-segregation are the cause of numerical chromosome instability (n-CIN) and aneuploidy. In this review, we will discuss recent evidence showing that these two forms of chromosomal instability can be mechanistically interlinked. We first summarize how replication stress causes structural and numerical CIN, focusing on mechanisms such as mitotic rescue of replication stress (MRRS) and centriole disengagement, which prevent or contribute to specific types of structural chromosome aberrations and segregation errors. We describe the main outcomes of segregation errors and how micronucleation and aneuploidy can be the key stimuli promoting inflammation, senescence, or chromothripsis. At the end, we discuss how CIN can reduce cellular fitness and may behave as an anticancer barrier in noncancerous cells or precancerous lesions, whereas it fuels genomic instability in the context of cancer, and how our current knowledge may be exploited for developing cancer therapies. To maintain a stable genome, at each cell division, a cell must accurately duplicate its genetic material and equally distribute the newly replicated chromosomes in each daughter cell during mitosis. Dysfunctions in one of these processes are the main causes of chromosomal instability (CIN), which is defined as an increased rate of chromosomal changes. As a form of genomic instability, CIN can be manifested as either numerical  or structural  termed numerical chromosome instability (n-CIN) and structural chromosome instability (s-CIN), respectively . The souIn the second scenario, the source of s-CIN is distinguished as premitotic, since structural aberrations mainly result from defects in the cellular processes that directly influence chromosome integrity, such as DNA replication and repair. However, these two types of CIN can be interlinked, and they often coexist in cancer cells .Strikingly, one other hallmark of cancer that, similar to CIN, can also be detected in precancerous and cancerous lesions and has a dual role in restraining and promoting cancer development and progression is replication stress . Recent In the following paragraphs, we analyze the main mechanisms underlying this connection, as well as explain how distinct CIN mechanisms can fuel each other.“Replication stress” is a broad term that refers to any condition that leads to the hindrance of DNA replication forks or the perturbation of replication dynamics. DNA replication is a highly regulated process that takes place during the S phase of the cell cycle, when the cell duplicates its genetic material. It starts at multiple loci called “origins of replication”, from which replication forks emanate and travel in opposite directions to allow for complete replication of the genome. Replication origins are established (licensed) in late mitosis and the G1 phase before being activated at the G1-S phase transition and fired during the S phase, following a spatially and temporally regulated program . The denReplication stress is the main driver of genomic instability in the early preneoplastic stages of tumor development ,18. IndeIt has become clear from these studies that different sources of stress often converge on a limited pool of vulnerable sites that are particularly sensitive to replication stress and, under these conditions, have the tendency to undergo breakage during mitosis . These sTo protect the genetic material from endogenous or exogenous assaults, eukaryotic cells have evolved the DNA damage response (DDR), a complex signal transduction pathway in which DNA damage is detected and activates the core DDR transducer kinases, the ataxia telangiectasia-mutated (ATM), ATM and Rad3-related (ATR) and DNA-PKcs to orchestrate the cell response to specific DNA lesions and alterations to the DNA structure and promote their repair . The celUnder replication stress conditions, the uncoupling of DNA polymerase and MCM helicase at stalled replication forks results in the formation of ssDNA that is instantly protected by an RPA coating, recruiting ATR and its partner ATRIP, which activate downstream effectors to stabilize the forks and activate the checkpoint . ATR sigAdditionally, under unperturbed conditions, ATR and CHK1 are activated to limit dormant origin firing in actively replicating sites. By stabilizing the PP1 phosphatase and RIF1 interaction, ATR and CHK1 prevent the phosphorylation of RIF1 and inhibit the assembly of the CMG helicase at replication origins . InteresThese findings are in agreement with those of a previous study, showing that, in the presence of ATR and CHK1, low levels of under-replicated DNA were able to escape surveillance and continue into mitosis; furthermore, this portion of “leaked” under-replicated DNA was increased after ATR depletion . It is uThe Cimprich laboratory has shown that the orientation of TRCs determines distinct DNA damage responses, with head-on collisions between transcription and replication machineries promoting R-loop formation and ATR activation, and co-directional collisions promoting R-loop resolution and leading to ATM activation, likely as a consequence of DSB formation, at R-loop-prone sequences .Previous studies have also suggested a role for ATM in the replication stress response, where ATM with the MRE11–RAD50–NBS1 (MRN) complex promotes the HR-mediated recovery of stalled or collapsed replication forks ,69. ATM The first hints about a connection between the premitotic and mitotic origins of CIN came from the identification of the role of factors known to be involved in DNA repair pathways, such as Bloom syndrome helicase (BLM) and FA proteins, during mitosis ,76,77,78In 2007, a study from Erich Nigg’s laboratory led to the identification of the Plk1-interacting checkpoint “helicase” (PICH), an SNF2 family ATPase that was found to decorate centromeric thread-like structures connecting sister kinetochores from metaphase to anaphase . In thatThese two studies defined a peculiar class of anaphase bridges, termed “UFB”, which are not stained by conventional DNA dyes, such as DAPI or Hoechst, and are devoid of histones, contrary to bulky chromatin bridges. While bulky anaphase bridges result from chromosome/chromatid fusion, UFBs arise from persistent sister chromatid DNA entanglement.Today, at least five different subclasses of UFBs are known based on their loci of origin and/or underlying DNA structures , as discc-UFBs are the most prevalent type of UFBs that form under physiological conditions as a consequence of the late decatenation and disjunction of centromeric sister chromatid DNA ,92. TopoIn contrast to the c- and r-UFBs observed under normal conditions, CFS-UFBs were discovered following replication stress induction by APH. Under replication stress conditions, under-replicated DNA persists into mitosis, forming CFS-UFBs, and FANCD2 and FANCI mark sister chromatid interlinked structures at their extremities, possibly to orchestrate their resolution by other factors ,76,77. ROn the other hand, HR-UFBs arise as a consequence of double-strand break repair and the subsequent formation of stable Holliday junctions. Persistent HR-UFBs can lead to segregation defects in cells defective for recombination intermediate resolution . HR-UFBsT-UFBs seem to possess some properties of CFS-UFBs, r-UFBs and c-UFBs. T-UFBs can be induced by replication stress (similar to CFS-UFBs), which affects the completion of replication of difficult-to-replicate loci. Impaired telomere replication in Werner’s syndrome (WS)-deficient cells induces the recruitment of the BTR complex to facilitate t-UFB resolution . T-UFBs Finally, another kind of UFB, which depends on RAD51 and is prevented from forming by 53BP1, has been observed in cancer cells and leads to a rupture of the sister chromatid axes followed by chromosome bridging, resulting in gross chromosomal rearrangements . This phDiagram depicting the different types of UFBs. UFBs that arise from persistent double-stranded catenanes are formed at centromeres (c-UFBs) or originate from rDNA loci (r-UFBs). Under-replicated DNA or unresolved replication intermediates induce common fragile site UFBs (CFS-UFBs). Recombination intermediates as Holliday junctions drive homologous recombination UFBs (HR-UFBs). Unresolved replication intermediates and double-stranded catenanes induce telomeric UFBs (t-UFBs) (cc-by ).When UFBs are not properly resolved before anaphase, the sister chromatids remain interlinked, which can lead to chromosome nondisjunction or breakage, visible as micronuclei containing whole chromosomes and/or chromosome fragments. In addition, when the DNA comprising the UFB is not properly protected or resolved during anaphase or telophase, it may be ruptured by the end of mitosis ,78. ThesFurther insights into the link between replication stress and CIN came from the discovery of the replication stress protection conferred by specialized polymerases that are able to replicate across noncanonical DNA and AT-rich sequences at difficult-to-replicate regions ,106. NotThis work confirmed the long-standing observation indicating the late replication timing of CFSs  and suppA subsequent study confirmed that replication stress links n-CIN and s-CIN in colorectal cancer cells . In chroInterestingly, another study reported that homologous recombination (HR)-deficient cells are characterized by endogenous replication stress that can have both local and global consequences on chromosome segregation . It showThe fidelity of the mitotic process is primarily surveilled by the spindle assembly checkpoint (SAC) that, together with other error correction mechanisms, prevents the segregation of unattached or incorrectly attached chromosomes in mitosis and delays mitotic exit ,128. LosEach sister chromatid builds a multilayered proteinaceous structure onto centromeric DNA that is locally restrained to zones of CENPA nucleosomes deposited in the previous G1 phase . This muThe ability of the SAC to detect and respond to replication stress/DNA damage in mitosis appears to be dependent on the severity of the stress, and, based on the experimental settings, different observations were made. Research conducted to study kinetochore detachment, kinetochore tension or asymmetric DNA strand segregation induced mitosis with unreplicated genomes (MUGs). This condition can be achieved by high doses of HU in combination with caffeine, cyclin A overexpression, HU-only treatment or by silencing the origin of replication protein DUP ,136,137.Recently, it was shown that strong replication stress in primary IMR90 fibroblasts leads to p53-dependent interphase cell cycle arrest. The very same treatment in p53-deficient IMR90 E6E7 cells leads to sustained Mps1-dependent mitotic arrest and mitotic death, which suggests that severe under-replication that escapes interphase surveillance can be sensed and retained in mitosis . In the Whereas the first studies show that even completely unreplicated cells can enter anaphase and exit mitosis, the second scenario shows that a high stress level leads to mitotic cell death, which seem like contradictory findings. We envision that, in