Normothermic regional perfusion (NRP) is a novel technique that aids organ recovery from donors after circulatory death (DCDs). However, ethical concerns exist regarding the potential return of spontaneous cerebral and cardiac activity (ROSCCA). This study aimed to determine the likelihood of ROSCCA in NRP‐DCDs of abdominal organs.Extracorporeal cardiopulmonary resuscitation (ECPR) for refractory out‐of‐hospital cardiac arrest (OOHCA) was identified as a comparator for NRP‐DCDs and as a validation cohort. A systematic search identified all articles relating to NRP‐DCDs and ECPR‐OOHCA. Rates of ROSCCA and survival outcomes (ECPR‐OOHCA only) were recorded and analysed according to the duration of no perfusion.In NRP‐DCDs, 12 of 410 articles identified by database searching were eligible for inclusion. There were no instances of ROSCCA recorded among 493 donors. In ECPR‐OOHCA, eight of 947 screened articles were eligible for inclusion (254 patients). Where the absence of perfusion exceeded 5 min in ECPR‐OOHCA, there were no survivors with a favourable neurological outcome.ROSCCA is unlikely following commencement of NRP and has not occurred to date. Strict observance of the 5‐min interval following asystole provides satisfactory assurance that ROSCCA will not occur following NRP. Normothermic regional perfusion (NRP) is a technique that aids organ recovery from donors after circulatory death (DCDs), leading to acceptable transplantation outcomesversus central) and flow rates (lower in NRP).NRP closely resembles other extracorporeal membrane oxygenation (ECMO) circuits that can be used for cardiopulmonary bypass, extracorporeal cardiopulmonary resuscitation (ECPR) or for ventilatory assistance in patients with refractory acute lung injury. ECMO provides mechanical ventilatory and circulatory support via an extracorporeal circuit incorporating a membrane oxygenator, centrifugal pump or roller, heat exchanger and perfusate reservoir. NRP and ECMO have differences, which include the anatomical location of cannulas ; and the potential for reperfusion to lead to return of spontaneous cerebral and cardiac activity (ROSCCA). The latter could invalidate the declaration of death, thereby threatening the fundamental tenet of deceased donation following cardiac arrest. ROSCCA can be assured categorically only by exclusion of the coronary and cerebral circulations from the NRP circuit. In abdominal organ donors, this is achieved by mechanical occlusion of the supracoeliac aorta most commonly using an inflated balloon, or by cross‐clamping. Nonetheless, the potential for ROSCCA to occur while using an inflated aortic occlusion balloon remains poorly understood and characterized. Concerns have been expressed that NRP, with or without inflation of an intra‐aortic occlusion balloon, provides an unsatisfactory assurance that ROSCCA would not occurThis study aimed to determine the likelihood of ROSCCA in NRP‐DCDs of abdominal organs, and the impact an inflated intra‐aortic occlusion balloon may have on the likelihood of ROSCCA. The ethical implications of this increasingly used organ preservation technique in an era of donor organ shortage are also explored.A validation cohort of patients who most closely resembled organ donors, but who received ECMO for therapeutic indications without an inflated intra‐aortic occlusion balloon, was identified to determine the likelihood of ROSCCA occurring in NRP‐DCDs. Patients receiving ECMO as part of resuscitative measures following cardiac arrest ) were considered an appropriate comparator for NRP‐DCDs. This is because ECPR is employed for refractory cardiac arrest where cessation of resuscitative efforts would lead to the inevitable declaration of death.The technical and mechanical components of NRP and ECPR are virtually identical, but two important distinctions exist. First, ECPR has resuscitative intent whereas the intent of NRP is preservation; and, second, there is no intra‐aortic occlusion balloon in ECPR.In OOHCA, a variable period of absent perfusion (no‐flow time) exists between the onset of cardiac arrest and the commencement of cardiopulmonary resuscitation (CPR) with chest compressions, either by a bystander or a trained professional. A period of low flow exists from the commencement of mechanical compressive CPR until the return of perfusion by either return of spontaneous circulation or ECPR.In the organ donation process, stand‐off time refers to the interval after declaration of death up to commencement of the donor perfusion and organ preservation process. A stand‐off time, commonly 5 min, is necessary in most jurisdictions to ensure that autoresuscitation does not take place and that the declaration of death is validA search of MEDLINE, Embase and the Cochrane Library electronic databases was performed to identify all articles relating to NRP for DCDs of abdominal organs. The following Medical Subject Heading (MeSH) terms were used and combined using Boolean operators: ‘extracorporeal membrane oxygenation’, ‘donors after cardiac death’, ‘non‐heart‐beating donors’, ‘donors after circulatory death’, ‘normothermic recirculation’, ‘normothermic perfusion’, ‘regional perfusion’, ‘liver transplantation’, ‘kidney transplantation’ and ‘pancreas transplantation’. A further search was carried out in an identical manner to identify all articles relating to ECPR‐OOHCA. The following MeSH terms were used: ‘extracorporeal life support’, ‘extracorporeal resuscitation’, ‘extracorporeal cardiopulmonary resuscitation’, ‘extracorporeal membrane oxygenation’ and ‘out‐of‐hospital cardiac arrest’. References of all identified papers were searched to ensure a comprehensive review. PRISMA guidelines were followedex situ perfusion process were also excluded. For the ECPR‐OOHCA search, articles that did not describe the relationship between no‐flow time and outcome were excluded. Publications reporting outcomes in ECPR for in‐hospital cardiac arrest were excluded, because the mean delay from cardiac arrest to commencement of CPR is much shorter than in OOHCA.All study designs including cohort studies, case–control studies and case series were considered eligible. Studies performed between January 1997 and June 2016 were included. Case reports, conference abstracts, review articles, animal studies and articles not written in English were excluded. For the NRP‐DCD search, studies of organ donation of non‐abdominal organs and those using an Data extraction was undertaken using a standard pro forma. Patient and donor age, perfusion flow rates, and rates of failure in establishing successful perfusion were recorded. The NRP and ECPR protocols were interrogated, and exclusion and inclusion criteria identified and compared. Where recorded, methods of preventing and identifying cerebral and cardiac perfusion in NRP‐DCDs were also identified. In ECPR‐OOHCA, the duration of no flow was recorded.TableSurvival outcomes  and cause of death following ECPR‐OOHCA were recorded and analysed according to the duration of absence of perfusion. Neurological outcomes were considered favourable if recorded as having a cerebral performance category of 1 or 2  survived to discharge, either in a persistent vegetative state or with significant neurological disability. It is not possible using the existing data to provide categorical assurance that ROSCCA would not occur in NRP‐DCDs in the absence of a proximally occluded aorta. The periods of no flow and low flow preceding the stand‐off time, in uncontrolled and controlled DCDs respectively, inevitably compound the ischaemic insult and further reduce the likelihood of ROSCCA.et al.Japan presents an interesting situation in that organ donation after brain death, although legal, is not acceptable culturally, but the country also has the largest worldwide practice of ECPR‐OOHCAOrgan donation from DCDs is a sensitive issue, with associated fears of NRP‐related ROSCCA that are both appreciable and justified, especially given the reported incidents of prolonged survival after brain deathAn inflated occlusion balloon in the proximal aorta has significant advantages, as perfusion can be commenced following a minimally invasive approach, and allows organ assessment before a definitive decision on organ procurement is made. In some instances, it is possible that the integrity of the inflated aortic occlusion balloon may have been compromised, but went unrecognized and unreported. However, balloon integrity can be confirmed by the presence/absence of a radial BP signal, both before and after commencing NRP. In the UK, cross‐clamping the thoracic aorta following rapid thoracolaparotomy is the preferred method for NRP‐assisted controlled DCD procurement of abdominal organs. However, thoracotomy ought not be a prerequisite for NRP, and minimally invasive NRP provides particular advantages in uncontrolled donors in societies where a presumed consent process is part of the legislation.It has been mooted that NRP‐DCDs without a proximal occlusion of the aorta would be more appropriately termed as donors after brain circulatory death because recirculation of blood occurs following NRP, and that this procedure should therefore require specific consentIn countries where ECPR‐OOHCA is practised routinely, the boundaries between ECPR for resuscitative purposes and NRP as an organ preservation technique may become blurred. Where a patient resuscitated by ECPR has a cerebral performance category of 4 or 5 but is dependent on ECMO for circulatory support, management of the potential organ donor should proceed identically to that for ECMO‐independent patients. However, ECMO‐independent patients existing in a vegetative state present a very challenging set of circumstances that requires careful individual consideration by families, clinicians and judicial representatives.The main limitation of this study is the small number of ECPR‐OOHCA papers that were eligible for inclusion to approximate NRP‐DCD donation. Only eight of 947 potential papers on ECPR‐OOHCA were eligible and a proportion of articles, especially those from Japan, were excluded; this could have contributed to unintended bias. The criteria for the declaration of death, in particular brain death, remain controversial across the spectrum of clinical, scientific and religious communities, including within societies where organ donation practices are well establishedThis study has demonstrated that ROSCCA leading to survival with favourable neurological outcome following ECPR‐OOHCA does not occur when the duration of no flow exceeds 5 min. This suggests a similar process in NRP‐DCDs, with a stand‐off time of 5 min. ROSCCA in NRP‐DCDs with occlusion of the proximal aorta has not been reported to date. The presence of an occlusion balloon provides an additional mechanical level of assurance in a very sensitive clinical scenario. This is particularly important as any adverse events would have a catastrophic impact on the public perception of organ donation and transplantation.Table S1 Demographics and outcomes from the normothermic donor after circulatory death (NRP‐DCD) programmesTable S2 Demographics and outcomes from the extracorporeal cardiopulmonary resuscitation for out of hospital cardiac arrest (ECPR‐OOHCA) programmesClick here for additional data file.


IL)‐1β and IL‐18, and additionally by inducing pyroptosis, a lytic cell death mode that releases additional inflammatory mediators. Microglia are the prominent innate immune cells in the brain for inflammasome activation. However, additional CNS‐resident cell types including astrocytes and neurons, as well as infiltrating myeloid cells from the periphery, express and activate inflammasomes. In this review, we will discuss current understanding of the role of inflammasomes in common degenerative diseases of the brain and highlight inflammasome‐targeted strategies that may potentially treat these diseases.Neuroinflammation and neurodegeneration often result from the aberrant deposition of aggregated host proteins, including amyloid‐β, α‐synuclein, and prions, that can activate inflammasomes. Inflammasomes function as intracellular sensors of both microbial pathogens and foreign as well as host‐derived danger signals. Upon activation, they induce an innate immune response by secreting the inflammatory cytokines interleukin ( Blood–brain barrier (BBB)a physiological barrier between the blood and the CNS parenchyma. The BBB is formed by endothelial cells that are joined by complex tight junctions and coated by a basement membrane and an additional membrane of astrocytic end‐feet, known as the glia limitans.Caspasesa family of cysteine‐dependent aspartate specific proteases that play a central role in inflammation and programmed cell death.Cerebrospinal fluid (CSF)contained in the ventricles of the brain and the cranial and spinal subarachnoid spaces. It provides a mean for transporting different molecules, including cytokines, neurotransmitters, and hormones, but also resides innate immune cells.CNS macrophagesnon‐parenchymal macrophages of the CNS that are located in meninges, choroid plexus, and perivascular spaces.Creutzfeldt–Jakob diseasefatal degenerative prion disease that can be sporadic, hereditary, or acquired. The acquired form of the disease is caused by exposure to the misfolded scrapie form of the PrP protein.Cuprizone‐induced demyelinationan experimental model to study local demyelination of the corpus callosum, which is induced by the administration of the copper chelator cuprizone (bis(cyclohexanone)oxaldihydrazone) to the food of mice during 5 weeks.Dopaminergic neuronsthe main dopamine‐producing cells of the CNS and essential for controlling key functions of the brain, including voluntary movement, reward processing, mood, and working memory.Encephalomyelitisgeneral term to describe inflammation in the brain or spinal cord. Autoimmune encephalomyelitis is caused by an abnormal immune response to a self‐antigen.Experimental autoimmune encephalomyelitis (EAE)the main rodent model of MS. EAE is actively induced by peripheral immunization with myelin‐specific proteins or peptides in combination with an adjuvant, or passively by transfer of encephalitogenic T cells.Familial Mediterranean Feverthe most common monogenic autoinflammatory disease and is characterized by periodic fevers with childhood onset, and frequently associated with serositis