the first scenario, the complete absence of one chromatid allows microtubules to attach to the remaining chromatid (merotelic attachment), and the checkpoint is satisfied after prolonged mitosis. This theory fits with the proposition that interkinetochore distance and tension are not needed to satisfy the checkpoint ,139. In Recent findings in yeast tubulin mutants with increased sensitivity to the DNA replication stress/DNA-damaging agents HU and 4-nitroquinoline 1-oxide (NQO) substantiate the previous suggestion. These mutants were shown to arrest in mitosis after treatments with high doses of HU followed by release in a normal medium. This arrest was not due to the classical activation of the SAC but to an Aurora B-dependent tension checkpoint, and the authors suggested that the observed phenotype may arise because tubulin mutants fail to detect some DNA damage in interphase that is carried over to mitosis. Such damage may cause tension defects, as the centromere, even if fully replicated, may contain centromeric lesions that compromise kinetochore assembly and kinetochore microtubule attachment .As nonreplicated chromosomes hardly arise in a physiological context and strong HU/APH-induced replication stress leads to mitotic death, we believe that the most severe threat comes from low replication stress that is not or only partially sensed in mitosis. We hypothesized that, under these circumstances, cells survive or at least exit mitosis, which we showed to be the case for noncancerous RPE1 cells challenged by low doses of APH. As RPE1 cells are supposed to be proficient in all checkpoints, we concluded that low replication stress remained below the threshold of detection in mitosis .An interesting study in yeast provides one possible explanation of how a sustainable amount of replication stress can attenuate/shut off SAC activation and, therefore, does not interfere with mitotic progression . SUMO-taIntriguingly, another target regulated by RNF4 (Slx5/Slx8) is FANCD2/FANCI. Extraction of the FANCD2/I complex from damaged foci was shown to depend on RNF4-dependent polyubiquitination and consequent proteasomal degradation. Such timely turnover at damaged sites seems to be required to guarantee downstream repair pathways to correctly take over . FANC-deAnother interesting study conducted in yeast found that DNA damage can lead to an “inflammation zone” at the chromosome, and when this zone is sufficiently close to a centromere, it can lead to epigenetic centromere changes; these changes are premitotically detected by the SAC (in this case Mad2), which then overlaps with the canonical Mec1/Tel1 (ATR/ATM) checkpoint and delays G2. This finding indicates an involvement of the SAC in cell cycle delay even earlier than originally anticipated and not only prior to anaphase . FinallyIn conclusion, we suggest that low replication stress is the most severe threat to genome integrity, as mitotic checkpoints are either blind to detecting it or can actively override it. In the end, such a mitotic exit can be accompanied by structural and numerical chromosomal aberrations ,142,148.The previously discussed checkpoint in mitosis is strongly interdependent with microtubule dynamics, as indicated by the finding that compromised microtubule stability and/or turnover possibly lead to erroneous kinetochore-microtubule attachments .Microtubule stability is posttranscriptionally regulated and depends on the stability of its own mRNA to ensure that microtubules can react promptly to diverse insults . This meIt has been shown that DNA repair factors can be recruited to mitotic spindles ,152,153 + cells alter microtubule dynamics by increasing microtubule plus-end growth. Merotely and chromosome mis-segregation occurred here in the absence of spindle multipolarity and was entirely due to microtubule alterations [Our own data from experiments with low-dose replication stress confirmed that mitotic microtubule stability is altered when cells are exposed to APH in the previous interphase . As we derations .Another study by Bakhoum and colleagues confirmed the abovementioned findings and provided a possible explanation for how DNA damage impacts microtubule stability. This is one of the few reports that observed prompt microtubule dynamicity changes after DNA insults within the same mitosis. The dominant chromosome segregation error was whole chromosome lagging due to increased microtubule stability. Slow decaying kinetochore microtubules were the ones affected by DNA damage, suggesting that the attachment to kinetochores is specifically stabilized. γ-H2AX, ATM and CHK2 signaling were implicated in stabilizing microtubule attachment after DNA damage. The downstream actors that were hyperphosphorylated under these conditions and directly interfered with microtubule stability were Aurora A and PLK1. In conclusion, a DNA damage-signaling cascade with ATM-CHK2-PLK1-Aurora A leads to microtubule stabilization and lagging chromosomes and connects the DNA damage response to the cytoskeleton .+) colorectal cancer (CRC) cells have increased microtubule plus-end growth, compared to nontumorous RPE1 and CIN− CRC cells, as shown by the microtubule plus-end protein EB3 tracking in space and time. Aurora A and CHK2 were identified as the main regulators of microtubule assembly in CIN+ CRC cells. CHK2-dependent phosphorylation of BRCA1 at the centrosomes limits the centrosomal pool of Aurora A, which complies with normal microtubule assembly rates generated by the centrosomes. Loss of CHK2 or Aurora A overexpression led to excessive accumulation of Aurora A on centrosomes and increased the microtubule assembly rates [Centrosomes are the main microtubule-nucleating center in cells and recruit DNA repair factors, which, in turn, can influence microtubule nucleation rates. The Bastian laboratory showed that chromosomally instable  cells into mitosis, despite the presence of nonreplicated DNA. Mitotic exit was accompanied by multipolar division, thus leading to aneuploid progeny . The autSince failures such as centrosome splitting or overduplications are phenotypes observed after the depletion of several different repair/replication proteins; the link between hampered replication/repair and impaired centrosome integrity may be indirect. We believe that the actual presence of repair/replication proteins at centrosomes is not necessarily required; rather, the absence of these proteins during interphase creates a cell cycle delay and/or DNA damage. As synchrony between DNA replication and centriole duplication has to be guaranteed for a correct number and integrity of the centrosomes, any defect that delays the cell cycle could interfere with the centriole duplication/centrosome maturation process. Direct evidence comes from our own experiments showing that, in RAD51- or BRCA2-deficient cells, spindle multipolarity was suppressed by the simple suppression of the intrinsic replication velocity deficiencies of these cells ,180 and Many experiments suggest that delaying interphase in S or G2 can lead to a continuous centrosome duplication cycle ,182,183.We think that centriole disengagement is a multicomponent phenomenon, where advanced centriole and centrosome maturation come together with increased microtubule stability during mitosis. Only this toxic combination drives centrioles to disengage and centrosomes to split. Both centrioles of the centrosome might, at this point, be surrounded by PCM and nucleate microtubules; in this case, microtubule forces may indeed tear the two centrioles apart. As it was shown that PCM integrity is altered after DNA damage , we specOne common idea is that centrioles, similar to sister chromatids, are held together by cohesion  and thatUnder physiological conditions, WAPL opens cohesin rings, specifically at the chromosome arms, in prophase (“prophase pathway”), and centromere cohesion, protected by shugoshin, remains intact until anaphase onset, when separase cleaves it. This two-step pathway of cohesion removal, which gives metaphase chromosomes their characteristic X-shape, is crucial to maintain genetic stability .Whereas we and others reported no significant increase in cohesion defects after moderate replication stress ,142, higTRF1, telomere-repeat binding factor 1, is part of the shelterin complex at telomeres and ensures correct replication at telomeres. However, it also has a largely unrecognized role in mitosis, as TRF1 depletion leads to accelerated mitotic timing, cohesion defects and merotelically attached chromosomes that induce elevated levels of n-CIN . TRF1 haIn yeast, age-related cohesion loss depends on the downregulation of cohesion subunits and is accompanied by the downregulation of DNA repair factors, which suggests that DNA damage accumulation with age is accompanied by the imperfect holding of sister chromatids together . OocytesIn conclusion, it seems that the cell in the most vulnerable cell cycle phase has found quite drastic means to respond to replication stress. Even when mitotic checkpoints can partially detect replication stress, it seems that, at least, low-dose stress is transmitted through the following interphase. Furthermore, we summarized evidence that replication stress can hamper the correct bipolar spindle assembly, microtubule stability and cohesion maintenance, all of which can lead to increased levels of merotelically attached chromosomes , which, Having considered how replication stress can lead to mitotic defects and chromosome mis-segregation, we now examine the evidence showing that mitotic defects and aneuploidy may, in turn, generate replication stress and DNA damage, thus creating a vicious circle .Chromosome segregation defects may directly lead to DNA damage and structural aberrations when lagging chromosomes become trapped in the cleavage furrow, which has been shown to provoke double-strand breaks and activation of the DNA damage response . AlternaIn addition to the direct effects of segregation errors on genome stability, aneuploidy itself may have genome destabilizing effects ,206. As One of the main consequences linked to disturbed proteostasis is the alteration of cellular metabolism and increased production of reactive oxygen species (ROS). A higher level of ROS and altered energy metabolism have been shown in disomic yeast strains, mouse embryonic fibroblasts (MEFs) and Drosophila ,213,214.The bidirectional interplay between replication stress and chromosome mis-segregation not only promotes genome instability and transformation but may also perpetuate CIN, thus providing a mechanism of evolution for cancer cells.Whereas replication stress and CIN play fundamental roles in driving tumor evolution, aneuploidy is known to restrain cell proliferation . One of The activation of p53 as a result of mitotic defects and aneuploidy has been attributed to different mechanisms , includiThese findings indicate that aneuploidy per se may not be sufficient to elicit p53 activation and that the proliferation defects of aneuploid cells may result from multiple converging stress signaling pathways . In addiTherefore, the propagation of CIN in cancer can arise through inactivation or bypass of the p53 pathway and the acquisition of mechanisms that allow tolerance of replication stress and aneuploidy-associated stressors, such as activation of the proteasome and autophagy and CIN adaptation ,229,231.Another key brake to the proliferation of aneuploid cells is the immune system . SantaguThe capacity of tumors with CIN to activate or suppress immune signaling and to drive metastasis is an emerging area in cancer biology and immunotherapy ,250. By Developing new methods to detect tumor CIN, as well as therapeutic strategies based on leveraging CIN to modulate tumor fitness and immunogenicity, are therefore of utmost importance in cancer treatment ,250.Over a century ago, the work of von Hansemann, who reported the presence of abnormal mitotic figures in tumors, and the experiments of Theodor Boveri showing the abnormal development of sea urchin eggs after dispermic fertilization, founded the hypothesis that an abnormal chromosome constitution (aneuploidy) produced by abnormal mitosis could be involved in the process of tumorigenesis (see ).However, aneuploidy does not always act as a driver of tumorigenesis and, instead, is mostly detrimental for cell and organismal fitness ,216,256.When we discuss CIN, we have to consider it in the precancerous and cancerous contexts; although CIN is known to fuel intratumor heterogeneity, it can be detected during early tumorigenesis. Specifically, the DNA damage and chromosomal aberrations that occur in precancerous lesions as a consequence of replication stress have been shown to activate the DNA damage response that helps to eliminate damaged cells or induce them into senescence (as proposed for oncogene-induced senescence) ,30. HoweAs replication stress and CIN fuel cancer evolution, the problem of treating tumors that are not merely the clonal expansion of one single mutated cell but of many mutated cells becomes more complicated. Multiple mutations reduce the benefits of an initially efficient treatment, as the nonclonal expansion of cells leads to rapid adaptation to drugs. Intratumor heterogeneity not only often makes chemotherapeutic treatment ineffective but also renders the search for biomarkers, such as differently expressed proteins or tumor signatures, a race against time. As a logical consequence, targeting DNA replication, transcription or mitosis and, thus, processes that are similar for many different clones within a tumor has been revealed as a potent strategy to attenuate unrestrained proliferation.Here, we briefly discuss how cancer treatment is approached today and what we believe is missing in the current discussions. The knowledge that replication stress fuels structural and numerical chromosome aberrations simultaneously could be and should be used to target cells with intrinsic replication stress. We believe that, regardless of the underlying cause of replication stress , a common phenotype links them during mitosis, making mitosis-targeting drugs attractive investments in this context.Classical methods to target replication stress and DNA damage include drugs that either increase DNA replication stress and/or lead to interphase DNA damage, such as cisplatin, gemcitabine, olaparib, doxorubicin or topotecan. Healthy cells should escape such treatment, as they are supposed to be not/low proliferating and/or proficient in pathways that repair the introduced DNA damage . In the The oldest and most efficient chemotherapeutic agent to date is paclitaxel—that is, the microtubule stabilizing compound Taxol , which i+ tumors [Pioneering studies from the late 1990s demonstrated that p53 activation/stabilization after DNA damage, such as UV irradiation, bleomycin or doxorubicin, decreased Map4 (microtubule-associated protein 4) expression. Decreased expression of this microtubule binder leads to less stable microtubules and increased sensitivity to microtubule destabilizing drugs such as Vinca alkaloids . Another+ tumors .We discovered a synthetic lethal situation with replication stress and Taxol, which increased the number of multipolar dividing cells even when used at very low doses. Since replication stress leads to premature centriole disengagement, we propose that Taxol, which already increases multipolarity on its own, shows additive effects with APH . We suspSpindle multipolarity due to centriole disengagement is the dominant and most intriguing phenotype acquired after replication stress. Indeed, we clearly observed that RPE1 noncancerous cells clustered spindle poles and ultimately divided in a bipolar manner. This data indicates an opportunity to interfere with clustering and force cells to divide in a multipolar manner, which should be detrimental. As we also observed that cells with multiple centrioles tend to disengage , and thaReplication stress and CIN are hallmarks of cancer that fuel each other during its evolution; however, they also represent an Achilles heel that can be exploited in cancer therapy. The identification of the molecular mechanisms underlying this bidirectional link can be useful to find biomarkers to selectively target cancer cells and to design new preventive or therapeutic strategies acting on the premitotic and postmitotic causes of CIN. The study of the molecular pathogenesis of CIN syndromes and of the mechanisms underlying their phenotypic heterogeneity will be key to informing these strategies. This knowledge can lead either to therapies attenuating the effects of replication stress in a precancerous setting or to exacerbation of the endogenous stress of cancer cells over a threshold level to induce mitotic catastrophe. Finally, the identification of CIN signatures and their relationship with the activation of inflammatory pathways and immune responses will be of increasing importance to stratify patients and select them for immune therapies.


Currently, treatment of bacterial infections focuses on choosing an antibiotic which matches a pathogen’s susceptibility, with less attention to the risk that even susceptibility-matched treatments can fail due to resistance emerging in response to treatment. Here, combining whole-genome sequencing of 1,113 pre- and post- treatment bacterial isolates with machine-learning analysis of 140,349 urinary tract infections (UTIs) and 7,365 wound infections, we find that treatment-induced emergence of resistance could be predicted and minimized at the individual-patient level. Emergence of resistance was common, yet driven not by de novo resistance evolution, but rather by rapid reinfection with a different strain resistant to the prescribed antibiotic. As most infections are seeded from the patient’s own microbiota, these resistance-gaining recurrences can be predicted based on the patient’s past infection history, and their expected risk minimized by machine learning personalized antibiotic recommendations, offering a means to reduce the emergence and spread of resistant pathogens. Urinary tract infections (UTIs) and wound infections are two of the most common reasons for prescribing antibiotics –3. TheseYet, even when an antibiotic is correctly prescribed to treat a pathogen sensitive to it (susceptibility-matched), treatment is a double-edged sword: it may clear the ongoing infection, but it may also select for resistant pathogens among the patient’s resident microbial population, limiting current and future treatment efficacy , 12. IndHere, to understand and predict personal risk of treatment-induced gain of resistance, we combined whole-genome sequencing of isolates from same-patient recurrent infections with analysis of a longitudinal dataset of UTIs and wound infections collected by Israel’s Maccabi Healthcare Services (MHS) between June 2007 and January 2019. We identified 215,732 MHS patients with at least one record of a UTI  and based on their outcome: recurrence with a sensitive or resistant infection, or no recurrence  . While sThe large number of correctly treated infections that subsequently gained resistance could be caused by three possible mechanisms: evolution of resistance through mutations (mutations); through dedicated resistance genes (resistance genes); or through reinfection with a different strain resistant to the antibiotic (strain replacement) . To distThe genomic analysis showed that while the same E. coli strain often persists in early UTI recurrences that do not gain resistance, resistance-gaining recurrences were caused by strain replacement. No cases were identified of resistance appearing through point mutations in the originally infecting strain. Analyzing strain relatedness, we find that while reinfection with a new strain was rare in recurrences that did not change resistance to the treatment , it was the dominant mode in infections gaining resistance  algorithms for personalized antibiotic recommendations which minimizes the predicted risk of treatment-associated emergence of resistance for both UTIs and wound infections . For eacAnalyzing all susceptibility-matched treated cases in the test-period, we found that in most cases there was an alternative susceptibility-matched antibiotic which had a lower patient-specific predicted risk of resistance emerging compared to the antibiotic prescribed by the physician . Choosing for each patient the antibiotic with the minimal ML predicted risk of emergence of resistance (ML-recommended) reduces the overall risk of emergence of resistance by 70% for UTIs and 74% for wound infections compared to the risk for physician-prescribed treatments . Since mIn conclusion, while much effort is invested in methodologies for matching antibiotic treatment to infection susceptibility, susceptibility-matched treatments often fail as they select for emergence of resistance via reinfection with different strains specifically resistant to treatment. The strong association between such treatment-induced selection for resistance and personal history of past resistant infections suggests a patient-specific strain reservoir. Given the known role that uropathogens and wound pathogens persisting in the patient’s microbiome have in seeding new infections , 31, 32 Supplementary Materials