and joint pain. It predominantly affects Mediterranean populations.Grand‐mal seizuresor tonic‐clonic seizures: seizures that are characterized by the loss of consciousness and violent muscle contractions. This seizure comes in two phases: a tonic phase followed by a clonic phase. The brief tonic phase features loss of consciousness and muscle stiffening, while in the clonic phase the muscles go into rhythmic contractions.Induced pluripotent stem cells (iPSCs)generated by genetic reprogramming of adult somatic cells. The advantage over other types of stem cells is that they are not derived from a human embryo.Ischemic infarctan infarct caused by the interruption of the blood flow in the brain due to occlusion of a cerebral vessel.Middle cerebral artery occlusionexperimental stroke model that involves the permanent or transient occlusion of the middle cerebral artery. The middle cerebral artery is one of the three arteries that supply blood to the cerebrum. In the “filament model”, a suture filament is (transiently) introduced into the internal carotid artery and forwarded until the tip occludes the middle cerebral artery.Neonatal‐onset multisystem inflammatory disease (NOMID)or chronic infantile neurologic cutaneous and articular (CINCA): the most severe cryopyrin‐associated periodic syndrome (CAPS). NOMID is characterized by neonatal‐onset skin lesions, chronic aseptic meningitis, and recurrent fever along with joint symptoms.Sepsisa life‐threatening systemic inflammatory response syndrome (SIRS) caused by the body's response to an infection.Spatial memoryan important cognitive function that allows us to recall three‐dimensional objects or places.Steatohepatitisa type of fatty liver disease and is characterized by the accumulation of lipids and the infiltration of inflammatory cells in the hepatic parenchyma.Striatadopamine neurons from the substantia nigra pars compacta project to the caudate and the putamen of the basal ganglia, together referred to as the striatum.Substantia nigra pars compactastructure in the brainstem that contains most of the dopamine‐producing neurons.et al, et al, The innate immune system is a rapid and coordinated cellular defense response that aims to eliminate the threat posed by both sterile and infectious insults. Recognition of these pathogenic agents is mediated by pattern recognition receptors (PRRs) that sense pathogen‐associated molecular patterns (PAMPs) and host‐ or environment‐derived danger‐associated molecular patterns (DAMPs). In the central nervous system (CNS), these PRRs are primarily expressed by microglia, astrocytes, and macrophages, but also oligodendrocytes, neurons, and endothelial cells express a repertoire of PRRs  that is responsible for the maturation and secretion of the inflammatory cytokines IL‐1β and IL‐18, and additionally induce pyroptosis   is the most common chronic inflammatory disease of the CNS. MS is characterized by a compromised blood–brain barrier (BBB) that leads to immune cell infiltration from the periphery and local microglia and astrocyte activation, which together promote inflammation, demyelination, and neurodegeneration  have significantly higher levels of inflammasome markers in their CSF, including ASC, caspase‐1, NLRP1, and NLRP3  induced higher levels of NLRP1, ASC, caspase‐1, IL‐1β, and IL‐18 in mice, and therapeutic neutralization of ASC was proposed to result in tissue sparing and functional improvement due to reduced inflammasome activation in neurons (de Rivero Vaccari Alzheimer's disease (AD) is the most common age‐related neurodegenerative disorder and is characterized by progressive memory deficits and cognitive impairment , which resulted in increased vulnerability to intracerebroventricular injection with human oligomeric Aβ1–42  is a fatal neurodegenerative disease that is caused by the progressive loss of upper and lower motor neurons and leads to muscle weakness and wasting  is a progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra pars compacta. Lewy bodies are pathological hallmarks of the disease that predominantly consist of intra‐neuronal aggregates of fibrillar α‐synuclein   is an autosomal dominant progressive neurodegenerative disease that is caused by the expansion of a trinucleotide CAG repeat in the 5′ coding region of the huntingtin gene, leading to the expression of an abnormal protein that gradually damages cells in the brain , canakinumab , and rilonacept (soluble decoy receptor for IL‐1β and IL‐1α) , or caspase‐1, caspase‐4, and caspase‐5 (human) releases an amino‐terminal pore‐forming GSDMD domain that oligomerizes and inserts in the plasma membrane, inducing membrane rupture and leakage of the cytosolic contents  conditions.Most research in the field has focused on the role of the NLRP3 inflammasome, largely neglecting the potential importance of other inflammasome complexes in CNS disorders. Future research will have to investigate and characterize less well‐known inflammasomes and expand the understanding of inflammasome signaling and its importance for neurodegeneration. The generation of new mouse models and reagents that allow selective targeting of the inflammatory mechanisms initiated by these inflammasomes will be crucial to this end. Also, although the importance of inflammasome activation has been demonstrated in many neurodegenerative diseases and experimental model systems, their specific role in the different CNS cell types is not always clear. Most research has focused on microglia, but other brain cell types also have functional inflammasomes which may contribute to disease pathology (Walsh NLRP1 gene, while mice have three isoforms of Nlrp1. Similarly, humans have two orthologues for caspase‐11, viz. caspase‐4 and caspase‐5 (Lamkanfi & Dixit, Major progress needs to be made in translating findings from animal studies to humans. However, key differences exist between human and mouse inflammasomes. Humans have only a single et al, et al, et al, et al, Finally, since inflammasome signaling is central to many neurodegenerative diseases, targeting their activation is regarded as a potential therapy to treat these diseases. Targeting the downstream IL‐1β and IL‐18 cytokines is probably not the best approach since these cytokines are needed for optimal immunity in the host. Moreover, cytokine‐targeting biologics have limited capacity to penetrate the blood–brain barrier. Therefore, modulating the activity of specific inflammasome sensors and cell type‐specific inflammasome targeting with small molecules will be vital in this context and may have better potential as therapies to treat patients that suffer from neurodegenerative diseases (Guo The authors declare that they have no conflict of interest.The cell type‐specific contribution of inflammasome signaling to neurodegenerative diseases, both peripheral and CNS resident.Direct evidence for inflammasome activation and signaling in patients with neurodegenerative disease.Characterization of the central activation mechanism and high‐resolution structure of inflammasome proteins.Development of compounds able to cross the BBB and that selectively inhibits those inflammasomes that contribute to the pathogenicity.