Organs-on-chips are broadly defined as microfabricated surfaces or devices designed to engineer cells into microscale tissues with native-like features and then extract physiologically relevant readouts at scale. Because they are generally compatible with patient-derived cells, these technologies can address many of the human relevance limitations of animal models. As a result, organs-on-chips have emerged as a promising new paradigm for patient-specific disease modeling and drug development. Because neuromuscular diseases span a broad range of rare conditions with diverse etiology and complex pathophysiology, they have been especially challenging to model in animals and thus are well suited for organ-on-chip approaches. In this Review, we first briefly summarize the challenges in neuromuscular disease modeling with animal models. Next, we describe a variety of existing organ-on-chip approaches for neuromuscular tissues, including a survey of cell sources for both muscle and nerve, and two- and three-dimensional neuromuscular tissue-engineering techniques. Although researchers have made tremendous advances in modeling neuromuscular diseases on a chip, the remaining challenges in cell sourcing, cell maturity, tissue assembly and readout capabilities limit their integration into the drug development pipeline today. However, as the field advances, models of healthy and diseased neuromuscular tissues on a chip, coupled with animal models, have vast potential as complementary tools for modeling multiple aspects of neuromuscular diseases and identifying new therapeutic strategies. Summary: Modeling neuromuscular diseases is challenging due to their complex etiology and pathophysiology. Here, we review the cell sources and tissue-engineering procedures that are being integrated as emerging neuromuscular disease models. Caenorhabditiselegans  protein  diseases have been linked to 870 mutations in over 80 genes , such as or MFN2 . This geN2  is another neuromuscular disease that introduces unique challenges for modeling in animals because it can be either inherited (10%) or sporadic (90%) . In ALS,bination . Animal bination , Drosophosophila , zebrafiosophila  and C. e elegans . Additio elegans . However elegans . Owing,  elegans .Drosophila and C. elegans because they lack an adaptive immune system. The causes of MG are still mostly unknown and treatment is limited to acetylcholinesterase inhibitors, immunosuppressants or a thymectomy  is a sporadic autoimmune disease in which auto-antibodies selectively destroy acetylcholine receptors, causing a reduction in NMJ signal transmission . A mouseymectomy , highligin vitro platforms, known as organs-on-chips, is an emerging solution to fill the gaps of animal models and holds promise for patient-specific neuromuscular disease modeling and drug development. As discussed below, these platforms are often developed using animal cells or cell lines that are easy to scale, and that can provide important proof-of-concept and basic physiological information. To address issues of human relevance, animal cells or cell lines can then be replaced with patient-derived cells, which can be acquired from a variety of sources. In the next section, we describe the cell sources for neuromuscular disease models, which can ultimately be integrated into the two- (2D) and three-dimensional (3D) engineered tissue platforms described in the following sections.Collectively, these examples highlight that neuromuscular diseases are very diverse and are characterized by many complex genetic and non-genetic etiologies and pathophysiologies. These complexities introduce many challenges for developing comprehensive animal models. Thus, new disease models that are more efficient and predictive are essential for accelerating our mechanistic knowledge of these diseases, as well as the discovery of effective therapies. Integrating patient-derived cells with microfabricated In vitro models can mitigate many of the limitations of animal models described above, such as human relevance and scalability. However, the usefulness of any in vitro model is highly dependent on the source and structural and functional maturity of its cells. This is especially complex when modeling neuromuscular tissues, which consist of both muscle cells and motor neurons. In this section, we will describe the types of muscle and motor neuron cell types available today for in vitro models, and weigh up their advantages and disadvantages.in vitro is a multi-step process. First, mononuclear skeletal myoblasts are seeded on a standard culture surface that is often coated with extracellular matrix (ECM) proteins, such as collagen or laminin. The myoblasts are then expanded in a high-serum growth medium until they reach confluence. The medium is then substituted with a low-serum differentiation medium that triggers the fusion of myoblasts into multi-nucleated myotubes, the in vitro surrogate to muscle fibers  or human induced pluripotent stem cells (hiPSCs) have recently emerged . These cMYOD1  has been generated by fusing neuroblastoma cells with embryonic motor neurons. NSC-34 cells retain the proliferative properties of the tumor cells while also exhibiting select neuronal properties, such as acetylcholine synthesis, neurotransmitter release and neurofilament proteins . This ceNGN2 , ISL1 and LHX3  (a silicone elastomer) stamps with ridges several micrometers in size . PDMS stSynthetic biomaterials are advantageous culture substrates because their mechanical and biochemical properties are highly controllable and reproducible. For example, polyethylene glycol and polyacrylamide (PA) are both biologically inert hydrophilic polymers that can be crosslinked into hydrogels with elastic moduli tuned to match the developing, healthy or fibrotic muscle tissue matrices . AnotherTo achieve consistent cell adhesion, researchers must functionalize the synthetic substrate with ECM proteins. This is generally considered an advantage because ECM ligand type and concentration can be specified. Because collagen accounts for up to 10% of the dry weight of muscle , severalSynthetic biomaterials are also compatible with many micropatterning techniques that can be used to introduce microscale features on the surface to spatially control cell adhesion and alignment , as showMicropatterned synthetic culture substrates are especially compatible with assays that quantify myotube contractility because the mechanical properties of the substrate are well defined, and myotube architecture can be controlled to increase the magnitude and reproducibility of contractile force production. Micropatterned PA gels are widely used as a substrate for traction force microscopy, a technique that quantifies forces generated by cells by tracking the displacement of fluorescent beads embedded in the hydrogel. Although traction force microscopy is more commonly used for cardiac myocytes , it has Contractility can also be quantified by culturing myotubes on flexible cantilevers, as in the muscular thin film (MTF) assay. The MTF assay entails first spin coating a glass coverslip with a layer of poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, followed by a layer of PDMS . The PDMMicrofabricated surfaces have also been developed to improve mixed co-cultures of myotubes and motor neurons. For example, photopolymerization techniques have been used to fabricate PA hydrogels with alternating soft and stiff stripes that mimic the rigidity of nervous tissue and muscle tissue, respectively . On thesNMJ maturation has also been achieved in mixed co-cultures by applying electrical stimulation using a bioreactor . This apCompartmentalized culture devices have also been microfabricated to physically isolate motor neurons and myotubes into separate chambers . These cAlthough engineered 2D neuromuscular tissues have many advantages from an assay perspective, they fundamentally lack the bundle-like architecture and cell-ECM interactions of native muscle fibers. To address this, researchers have developed several approaches to engineer miniature 3D muscle bundles . These tOver the past two decades, approaches for engineering 3D muscle bundles have been advanced and refined. Several different types of culture chambers with anchor points have been fabricated , includiMicrofluidic devices have also been fabricated to engineer and maintain 3D muscle bundles . These sOne common approach to innervate 3D muscle bundles is to directly seed them with spheroids of motor neurons . These sSimilar to 2D models, compartmentalized microdevices have been developed to culture motor neuron spheroids and engineered muscle bundles in separate compartments connected by axon-permissive channels  Osaki e. In thesin vitro models of neuromuscular tissues are far from achieving adult-like maturity, especially when based on hiPSC-derived muscle cells and motor neurons. Furthermore, in vitro models of neuromuscular tissues lack the supporting cells known to be important regulators of NMJs in health and disease, such as Schwann cells (in vitro models also currently lack immune cells, despite the established role of neuroinflammation in many neuromuscular diseases (in vitro models are most powerful when implemented hand-in-hand with animal models, which have less human relevance but more advanced motor unit structure and physiology. Together, these complementary model systems are likely to pave the way for more effective and personalized therapies for these debilitating diseases.Recently developed approaches to model healthy and diseased neuromuscular tissues on a chip have the potential to capture the vastly heterogeneous genotypes and phenotypes of individuals with a variety of neuromuscular disorders. Newer technologies, such as 3D bioprinting, which is a form of additive manufacturing that uses cells and other biomaterials as ‘inks’ to print living structures , will prnn cells . Most indiseases . Howeverdiseases , to prob


Current medical professions involve an extensive knowledge of the latest validated scientific data to implement disease diagnosis, therapeutic strategies, and patient care. Although clinicians can refer to a growing number and type of information sources to keep current with new scientific achievements, there are still various concerns about medical information validity, quality, and applicability into clinical practice. Novel strategies are required to identify physicians’ real-life needs with the final aim to improve modern medical information delivery.Our research used an innovative tool to collect real-time physician queries in order to investigate information needs and seeking behavior of Italian neurologists treating patients with multiple sclerosis (MS) and migraine.The study was designed as an exploratory mixed methods  study involving 15 consecutive days of observation. A total of 50 neurologists  were recruited. Data were collected using an instant messaging mobile app designed for this research. At each information-seeking event, moderators triggered a computer-assisted personal interview including both semistructured interview and close-ended questions. Interactions and physician queries collected using the mobile app were coded into emerging themes by content analysis.P=.01). MS specialists were 53% more likely to engage in one information-seeking event compared with migraine neurologists . MS specialists tended to be more interested in patient-related content than migraine clinicians , who conversely more likely sought information concerning therapy management . Compared with MS clinicians, migraine specialists had a harder time finding the required information, either looking at online or offline channels  and less frequently used offline channels . When multiple sources needed to be consulted to retrieve an information item, a reduced satisfaction rate was observed both among migraine and MS specialists (single source vs multiple sources P=.003).Neurologist queries were relevant to the following major themes: therapy management  