Viruses with a small genome encode only a few immunomodulatory proteins, but these are often multifunctional, such as the NS1 protein from influenza A virus, which inhibits RNA sensing in multiple ways. Intracellular bacteria and protozoa can also be detected by nucleic acid sensors. However, as the type I interferon response is not always beneficial for the host under these circumstances, some bacteria subvert, rather than evade, these signalling cascades for their own gain.Intracellular DNA and RNA sensors play a vital part in the innate immune response to viruses and other intracellular pathogens, causing the secretion of type I interferons, cytokines and chemokines from infected cells. Pathogen RNA can be detected by retinoic‐acid inducible gene I‐like receptors in the cytosol, whereas cytosolic DNA is recognized by DNA sensors such as cyclic GMP‐AMP synthase (cGAS). The resulting local immune response, which is initiated within hours of infection, is able to eliminate many pathogens before they are able to establish an infection in the host. For this reason, all viruses, and some intracellular bacteria and protozoa, need to evade detection by nucleic acid sensors. Immune evasion strategies include the sequestration and modification of nucleic acids, and the inhibition or degradation of host factors involved in innate immune signalling. Large DNA viruses, such as herpesviruses, often use multiple viral proteins to inhibit signalling cascades at several different points; for instance herpes simplex virus 1 targets both DNA sensors cGAS and interferon‐ In addition, many cell types can also recognize when they have been infected themselves by an intracellular pathogen using intracellular PRRs that constantly survey the cell's contents for signs of infection. Intracellular PRRs raise the alarm either after sensing the presence of PAMPs directly, or after detecting the indirect effects of infection or tissue damage as danger‐associated molecular patterns.Our innate immune system provides a rapid, if relatively non‐specific, response to invading pathogens. The cornerstones of innate immunity are the pattern recognition receptors (PRRs), which detect pathogen‐associated molecular patterns (PAMPs) during infection. PAMPs are key molecular features shared by classes of pathogens that are usually absent from healthy cells, and include, for instance, bacterial or fungal cell wall components, or viral nucleic acids that would not usually be found in the host cell. PAMPs can be recognized Intracellular nucleic acid sensors are PRRs that can perform both those functions: they can recognize pathogen‐derived DNA or RNA species inside the cell, and also detect nucleic acids as danger signals, for instance when infection leads to the leakage of mitochondrial DNA or RNA into the cytosol. Intracellular nucleic acid receptors are particularly important in the detection of viruses, which lack many other conspicuous features such as the unique cell wall components of bacteria or protozoa. However, nucleic acids are starting to emerge as key mediators during the innate immune response to a range of pathogens, including intracellular bacteria and protozoa.κB (NF‐κB) and interferon regulatory factor 3 (IRF3). Many pathogens also block innate immune responses at the level of these downstream signalling nodes, or by inhibiting the function of interferons and cytokines after they have been secreted, and we refer the reader to further reviews on this topic.In this review, we will focus on the intracellular DNA and RNA sensors that induce a transcriptional response leading to the production of interferons, cytokines and chemokines. The rapidly inducible transcriptional programme is a key factor in the early elimination of pathogens and for the initiation of appropriate local immune responses that ultimately determine the outcome of an infection. For this reason, it can be assumed that all pathogens need to evade or subvert the host's innate immune responses to be able to establish an infection. Here, we will describe the strategies that intracellular pathogens use to hide from the cell's nucleic acid receptors, and to disable or subvert their function. Ultimately, the activation of signalling cascades by nucleic acid sensors and many other PRRs converges on the activation of transcription factors such as nuclear factor‐The RIG‐I‐like receptors (RLRs) retinoic‐acid inducible gene I (RIG‐I) and melanoma differentiation‐associated protein 5 (MDA5) recognize exogenous RNA in the cytosol of infected cells. RIG‐I detects short stretches of dsRNA as well as RNA species containing a triphosphate or diphosphate moiety at their 5′ end, and MDA5 recognizes long dsRNA molecules and higher‐order aggregates of viral dsRNA.κB, which co‐operate in the activation of the interferon‐β promoter. RLR signalling is further regulated by the addition of K48‐linked and K11‐linked ubiquitin chains, which mediate the degradation of RIG‐I and MAVS.Following detection of specific RNA ligands in the cytosol, RIG‐I and MDA5 undergo post‐translational modifications such as dephosphorylation and polyubiquitylationFlaviviridae and Coronaviridae families.Caliciviridae family, such as norovirus.Legionella pneumophila, Salmonella typhimurium and Shigella flexneri.The RLRs have been implicated in the detection of many viruses, as well as other intracellular pathogens. Most viral RNA polymerases generate transcripts with 5′ triphosphate ends and many viruses also generate dsRNA during their life cycle. Hence, both RIG‐I and MDA5 are predicted to be able to detect the dsRNA genomes of reoviruses and the dsRNA replicative intermediates of the positive‐strand ssRNA viruses of the Many mammalian cell types can also detect exogenous DNA. Bacterial, viral or synthetic dsDNA, and even dsDNA isolated from mammalian cells, can be sensed, if it gains access to the cytosol and is over 40–50 bp in length.γ‐inducible protein 16 (IFI16), which co‐operates with cGAS in the activation of STING in human cells.β production. Other DNA sensors include DNA‐dependent activator of interferon‐regulatory factors (DAI),Additional DNA binding proteins have been proposed to function as DNA sensors.Plasmodium falciparum and intracellular bacteria including Mycobacterium tuberculosis and Listeria monocytogenes.The cytosolic DNA sensing pathway has been shown to be crucial for the detection of many different DNA viruses and retroviruses.Mycobacterium tuberculosis and Listeria monocytogenes.During bacterial infection, the DNA sensing adaptor STING has a second function and can act as a PRR in its own right. STING can sense bacterial signalling molecules such as cyclic di‐GMP and cyclic di‐AMP.Pathogens and their hosts co‐evolve, and there are clear signs of selective pressure on host factors such as DNA and RNA sensors on the one hand and pathogen‐encoded immune evasion proteins on the other.Mycobacterium tuberculosis is responsible for the exposure of bacterial DNA to cGAS in macrophages, but also secretes virulence factors that interfere with host functions.Pathogens that reside inside host cells are already sheltered from the immune system to some extent, by avoiding exposure to antibodies or complement components. However, due to the existence of intracellular PRRs, additional camouflage strategies are essential for the establishment of infection. Intracellular bacteria and protozoan parasites often co‐opt vacuoles as a niche for replication, which prevents pathogen‐derived nucleic acids and other PAMPs from being exposed to the cell's PRRs. Hence, detection of intracellular nucleic acids during bacterial infection often requires the presence of bacterial secretion systems. For instance, the type VII secretion system ESX from Many pathogens aim to keep PAMP levels at a minimum: coronaviruses for instance quickly degrade any excessive dsRNA formed during the viral life cycle,Picornaviridae, Caliciviridae and Astroviridae covalently attach a protein, Vpg, to the 5′ end of viral RNAs, which prevents RIG‐I‐mediated recognition.Bornaviridae and Bunyaviridae families involves the cleavage of the first nucleotide by a viral endonuclease to generate a monophosphate group at the 5′ end.Herpesviridae and Papillomaviridae families and retroviruses such as human immunodeficiency virus, co‐opt the cell's own capping machinery to protect the 5′ end of their mRNAs with a 7‐methylguanosine cap, while others, including IAV, use ‘cap snatching’ to remove the capped 5′ ends from cellular mRNAs and incorporate them into viral RNA transcripts.O‐methylation of the first RNA nucleotide next to the cap, which is found in cellular mRNAs and further decreases recognition by RIG‐I.As viral RNA polymerases generate RNA species with a 5′ triphosphate moiety, many viruses employ additional strategies to modify their RNA, so that it is not recognized efficiently by RIG‐I. For instance, So far, it is not known whether DNA modifications could prevent recognition by cGAS or other DNA sensors. It has been proposed that IFI16 may be able to distinguish viral DNA from the cell's own genome by recognizing longer (>40 nucleotides) stretches of free DNA,All intracellular pathogens encode virulence factors that interfere with the host's signalling cascades. Viral immunomodulatory proteins are usually expressed as immediate early genes, or are even part of the viral particle, so that they are able to inhibit the host's signalling cascades as soon as PAMPs are detected early in infection. Intracellular bacteria employ secretion systems to transport effector proteins through the vacuolar and bacterial membranes into the host cell, whereas protozoans can secrete virulence factors via exosomes or other membrane‐bound vesicles. A vast repertoire of virulence factors that influence the cell's innate immune signalling cascades have been described, particularly in viruses, which often dedicate a large portion of their genome to interfering with the host's innate immune response. Although many viruses encode only a few proteins, these are often multi‐functional and target several key host factors. Large DNA viruses, such as poxviruses and herpesviruses, encode a larger repertoire of dozens of immunomodulatory proteins that interfere with anti‐viral signalling cascades in the cell at multiple points.Many pathogens have evolved similar strategies to inhibit the innate immune signalling cascades in the host cell: virulence factors act to either eliminate host signalling factors by degradation, sequester them or block their function in the signalling cascade. As we learn more about the regulation of innate immune signalling cascades that detect pathogens, more examples of counterstrategies employed by pathogens also emerge.pro from foot‐and‐mouth‐disease virus also cleaves Lpg2, a co‐factor for MDA5 activation.Since the discovery of the intracellular RNA sensing system, many immune evasion proteins that target RLRs or MAVS have been identified see Fig. , mostly Even under conditions where the expression of RIG‐I and MDA5 is intact, the RNA sensors can be prevented from carrying out their signalling function by viral proteins that bind to them. The HSV‐1 protein UL37 interacts with RIG‐I and causes the de‐amidation of its helicase domain, which renders RIG‐I unable to sense RNA ligands.Vibrio vulnificus. Cyclo(Phe‐Pro) specifically binds to RIG‐I and prevents it from being ubiquitylated by TRIM25,The activity of RIG‐I and MDA5 is tightly regulated in the cell: In the absence of infection, the CARD of the RNA sensors are kept in a phosphorylated state, and activation involves their dephosphorylation catalysed by protein phosphatase 1.ε to prevent the translocation of RIG‐I to MAVS.pro proteases, the Z and N proteins of some arenaviruses, NS2 from respiratory syncytial virus, NS1 from IAV, NSP1 from rotavirus and US11 from HSV‐1.A multitude of viral immunomodulators prevent RIG‐I and MDA5 from interacting with its adaptor protein MAVS, so blocking the nucleation event that allows MAVS to form higher‐order signalling assemblies. Dengue virus NS3 binds 14‐3‐3MAVS is also targeted directly by many viruses. MAVS‐interacting proteins include the metapneumovirus protein M2‐2,Salmonella typhimurium uses its effector protein SopA, an E3 ubiquitin ligase, to ubiquitylate the host proteins TRIM56 and TRIM65, which in turn promote RIG‐I and MDA5 signalling.Although there are abundant examples of inhibition of RNA sensing by pathogens, and in particular viruses, there is one instance where a pathogen actually enhances signalling by RIG‐I and MDA5. The food‐borne pathogen Even though the cGAS/STING DNA sensing pathway was only discovered relatively recently, several viral and bacterial virulence factors that target this pathway have already been discovered, see Fig. Surprisingly, not only DNA viruses but also some RNA viruses have been found to antagonise STING – even though they do not produce DNA or cyclic dinucleotides during their replication cycle. It has been reported that STING can function in a cGAS‐independent manner during the detection of viral membrane fusion, for instance during infection with IAV.Shigella effector protein IpaJ inhibits STING translocation from the endoplasmic reticulum to endoplasmic reticulum–Golgi intermediate compartment,Yersinia YopJ protein also blocks STING trafficking and causes its de‐ubiquitylation.Mycobacterium tuberculosis secretes the cyclic di‐nucleotide phosphodiesterase CdnP , which degrades cGAMP and bacterial cyclic dinucleotides, thus inhibiting both DNA‐ and cyclic‐dinucleotide‐induced STING activation.Streptococcus pyogenes, subverts, rather than evades, STING signalling: it uses its M protein to activate STING, resulting in the production of the anti‐inflammatory cytokine interleukin‐10 downstream of type I interferon signalling.While the production of type I interferons is not always an effective strategy to limit bacterial infections, the activation of STING by bacterial DNA or cyclic dinucleotides can also promote autophagy to clear bacteria from the infected cells. For this reason, some intracellular bacteria also inhibit STING.All pathogens, having co‐evolved with their hosts, employ elaborate strategies to strike a fine balance between the requirements of their own life cycle, the evasion of host defences, and effects on pathology for the host, which can influence further transmission and hence the evolutionary fitness of a pathogen. As in so many fields in biology, much can be learnt from insights into the host–pathogen interactions that interfere with our cells' defences. Immunomodulatory proteins from pathogens can point us to their targets, which are often key signalling nodes that play a role during infection. Although immune evasion has long been studied in human cells and mouse models, the investigation of innate immune signalling in reservoir hosts such as bats or birds, is an emerging field of research. The variation of these interactions during pathogen evolution often underlies the ability of pathogens to cross species boundaries or to adopt new traits in transmission or virulence during emerging infections. Knowing more about the functions of individual immunomodulatory proteins is also crucial for the rational development of vaccine vectors, as for instance the removal of individual immune evasion factors can aid safety and immunogenicity of a live vaccine. It is becoming increasingly clear that nucleic acid PAMPs and their PRRs also perform key roles during sterile inflammation, autoimmunity and even cancer treatment, so the lessons we learn from our pathogens may ultimately have applications for the treatment and prevention of both infectious and non‐infectious diseases that involve the immune system.The authors declare no competing interests.