and drug-related information , followed by diagnostic strategies and procedures . Quantitative analysis indicated online resources were preferentially used by clinicians  compared with offline sources . A multichannel approach, in which both online and offline sources were consulted to meet the same need, was adopted in 33% (65/198) of information-seeking events. Neurologists more likely retrieved information from online relative to offline channels (F=1.7; This study provides a detailed description of real-life seeking behavior, educational needs, and information sources adopted by Italian MS and migraine neurologists. Neurologist information needs and seeking behavior reflect the specific characteristics of the specialty area in which they operate. These findings suggest identification of time- and context-specific needs of clinicians is required to design an effective medical information strategy. Modern health care professionals need to consult increasing amounts of scientific content to keep current on medical science advances . It has Whereas continuing medical education is a prominent (and often mandatory) source of medical knowledge for most physicians , it has Medical Information departments of pharmaceutical companies often deliver up-to-date, balanced, and evidence-based information on a peer-to-peer basis, answering unsolicited medical requests through different channels ,17. A reIn this study, we investigated the information needs and seeking behavior of Italian neurologists treating patients with multiple sclerosis (MS) and migraine in order to inform the content and layout of Medical Information services in our department. MS and migraine therapeutic management have evolved rather differently over the last few years. In fact, in the field of MS there were important advances, with increasing disease-modifying therapies becoming available for both progressive and relapsing-remitting MS treatment . TherefoInformation-seeking behavior is a complex phenomenon that is contextually shaped by personal needs, learning styles, available resources, and affective components among other factors ,25,26. WThis research describes the real-life seeking behavior, educational needs, and information sources of Italian MS and migraine neurologists. To the best of our knowledge, these aspects have never been explored before in this context. Our data provide an initial, exploratory step in understanding the specific information needs of neurologists and give insights on the motivation, response, and gaps in the landscape of information available for this group of users. These results could be used to design novel Medical Information strategies aimed to deliver personalized, accurate, consistent, and timely information with an omnichannel approach, allowing health care professionals to make informed decisions that can improve patient care.To evaluate physician eligibility, a screener questionnaire was administered to 72 clinicians working in different health centers and hospitals throughout the Italian territory. Based on the screener results, we enrolled 50 neurologists, of whom 25 were MS specialists and 25 migraine specialists. Recruitment was planned to equally represent physicians from all Italian geographical macroregions in each specialty area.The research was designed as an exploratory mixed methods  study and involved 15 consecutive days of study. The observation was conducted through the Physician Line app, an instant messaging software app consisting in an instant messaging phone app based on WhatsApp that allowed physicians to share and describe the information needs experienced during their daily clinical practice and information sources used to retrieve the information needed. Participants were instructed on the Physician Line app functionalities by means of a kick-off video presentation. To improve response rate, respondents were rewarded with a cash incentive. Two expert researchers in the field of qualitative research (BG and MA) were able to initiate an interaction every time a physician sent a text message in the Physician Line app. Therefore, there was a direct interaction between physicians and moderators during the interview conducted by these means. In fact, each information-seeking event triggered a computer-assisted interview, during which a semistructured interview and two close-ended questions were administered to the physicians through the Physician Line app. The semistructured interview included 5 items capturing physician motivations and behavioral patterns . At the Due to the nature of the research, no ethics committee approval was required . In fact, the study did not involve patients or lay citizens and no health intervention had been administered to participants. This study was conducted in compliance with the European Union General Data Protection Regulation 2016/679 and in line with well-established regulatory practices and procedures governing marketing research, including the Market Research Society code of conduct (2019 revision) and the Italian Code of Professional Ethics (curated by ASSIRM 2016 revision).Physicians actively chose to participate to the study. Records collected by Doxa Pharma Srl include data retention policies, data privacy statements, permission to take part in a data collection exercise, and agreement to the processing of personal data. The interview questions were not aimed at investigating sensitive issues like religious or political beliefs or sexual orientation. Doxa Pharma Srl ensured respect of confidentiality of collected information and pseudonymization of individual answers before primary data abstraction and analysis.Physician queries and their interactions with the moderators collected with the Physician Line app were subsequently evaluated by content analysis. Following the sense-making approach, we predefined an ontology of information seeking entailing the concept of an information-seeking event described by a set of domains including situations, gaps, and tactics, namely motivation and triggers, context, information gap content, information sources, and information search strategy. An information-seeking event was defined as any action carried out by a physician in order to meet an information need .Each moderator abstracted relevant themes from the Physician Line app transcript with a mixed deductive-inductive content analysis of the material transcripts. Moderators precoded the transcript by highlighting codable words, sentences, or paragraphs. An initial distinction was made to discriminate motivation and triggers of information seeking and query content. The transcript was then open-coded by assigning descriptive labels to transcripts excerpts under these first two categories. Both motivation and triggers and content were further coded as follows. After consolidating redundant codes, a matrix was generated by including all codes emerging from the discussion. The codes were inductively grouped into broader categories by observing similarities of content and meaning. When disagreement occurred among coders, the item was discussed until a common taxonomy was achieved.In a second stage, each information-seeking event was coded (for each specific motivation and content) concerning source, context, and event time (based on recording metadata). Coding of information sources was based on a predefined coding scheme . We computed confidence intervals for information-seeking event rates based on the Poisson distribution. Furthermore, we used 2-way analysis of variance to evaluate differences in information retrieval and satisfaction scores across medical specialty and information channel used. Finally, differences in proportion of content type searches across specialties were assessed by Fisher exact or chi-square tests where appropriate. Analysis was conducted with SAS 9.4 (SAS Institute Inc).Participants worked in different settings, encompassing small private centers and large public organizations integrating multiple operative units aimed at providing health care for a wide catchment area. Physician characteristics are reported in Use of the instant messaging phone app Physician Line allowed real-time collection of relevant data without affecting the clinicians’ daily working routine; the tool was well accepted and provided physicians the opportunity to conveniently communicate with moderators.Physician queries concerned 8 categories relevant to marketed and investigational drugs, clinical management, disease epidemiology and physiopathology, pharmaceutical companies and their activities, diagnostic procedures, patient-related topics, congress and educational opportunities, and other minor categories. Overall, 37 items could be consistently coded and classified in these 8 categories .We identified different neurologist motivations to engage in information seeking which were sorted into 2 categories. The first category included exogenous motivations , external events triggering research for further information. Such events can be tentatively classified into passive and active. Specifically, passive triggers were defined as any activating content from newsletters, websites, marketing activities, or institutional or scientific communications which motivated further information seeking in the absence of a specific, preexisting information need.I am reading a paper on NEJM about a phase 2 trial of <Drug_name> among MS patients. I got there because I just received the NEJM weekly newsletter and this topic was relevant to my practice.N1Conversely, active triggers were deemed to involve questions raised by patients and colleagues; emerge as noteworthy themes while the physician was actively engaged in seminars, grand round discussions, informal discussions; or were raised by clinical problems emerging in the course of a medical encounter.One patient asked me about <Drug_name>. She has found the list of investigational drugs on Wikipedia and learned that the drug was under review by the FDA. She entered a secondary progressive course and she was worried about the clinical worsening of the disease.N2We also found that the need for professional growth and general scientific update rather than immediate, contingent problem-solving issues may represent a strong motivational driver for information-seeking among neurologists. In contrast to exogenous triggers, we called such experiences endogenous motivation.Nowadays we, as physicians, must keep current on new drugs and scientific developments. Our field is growing in complexity with new insights into disease pathology and novel therapeutic options. For this reason, I browse PubMed on a weekly basis.N3Physicians used both online and offline resources . More specifically, 120 information-seeking events  were sent by MS specialists, while 78  were sent by migraine neurologists. Hence, MS specialists were 53% more likely to engage in one information-seeking event compared with migraine specialists . Each information-seeking event included an average of 1.98 different information searches (95% CI 1.79-2.18), for a total of 392 needs recorded.P=.05); on the