Lancet Diabetes Endocrinol 2016;4:1025–36). Understanding how, and to what extent, maternal body mass index (BMI) and weight gain during pregnancy contribute to adverse outcomes for mothers and their offspring is therefore vital to informing future health policy.Obesity is one of the biggest challenges facing global reproductive health. Women in the UK and USA are today more likely to be obese or overweight at booking than normal weight, and many low‐ and middle‐income countries (LMICs) seem destined to follow suit  confirm strong correlations between pre‐pregnancy maternal BMI and the risks of gestational hypertension, pre‐eclampsia and gestational diabetes. Over one‐third of such complications in the study population were considered attributable to maternal overweight and obesity. The risk of large size for gestational age (LGA) at birth increased similarly across all categories of pre‐pregnancy BMI and gestational weight gain, although these data should be interpreted in the context of a continuing debate regarding the customisation of fetal growth charts. It remains uncertain how maternal height and weight influence fetal growth potential, and whether LGA babies born to mothers who are obese or mothers with excessive weight gain carry the same short‐ and long‐term health risks as LGA babies born to mothers who are normal weight. Preterm birth was also more common among women who are obese and past literature has suggested that this association is strongest for extremely preterm delivery , whether spontaneous or iatrogenic.In an individual participant data meta‐analysis of over 265 000 births, Santos et al. . Notably, the overall burden of pregnancy complications is similar in overweight and obese groups . This calls into question traditional models of care targeting women with a booking BMI above 30 kg/mObes Rev 2015;16:758–70).The authors acknowledge that the data were derived from cohorts who were largely white; however, comparable findings have been reported in LMICs with varied ethnic populations . Regardless of BMI, all women have the capacity to make their pregnancy healthier and should be empowered to do so through improved access to lifestyle advice and interventions that are tailored to individual needs.Being healthy entails more than just not being obese, and the study also draws important attention to the risks of small size for gestational age and preterm birth, particularly amongst underweight women with inadequate weight gain during pregnancy. These findings strengthen the argument for novel public health approaches to optimise maternal health with a shift in focus towards pre‐conception and interpregnancy interventions delivered in the community, as outlined in a recent review (Hanson et al. None declared. Completed disclosure of interests form available to view online as supporting information. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Climate change, energy use and food security are the main challenges that our society is facing nowadays. Biofuels and feedstock from microalgae can be part of the solution if high and continuous production is to be ensured. This could be attained in year‐round, low cost, outdoor cultivation systems using strains that are not only champion producers of desired compounds but also have robust growth in a dynamic climate. Using microalgae strains adapted to the local conditions may be advantageous particularly in Nordic countries. Here, we review the current status of laboratory and outdoor‐scale cultivation in Nordic conditions of local strains for biofuel, high‐value compounds and water remediation. Strains suitable for biotechnological purposes were identified from the large and diverse pool represented by saline (NE Atlantic Ocean), brackish  and fresh water (lakes and rivers) sources. Energy‐efficient annual rotation for cultivation of strains well adapted to Nordic climate has the potential to provide high biomass yields for biotechnological purposes. EPAeicosapentaenoic acidDHAdocosahexaenoic acidNPQnon‐photochemical quenchingPSIIphotosystem IIPUFApolyunsaturated fatty acidsTAGtriacylglycerol2 to produce biomass in a similar way as in plant photosynthesis. Properties that make microalgae superior to classical energy crops  are the following: (1) 5–20‐fold more productive per ha basis; (2) can grow on non‐arable land and are able to use various water sources ; (3) allow multiple harvests because they double biomass in 24 h and can be produced throughout the year; (4) store lipids, mainly as triacylglycerols (TAGs), which are readily available for biodiesel production; (5) reduce greenhouse gas emissions, especially when using exhaust CO2 . The geographic location of Nordic countries covers two large masses of water: the saline waters of the NE Atlantic (including the North Sea) and the brackish waters of the Baltic Sea. Although apparently not the ideal place for large scale cultivation, microalgae could be part of the solution for sustainable energy and food production in Nordic countries. A global evaluation of biofuel production from microalgae using a validated outdoor photobioreactor shows that even Sweden can supplement 30% of the transportation fuel consumption by cultivating microalgae on 20% of the non‐arable land , our focus is on studies using eukaryotic microalgae.The five Nordic countries span the geographic area between 40 and 80°N and include zones with continental, subarctic and artic climate. The continental climate is milder than that of other countries at the same latitude  due to vicinity of the ocean and the effect of Gulf Stream bringing warm waters from the coast of Florida.2 fixation (50 Gt C year−1)  while during the polar winter, the sun is under the horizon for the entire duration of a day. For this particular area, light is the most important factor that controls the distribution and seasonal occurrence of various species  and high light, which is highly deleterious for photosystem II (PSII), artic microalgae respond by a constitutive induction of non‐photochemical quenching (NPQ)  and increased EPA content in laboratory scale experiments  they were outperformed in terms of EPA production by a commercial strain of P. tricornutum  and docosahexaenoic acid , in their membranes. These fatty acids are essential for human health by preventing cardiovascular and inflammatory diseases. A screening pipeline was employed at University of Bergen, Norway, on a collection of 150 microalgae clones isolated from various locations in the North Atlantic (60–80°N) . The study aims for a rotational and year‐round strategy of outdoor algae cultivation. This approach has been successfully implemented in Japan  and at around 20°C, which is the