other hand, MS specialists tended to be more interested in patient-related content compared with migraine specialists .Overall, the majority of physician expressed at least one need concerning therapy management , followed by drug-related content , diagnostic strategies and procedures , disease-related content , congresses and educational opportunities , patient-related content  and other/miscellaneous including administrative issues, pharmacoeconomics , pharmaceutical companies , topical issues , and unclassified content . Distribution of major information needs was slightly different across specialties . MigrainP=.02).As shown in P=.03). An explanation of this behavior can be found in the analysis of Physician Line app data, which revealed that multichannel search was adopted for complex issues needing articulated responses or when the desired information was not available in a single authoritative source. Especially in the migraine field, this implied adopting an iterative tree-like search strategy, in which answers to the original questions raised further information needs that triggered further information-seeking behaviors.Overall, an initial adoption of a Web search query did not exclude subsequent use of offline channels and vice versa; indeed, online and offline channels were frequently used in combination (33% [65/198] of the information seeking events), adopting a multichannel approach. Of interest, migraine specialists more likely engaged a multichannel search than MS specialists  and diagnostic procedures . Conversely, public search engines tended to be used more frequently than professional scientific repositories to find out about congresses  and patient-related topics . Finally, professional and general search engines were equally used to look for information about drugs (18% [17/96] vs 19% [18/96]) and “clinical management” (20% [34/169] vs 19% [32/169]). Websites of scientific societies or institutions were more likely searched to retrieve information about congresses , disease epidemiology and physiopathology , and diagnostic procedures  compared with other themes .We found tentative evidence that different sources of information were selected to satisfy different information needs . For exaF=1.7; P=.01). Migraine specialists had a harder time finding answers to their questions compared with MS neurologists, either looking at online or offline channels .Data collected with the Physician Line app indicate that the information needed was available in at least one resource for most physicians. Average information retrieval rating was 3.27 (SD 0.99) indicating that physicians most often retrieved the information they needed. In very few instances, clinicians reported they did not find the information needed . In particular, migraine specialists were significantly less satisfied about offline channels compared with MS neurologists (P=.02). Conversely, satisfaction about information retrieved through online channels was similar in both specialty groups (P=.69). Finally, we observed reduced satisfaction when multiple sources were interrogated for a given information need . Such difference was observed among MS (P=.006) and migraine specialists (P=.005).Physicians reported they were generally satisfied with the information retrieved A and 4B.This research used an instant messaging phone app to investigate real-life needs, learning triggers, and information sources of Italian neurologists treating patients with either MS or migraine. This exploratory study provides a first time preliminary profiling of neurologists that could be useful for subsequent development of personalized approaches to medical education.An interesting finding of this study is that seeking behavior is different between MS and migraine specialists, suggesting that the specific clinical scenario of specialization determines a distinctive pattern of interests, information needs, issues to be addressed, and preferred information sources. There are significant differences between current therapeutic management of MS and migraine, mainly due to the recent advances in the treatment strategies for MS. Our data on information needs and seeking behavior of neurologists seem to reflect the differences occurring in the therapeutic area in which physicians operate. Indeed, compared with MS specialists, migraine neurologists were less prone to engage in information-seeking behavior and, when they did look for medical information, they more frequently asked questions about therapy management rather than other topics. Furthermore, migraine physicians often needed to consult multiple information channels to find answers and more frequently relied on the use of public search engines rather than professional scientific repositories. Finally, satisfaction of migraine neurologists with offline channels  was significantly lower than that of MS specialists.A deeper analysis of resource use according to information needs indicates that neurologists have developed need- and time-specific research patterns that depend on the type and content of the piece of information searched for. Indeed, when starting new information seeking, physicians choose the most effective search process as well as the most suitable information source to answer that particular question. This requires neurologists to estimate the properties, reliability, weaknesses, and strength of each available channel. Moreover, the choice to use online or offline channels or a combination also depends on the environment in which the information need occurs . For example, consistent with previous research ,12,28, iThe complexity around information seeking and learning modalities recently gave rise to the principles of personalized education (PE). Like the emerging framework of PM ,29,30, PThis study was based on the use of the Physician Line app, a novel data collection tool that enables real-life observation and sharing of the information needs experienced by physicians. This unique feature allows granular, simultaneous data collection, thus minimizing recall bias. Therefore, the Physician Line app offers the opportunity to obtain results similar to those provided by ethnographic research, in which moderators interact with participants in their real-life environment. Moreover, this tool allows the identification of time- and context-specific information needs. Taken together, these observations indicate that the Physician Line app may represent a valuable strategy for identifying the educational needs and information search strategies of health care professional.Some limitation of this research also needs to be acknowledged. First, the exploratory design involves a small sample size and a short observation period. Further, we lacked potentially important personal and contextual data that could bring further insight into information-seeking profiles among MS and migraine specialists.Our research shows that Italian neurologists practicing in different therapeutic areas experience different information needs, adopt different seeking behaviors, and refer to different information sources during their clinical practice and/or professional development. These findings suggest that an effective information delivery approach requires customization of both the information to be provided and the communication methods to be adopted, which must be tailored to the specific situations, needs, and requirements of health care professionals.Based on this, identification of time- and context-specific needs as well as physician’ profiling appear to be essential steps to design personalized information delivery and medical education strategies for neurologists involved in different subspecialty therapeutic areas. This requires novel instruments enabling us to thoroughly assess physicians’ real-life information needs. This proof-of-concept study investigated the suitability of the Physician Line app as a tool to collect relevant data without affecting clinicians’ daily working routine and providing, at the same time, the opportunity for interaction between physicians and moderators. Based on these preliminary results, the Physician Line app appears to be a valuable tool enabling identification of specific context-by-user information needs, optimization of educational activities, and design of user-centered learning, in accordance with PE principles. Further research should be designed to evaluate the potential of this novel instrument for data collection in a larger sample population. Delivery of personalized, accurate, consistent, and timely information to physicians is essential to improve patient care.


Conventional therapies for cancer treatment have posed many challenges, including toxicity, multidrug resistance and economic expenses. In contrast, complementary alternative medicine (CAM), employing phytochemicals have recently received increased attention owing to their capability to modulate a myriad of molecular mechanisms with a less toxic effect. Increasing evidence from preclinical and clinical studies suggest that phytochemicals can favorably modulate several signaling pathways involved in cancer development and progression. Combinations of phytochemicals promote cell death, inhibit cell proliferation and invasion, sensitize cancerous cells, and boost the immune system, thus making them striking alternatives in cancer therapy. We previously investigated the effect of six phytochemicals , at their bioavailable levels on breast cancer cell lines and were compared to primary cell lines over a period of 6 days. This study showed the compounds had a synergestic effect in inhibiting cell proliferation, reducing cellular migration and invasion, inducing both cell cycle arrest and apoptosis. Despite the vast number of basic science and preclinical cancer studies involving phytochemicals, the number of CAM clinical trials in cancer treatment still remains nascent. In this review, we summarize findings from preclinical and clinical studies, including our work involving use of phytochemicals, individually as well as in combination and further discuss the potential of these phytochemicals to pave way to integrate CAM in primary health care. Health challenges pose major burdens on population economic standards, life expectancy and mortality rate. Cancer is a major contributor to these global pressures with epidemiological evidence presenting a record of more than 14 million new cases and a mortality rate of approximately 8 million every year Some bioactive molecules have been isolated, characterized and identified as phytochemicals. These phytochemicals are classified based on their chemical composition and structure to carotenoids, organosulfur compounds, and phenolics. Accumulating evidence shows that many of these phytochemicals can target a wide variety of signalling molecules implicated in cellular growth, proliferation, differentiation, and death in a series of cell types In this review, we will focus on several bioactive phytochemicals as well as their target effectors in cancer.The variations in treatment response suggest intrinsic or acquired therapeutic resistance exists in a subset of cancer patients. This frequently leads to treatment failures, disease progression, and often mortality. Cancer can be treated with both invasive and non-invasive treatment modalities such as surgery, chemotherapy, radiation therapy, as well as other therapeutic modalities including gene therapy, immunotherapy, hormone therapy, photo-dynamic therapy, targeted therapy, palliative care, and a combination of these (e.g. radiosurgery) An ideal chemotherapeutic anticancer agent would selectively kill tumor cells but not normal cells. However, most conventional anticancer drugs display non-specificity, causing many undesired toxic effects and patient discomfort oC) or hyperthermia that is achieved within cancer cells and tissues, the renaissance of this method is mainly based on the modern electromagnetic heating techniques and is usually achieved by using microwaves to heat the tumor Many targeted therapies, including monoclonal antibodies and pro-inflammatory cytokines, have been developed to target key pathways in tumorigenesis. For instance, interleukin-2 (IL-2) plays an essential role in the immune response by stimulating proliferation of T-cells as well as inducing the generation of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells; which in turn attack tumor cells. However, despite all these immunity-boosting effects, administration of IL2 in therapeutically active doses is still hampered by excessive toxicities Despite its effectiveness, conventional chemotherapy has many side effects. For instance, cutaneous hypersensitivity reactions are observed in patients receiving treatments based on platinum alkylating agents, topoisomerase and mitotic inhibitors; Hyperpigmentation is prevalent with the use of alkylating agents such as ifosfamide, cyclophosphamide, and thiotepa. Hand and foot syndrome is prevalent in liposomal doxorubicin, daunorubicine and 5'-fluorouracil The development of biopharmaceuticals as an alternative hybrid therapy also poses limitations. Biologicals are complex anticancer macromolecules such as monoclonal antibodies, antibody fragments and anti-body-drug conjugates. These therapies have clinically displayed low efficacy and weak ability to penetrate solid tumors. Other therapies such as drug delivery systems (DDS) are designed with conventional active drugs molecules attached to biological carriers such as liposomes, nanoparticles or biodegradable polymers One of the major disadvantages of conventional therapies is the recurrence, as not all cancer stem cells are eradicated from the body. In addition, the development of multi-drug resistance (MDR) is a significant clinical challenge. Also, in many countries, social and economic burdens incurred through treatment costs remain among the many hurdles to be overcome for a global reduction in cancer incidence and mortality. Therefore, there is a pressing need to identify novel approaches and therapies that may offer relatively cost-effective regimens with less undesired side effects Complementary and alternative medicine (CAM) involves approaches and compounds that are not classically considered as conventional therapies et alone. In general, all types of cancer cells exhibit aberrant gene expression due to mutations in the epigenome because of epigenetic modifications involved in cell proliferation, differentiation and survival Most phytochemicals are required in high doses to be effective; these doses are often not achievable through diNutrigenomics, another promising cancer chemo-preventive strategy, involves the modulation of gene expression in response to dietary compounds. Therefore, a holistic and intuitive approach for chemo-preventive therapy will make use of these bioactive dietary compounds to regulate epigenomic changes and prevention Chemopreventive dietary phytochemicals are naturally occurring bioactives found in fruits, vegetables, plants, and spices. They possess anti-inflammatory, anti-oxidative, anti-proliferative and pro-apoptotic properties, and are particularly known to inhibit the growth of various cancer cells. Indeed, a steady inverse effect of increased vegetable and fruit consumption with reduced incidence of oral cancer has been reported via molecular interaction with adjuvant substance (potentiation). They exhibit a combined efficacy which is equivalent to the sum of individual effects (additive), or combined efficacy, which is greater than the sum of individual effects (synergistic), and lastly the combined efficacy which is less than the sum of individual effects Through drug-interaction, the pharmacological effects of dietary phytochemicals demonstrate a positive interaction promoting potency of the bioactive compound Several epidemiological and case studies have identified the molecular activities of selected phytochemicals that target oncogenic pathways and exhibit chemotherapeutic and/or chemosensitizing effects on cancer cells Table . One arein vitro. A main draw-back of these studies is the concentration of compounds used typically exceeds their 'bioavailable' concentration: the serum levels achieved by oral intake of extracts (as practiced in CAM) are much less than used in in vitro studies.A common property of these majority compounds is their anti-oxidant/free radical eliminating ability. However, few of them infuse high free radical formation to result in the killing/elimination of cancer cells. While several research has analyzed effects of individual compounds derived from grape seeds/skin, tea polyphenols, etc., few research studies determined the combined effects of these compounds when used in synergistic, additive or antagonistic combinations. Most studies have evaluated the effects of individual compounds on a variety of cancer cells Curcuma Longa plant. Howells et alet al. identified several putative novel molecular targets of curcumin using gene expression profiling in vitro studies with curcumin in the 10 μM range or below might have human physiological relevance. Its role as a chemopreventive agent may lie primarily within the gastrointestinal tract where its concentration is not dependent upon systemic absorption. In spite of low bioavailability, several animal studies have demonstrated the anticancer activity of curcumin in cervical via modulation of specific carcinogenic biomarkers such as cyclooxygenase-2 (COX-2), nuclear factor kappa (NF-kB), tumor necrosis factor alpha (TNF-α), cyclin D1 and STAT-3 via a Pirin-dependent mechanism Curcumin, the derivative of turmeric is extracted from the roots of the via the mitogen‑activated protein kinase‑signaling pathway et al. showed efficacy of dietary curcumin (used at 2% dose) or a combination of curcumin (1%) and PEITC to inhibit high grade carcinoma and increase apoptosis in a transgenic adenocarcinoma of the mouse prostate model accompanied by downregulating the Akt signaling pathway Recent evidence shows that curcumin targets stem cells of pancreatic cancer Polygonum cuspidatum) is a powerful antioxidant. Resveratrol is sometimes used therapeutically for treating inflammation and bacterial infections. It has Cox-1 inhibitory activity In-vitro investigations demonstrated its anticancer potential in a wide variety of tissues including breast, colon, pancreas, stomach, prostate, head and neck, ovary, liver, lung, and cervix Resveratrol, a phytoalexin extracted from grapes, red wine, peanuts and berries with highest concentrations is found in Japanese knotweed  Folate and Folic acid are extracted from asparagus, green leafy vegetables and broccoli Lycopene, a carotenoid extracted from vegetables and fruits is found in high concentration in tomatoes Sulforaphane (SFN) is found in high concentrations in cauliflower, broccoli, and cruciferous vegetables. SFN was shown to suppress tumor angiogenesis by reducing cell viability, migration and tube formation in HUVEC-epithelial cell lines possibly through inhibition of signalling between STAT3/HIF-1α/VEGF et al. Indole-3-carbinol (I3C), a phytochemical extracted from cruciferous plants such as broccoli, cabbage and cauliflower, is known to prevent cancer development Punica granatum (pomegranate), exhibits anti-proliferative capacities and promotes apoptosis in prostate cancer cells via the cyclin kinase inhibitor-cyclin cdk machinery. Ellagic acid down regulates Bcl-XL and BcL-2; cyclins D1, D2, cdk 2, 4 and 6 Ellagic acid, found in , an isoflavone found primarily in soybeans, modulates multi-signalling targets in cancer cells. This bioactive compound modulates the cell cycle and apoptosis process of prostate, breast and lung cancer cell lines by inhibiting NF-kB and Akt signalling pathway and promotes G2/M and G0/G1 arrest in various cancer cell lines L and BcL-2 and upregulates pro-apoptotic Bax, Bak, and Bad proteins in-vivo and in-vitro models reduced cell growth and promoted apoptosis when compared to chemotherapeutic treatments alone GenisteinDiallyl disulfide found in cauliflower, broccoli and cabbage was shown to promote G2/M cell cycle arrest by generating ROS and reducing the HDAC activity in cancer cells Many of these herbs and their extracts are further mixed and prescribed in Asian countries for chronic diseases such as arthritis, diabetes and cancers. There have been many claims by the practitioners in India, China and other asian countries, of having cured cancers. The senior author has met several of them, visited their 'clinics', seen their 'patients' who have been claimed to have been rescued from terminal stage neuroblastoma and adenocarcinomas. A common feature of these practitioners has been a reluctance to subject their herbal preparations to clinical trials using accepted parameters . In the US, they are not approved by the regulating bodies such as FDA for treating specific diseases, but are sold extensively over the counter as nutritional dietary supplements. Research in this exciting area has been intense and has succeeded in identifying several active compounds (such as the ones described in this review), purification, characterization and structure elucidation of these compounds and structure-function studies leading to identification of more potent analogues. Molecular mechanisms of action of these compounds is being investigated widely The preceding discussion of literature on these various phytochemicals demonstrates that 1) each of these chemicals has multiple effects and targets multiple pathways in the cancer cell, 2) an additive or synergistic action was noted upon combination of two and sometimes three compounds in vivo. The main objective of our proposal is to elucidate unique molecular mechanisms that contribute to increased effectiveness of these phytochemicals when used in combination to inhibit ovarian epithelial carcinoma. This data prompted us to generate several hypotheses that: 1) Cell proliferation inhibitory activity of phytochemical combination is the result of synergistic and additive actions of the individual selected phytochemicals; 2) combination of these phytochemicals activates gene(s) that are exclusive to the combination; 3) identification of these gene(s) unique to the combination, have the potential to be used for the design of targeted therapy of BC and 4) this combination of five will be effective in chemoprevention and chemotherapy of various cancers in in vivo. Our ongoing in vivo studies will shed light on the possibility of establishing specific phytochemical combinations to treat specific cancers. More interestingly, our genomic studies, using microarray or RNAseq technologies combined with functional studies will allow us the identification and validation of candidate genes that will pave the way towards the design of effective targeted therapeutic cancer strategies.Our recent work demonstrated synergism between several compounds Cancer is a complex disease with various aetiologies, and conventional therapies cause multiple side effects, representing a serious economic challenge. There are more than 5000 phytochemicals found in fruits, grains and vegetables with a large amount still undiscovered