average summer temperature . Moreover, if closed photobioreactors are used, the temperature inside can reach 40°C during the summer  seemed to be carbon limitation. Although CO2 solubility is increased in cold water, the gas–liquid mass transfer is lower, so that the addition of concentrated CO2 (1–2.5%) becomes essential for high biomass production  overcome the adverse effects of low temperature on biochemical reactions by encoding enzymes with high catalytic efficiency and by increasing the level of membrane unsaturation , increased its biomass and even its lipid content when grown with 20–25% CO2   have mostly been investigated for wastewater treatment, but also for biomass accumulation and lipid production. Locally isolated microalgae and even microalgae‐bacteria consortia have been used with success at laboratory scale for removal of nitrate and phosphorus from wastewater and for production of biomass and lipids Table , b. The Scenedesmaceae family (UHCC0027) as the best producer of biomass and lipids. The potential of this strain to grow under the Nordic climate was further investigated by Jämsä et al. bioreactors exemplified by flat panels and tubular reactors  and flagellates  for oyster larvae feed; and Simris Alg AB cultivating microalgae for PUFA and fish oil using tubular photobioreactors in greenhouses. Such companies do not generally use waste resources for their cultivation due to the high‐quality demand on the products. In Iceland, however, flue gas from a geothermal power plant is filtered to reduce the concentration of H2S, and thereafter used for cultivation of local microalgae in geothermal brine, consisting of a mixture of one‐third freshwater and two‐thirds marine water with high silica content supplied with additional nutrients. Geothermal electricity is used for heating. Among diatoms and other microalgae, the coccoid cyanobacterial species Cyanobacterium aponinum is grown in tubular photobioreactors at the Blue Lagoon for skin care products  originally isolated from Southern Sweden was inoculated from start but it was later shown that local consortia dominated by Chlorella‐like microalgae soon developed in the Tethys ponds and grew well in the flue gas. Green algae of the Monoraphidium‐type dominated in a nearby raceway pond cultivated simultaneously. The study aimed for lipid production but most importantly showed that the special cultivation technique with harvesting only once a year was energy efficient .Several pilot scale cultivations at industrial locations in the Nordic countries have been or are currently being performed. Common to all these cultivation sites is the use of waste resources for different remediation purposes, often combined with studies on production of biomass for bio‐based bulk compounds, mainly lipids, carbohydrates and proteins. The Tethys cultivation ponds discussed above was tested outdoors in natural light at the Nordic Paper Bäckhammar AB pulp and paper mill factory in south‐central Sweden for 3 years. Heat from the wastewater treatment plant at the factory and flue gas from the soda boiler was used in the study. The freshwater green algae strain Tetradesmus, local communities evolved and Dictyosphaerium was the dominating genera after 3 years of cultivation. A potential for reduction of pharmaceuticals was demonstrated  closed tank used for cultivation. The main aims are CO2 neutrality for the factory and production of bioactive compounds, PUFA and essential amino acids mainly for fish fodder. They have plans to expand to industrial scale .Waste heat and flue gas containing COMunicipal and industry wastewater are used at the Kungshamn (near Gothenburg) facility of the Swedish Algae Factory to cultivate microalgae and produce biomass for fish feed and organic fertilizer. The wastewater is used in a circular economy concept, where no waste is generated and the biomass utilizations are diverse. Relying on the natural properties of diatoms adapted to low light, the nanoporous silica shell of cultivated microalgae is used to coat solar panels and by that increase their efficiency with 4%. The nanoporous silica also confers UV‐B protection extending microalgae shell's use for UV‐resistant paints, plastics and cosmetics.https://www.co2bio.no/) aims for production of biomass rich in PUFA in a 200 m2 greenhouse with tubular photobioreactors supplied with sea water and CO2 from the Technology Center in Mongstad.A national algae pilot plant has also been started at Mongstad, near Bergen in western Norway. Here the consortium CO2Bio AS   can change with seasons , that m2 and inorganic nutrients, with low‐grade waste heat as an option to extend the growth season. Such access implies localization within effective transport distance of industries, power plants and areas with higher population density. Implementation within a near future will probably have a primary, or at least equal, focus on remediation of waste resources and on production of biomass for fuels or chemicals. A change in focus towards production of biomass as primary purpose will likely follow as society and industries shift to a higher ratio of bio‐based raw materials and the direct as well as indirect costs of environmental impact increase. The lower overall population density in Nordic countries as well as access to large bodies of water makes implementation of large‐scale microalgae cultivation feasible without competition with food production and arable land. Large collaborative consortia between the Nordic countries, such as NordAqua, promote interchange and cooperation in the field of biomass production and will lead to improved and diversified industrial applications of micro‐ and macroalgae in Nordic environments.Sustainable large‐scale production of biomass from microalgae will require access to waste resources, such as sources of elevated concentrations of CONannochloropsis, through genome editing using CRISPR‐Cas9 (Ajjawi et al. Despite the large biodiversity of marine microalgae species and the almost unlimited availability of sea water, there is a shortage of commercial marine strains. Culture collections of native microalgae strains present the excellent opportunity for exploring their potential for biotechnology. While performing the task of selecting high producing and robust strains, the availability of rapid and reliable methods for screening is also of outmost importance. Nature‐inspired strategies and the use of genetic engineering can be used to improve the quantity and the quality of biomass. The doubling of the lipid content in O.C. and S.E. wrote the manuscript with input from all the co‐authors. All authors provided critical feedback that shaped the final form of the manuscript.