The use of extracorporeal cardiopulmonary resuscitation (ECPR) to restore circulation during cardiac arrest is a time-critical, resource-intensive intervention of unproven efficacy. The current COVID-19 pandemic has brought additional complexity and significant barriers to the ongoing provision and implementation of ECPR services. The logistics of patient selection, expedient cannulation, healthcare worker safety, and post-resuscitation care must be weighed against the ethical considerations of providing an intervention of contentious benefit at a time when critical care resources are being overwhelmed by pandemic demand. Extracorporeal cardiopulmonary resuscitation (ECPR) describes the emergent use of extracorporeal membrane oxygenation (ECMO) to restore circulation in patients during cardiac arrest . OptimalThe coronavirus disease 2019 (COVID-19) pandemic poses additional challenges to the safe and appropriate use of ECPR. Prioritising healthcare worker safety whilst facilitating expedient cannulation is fraught with complexity and presents a considerable barrier to the effective implementation of ECPR in this setting . ApproprInterventions pertaining to the resuscitation of a patient in cardiac arrest are time critical. When cardiac arrest occurs, blood flow ceases and the resulting “no-flow” state rapidly produces irreversible neurological and multiorgan damage, if not promptly ameliorated. Conventional CPR (CCPR) produces a “low-flow” state that can temporarily sustain organ function. However, the longer the patient remains in cardiac arrest receiving CCPR, the less likely the patient is to achieve return of spontaneous circulation (ROSC), even when advanced life support measures are applied .The application of ECMO to maintain organ perfusion is a well-established technique extrapolated from the use of cardiopulmonary bypass in cardiothoracic surgery, where an anesthetised patient is cannulated and mechanical circulatory support is initiated before the heart is arrested. In the case of ECPR, the patient is unconscious due to loss of cardiac output, and in this uncontrolled situation, the cannulation and establishment of mechanical circulatory support must occur rapidly. The American Heart Association (AHA), in their 2019 update, support consideration of ECPR for those failing CCPR, where it can be “expeditiously implemented, and supported by skilled providers” . Howeverin-hospital cannulation, whereby patients suffering an IHCA or OHCA who fail to achieve ROSC with standard CCPR and advanced cardiac life support (ACLS) may be cannulated for ECPR. The site of cannulation is often the emergency department (ED), or cardiac catheterisation laboratory for OHCA, or the intensive care unit (ICU), operating theatre, or cardiac catheterisation laboratory for patients with IHCA. The second is pre-hospital cannulation of patients suffering OHCA refractory to ACLS and who are attended at scene by a mobile ECPR cannulation team. Current data suggest that time to cannulation is a more important determinant of ECPR outcome than the site where cannulation occurs per se [There are currently two main models of ECPR provision  Fig. 1)1). The fs per se , and thas per se .Fig. 1CIdentifying the group of patients who might benefit from ECPR is difficult. Having a reversible (predominantly cardiac) underlying aetiology for the arrest , the recThe timing of transition from CCPR to the institution of ECPR is not universally agreed on. This conversion may be facilitated by the application of a mechanical CPR device to continue chest compressions whilst cannulation for ECPR is performed. Following restoration of organ perfusion with ECPR, a targeted intervention to address the underlying aetiology of arrest must be performed. Cardiac arrests of presumed cardiac origin have been disproportionately represented in ECPR studies; hence, high rates of angiography and subsequent percutaneous coronary intervention (PCI) are often seen . This reSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may lead to a multisystem illness, COVID-19, in many patients. The majority of infected individuals either are asymptomatic or suffer a mild respiratory tract infection. Approximately 15% of individuals with COVID-19 will develop a more severe illness, and 5–6% will develop critical illness characterised by severe respiratory failure with acute respiratory distress syndrome (ARDS) . AdditioCentres with lesser experience or without established ECPR programmes are discouraged from initiating ECPR for OHCA during surge situations.non-COVID-19 patients depending on resource availability, whilst ECPR use in COVID-19-positive patients requires reflection on the risk-benefit ratio.Experienced centres may offer ECPR for IHCA for highly selected Emergency conversion of venovenous ECMO to venoarterial ECMO in the setting of a cardiac arrest in a patient receiving venovenous ECMO or during cannulation is not recommended—due to the poor outcomes anticipated.ECPR is complex, and efficient deployment relies on finely honed processes that may be significantly impacted by pandemic conditions. In acknowledging these circumstances, the current guidance on ECPR provided by ELSO  states tThere are two scenarios that might be used to describe the aetiology of cardiac arrest during the COVID-19 pandemic. First, a cardiac arrest occurring in a patient who does not have COVID-19 would be presumed to be due to one of the currently understood aetiologies of OHCA and IHCA. These patients would be eligible for consideration of ECPR based on currently used criteria where resources are not constrained by the pandemic. The second is cardiac arrest in a patient who is known, or suspected, to have COVID-19. In this case, the aetiology of cardiac arrest may be related to the effects of the SARS-CoV-2 virus, as abovementioned, or the virus may simply be a bystander. In all circumstances, as community transmission of COVID-19 increases, it will become difficult to differentiate patients at presentation with respect to infectious status and the default will be a presumption of positivity. Irrespective of their infectious status and the aetiology of the cardiac arrest, the resource constraints imposed by the pandemic may limit usual processes of care. Delays in CCPR initiation due to the reluctance of members of the public and healthcare workers to initiate out-of-hospital resuscitation attempts, recognising those patients who might benefit from ECPR, requirements for donning personal protective equipment (PPE), impaired ambulance response times, and lack of critical care resources, may preclude the use of ECPR even in those who would otherwise be eligible.Hypoxaemic respiratory failure leading to cardiac arrest appears to be common in COVID-19 patients. In a retrospective cohort study of 136 patients suffering IHCA in Wuhan, China, 87.5% of arrests were due to a respiratory aetiology. The vast majority occurred outside of an ICU setting, and shockable rhythms were observed in only 5.9% of this cohort. Survival outcomes were dismal in this cohort with only one patient surviving to 30 days with a favourable neurologic outcome  , 21). ItIn a true surge crisis, critical care demand outstrips capacity, and it becomes untenable to provide ECPR and post-resuscitative support. Establishing even basic CCPR may be hindered by delayed response times, time to allow PPE donning, and system pressures diverting the resuscitation team. Exceptions would include patients who arrest during coronary angiography, for instance, in which case rapid cannulation is possible, the aetiology may be more amenable to reversal, and there is the added benefit of advanced imaging available to confirm cannula placement should ECPR be initiated. Continuing access to ECPR during the COIVD-19 pandemic requires adherence to surge-specific protocols and prompt involvement of senior decision-makers at the time of an arrest in order to promote acceptable outcomes. Local tools could be developed to aid with rapid assessment of ECPR candidacy and feasibility .As with CCPR, ad hoc decision-making regarding ECPR should be discouraged. Goals of care and resuscitation status should be addressed transparently with patients and surrogate decision-makers, so that any limitations dictated by patient or system factors are explicit. The nature of ECPR often precludes such discussions from happening in real-time, only adding to the burden of responsibility on clinicians. The ECPR team response in COVID-19 Fig.  is complProvision of ECPR should ideally be an interdisciplinary decision and is best prepared for via high-fidelity simulation and streamlined cannulation teams , 20. Forsaturation are fluid judgements, relying on continual cycles of appraisal, integrating real-time data, and epidemiological projections [A fundamental principle underpinning all pandemic responses is the maximisation of benefit from scarce resources . Resourcjections . MaximisBeyond the immediate intervention, there are ongoing costs to convalescence. Although survivors of ECPR may demonstrate favourable neurologic outcome, the COVID-19 cohort cannot be assumed to be typical. The potential for generating dependent survivors is a burden ECPR may impose, and the benefits of ECPR may be overestimated in the COVID-19 cohort. There may be survivors with ECPR during the COIVD-19 pandemic, but at what individual and health opportunity cost? If resources are committed to ECPR early during a surge, ongoing availability of ICU resources may be further limited to other patients, infected or not , who havwith cardiac arrest, not death [The right to withhold life-sustaining treatment varies globally . In Denmot death . In someot death . With reot death , discernot death .Some centres routinely mandate ethicist consultations in the withdrawal of ECMO support . The urgObservational data suggests that ECPR provides an improved opportunity for favourable neurological survival in highly selected patients experiencing cardiac arrest compared with CCPR. Whilst there is a need for prospective study and high-quality randomised trials in this area , the cur