Chronic myelogenous leukemia (CML) is commonly treated with tyrosine kinase inhibitors (TKIs) that inhibit the pro‐leukemic activity of the BCR‐ABL1 oncoprotein. Despite the therapeutic progress mediated by TKI use, off‐target effects, treatment‐induced drug resistance, and the limited effect of these drugs on CML stem cells (SCs) are major drawbacks frequently resulting in insufficient or unsustainable treatment. Therefore, intense research efforts have focused on development of improved TKIs and alternative treatment strategies to eradicate CML SCs. Alongside efforts to design superior protein inhibitors, the need to overcome the poor therapeutic effect of TKIs on CML SCs has led to a renaissance of antisense strategies, as they are reported as effective in more primitive cell types. Despite the greater drug design flexibility offered by antisense sequence variability and remarkable chemical improvements, antisense drugs exhibit unacceptable levels of off‐target effects, precluding them from large‐scale clinical testing. Recent advances in antisense drug design have led to a pioneering mRNA recognition concept that may offer a helping hand in eliminating off‐target effects, and has potential to bridge the gap between research and clinical practice. Since its identification, the BCR‐ABL1 oncoprotein, a hallmark of CML cells, has become the primary molecular target for the development of selective therapeutics for patients with CML.BCR‐ABL1 mRNA rapidly became a potential treatment alternative for patients with CML. Compared to the limited, structure‐based design of protein inhibitors, antisense strategies take advantage of far greater target sequence variability for the design of specific antisense drugs. The benefits of sequence‐mediated specificity, allowing selective therapeutic intervention in CML cells, are so great that antisense strategies may be considered one of the most promising future pharmacological prospects for CML treatment since the approval of TKIs.A completely different treatment approach is offered by antisense strategies that enable control of disease‐associated genes through interaction with mRNA.Interestingly, despite the evident therapeutic potential of antisense strategies, only a minimal number of antileukemic drugs exploiting mRNA interference have reached clinical trials.BCR‐ABL1 suppression developed to date, 2 of which have even been tested in clinical trials,BCR‐ABL1 gene fusion junction.BCR‐ABL1, in the vast majority of studies, binding to off‐target mRNAs resulted in nonselective effects also in BCR‐ABL1‐negative cells. Interference with nontargeted homologous mRNAs, and consequent suppression of proteins outside of therapeutic intent, is a source of clinically relevant side effects and adverse events. Hence, addressing the issue of insufficient selectivity could significantly accelerate the entry of antisense strategies into clinical practice, and innovative solutions for elimination of this phenomenon are in high demand. A promising target recognition concept was recently reported with the intention of addressing this fundamental problem,BCR‐ABL1 mRNA recognition, and hence selective therapeutic intervention, exclusively in CML cells.Antisense systems for BCR and ABL1 mRNAs flanking the gene fusion junction can occur only when these sequences are separated from one another by a specific distance, which is defined by the linker. This simple but strict requirement significantly reduces the probability of stable interference with off‐target mRNAs, and thereby may provide dramatically enhanced selectivity towards CML cells.The solution addresses the basic design of the antisense construct, which is engineered to recognize the target mRNA via 2 antisense oligonucleotides connected by a size‐specific linker Figure .44 SimulBCR and ABL1 gene sequences, or with homologous sequences of off‐target mRNAs, will be thermodynamically unstable and energetically unfavorable because of the increased degree of conformational freedom and the absence of cooperative hydrogen bonds, respectively (Figure BCR‐ABL1 mRNA (Figure BCR‐ABL1 mRNA in CML SCs. The hope for a permanent cure for CML has been further advanced by recent reports of the phenotype of CML SCs.BCR‐ABL1‐independent signaling pathways which can contribute to escape from drug‐induced apoptosis.FOXO, BCL6, and CXCR4, or proteins involved in the WNT, Hedgehog and JAK signaling pathways have already been identified as relevant in this respect.This selective antisense strategy is based on the assumption that partial interaction of the antisense construct with wild‐type The vision of CML SCs eradication got even more realistic contours after the cell surface marker CD26 has been identifiedConsidering the history and evolution of treatment strategies for CML, and irrespective of the tortuous path toward treating CML without side effects, the therapeutic potential of the novel mRNA targeting approach described above is clearly worthy of thorough investigation. The progressive nature of this antisense concept is underlined by its theoretical applicability to any disease where the presence of a particular protein has causal role in pathophysiology.status quo in oligonucleotide‐based therapeutics will emerge over time. The fascinating possibility of leukemia treatment using a specific agent that can completely spare healthy cells remains a potent stimulus to the continued exploration of this approach and would facilitate its implementation into clinical practice.The extent to which this novel strategy will be able to alter the The authors declare no potential conflicts of interest.


Although most recharge estimation studies apply multiple methods to identify the possible range in recharge values, many do not distinguish clearly enough between inherent uncertainty of the methods and other factors affecting the results. We investigated the additional value that can be gained from multi‐method recharge studies through insights into hydrogeological understanding, in addition to characterizing uncertainty. Nine separate groundwater recharge estimation methods, with a total of 17 variations, were applied at a shallow aquifer in northwest Ethiopia in the context of the potential for shallow groundwater resource development. These gave a wide range of recharge values from 45 to 814 mm/a. Critical assessment indicated that the results depended on what the recharge represents , and spatial and temporal scales, as well as uncertainties from application of each method. Important insights into the hydrogeological system were gained from this detailed analysis, which also confirmed that the range of values for actual recharge was reduced to around 280‐430 mm/a. This study demonstrates that even when assumptions behind methods are violated, as they often are to some degree especially when data are limited, valuable insights into the hydrogeological system can be gained from application of multiple methods. Article impact statement: Analysis of common recharge methods shows the value of extracting additional information to improve hydrogeological understanding. Estimates of groundwater recharge allow quantification of renewable groundwater resources and can be used to indicate aquifer vulnerability to contamination or drought, assess groundwater contribution to streams (baseflow) and wetlands, and identify the implications of changes to land use, land cover or climate Misstear . Severalworeda, a local administrative district), in the context of an investigation into the resilience of shallow groundwater resources used for irrigation by rural communities. Following recommended approaches, for example, Scanlon et al.  before contributing to the aquifer  may have occurred since the water was recharged  recharge dominates. In arid regions characterized by deep water tables and losing rivers, recharge is usually focussed (or indirect) along river corridors with rates generally limited by water availability at the surface Allison . The facVarious techniques are available for estimating recharge, the selection of which is not straightforward  has an area of approximately 900 km2 and a population of around 175,000 of which 135,000 are rural , while the east drains via the Gilgel Abay river into Lake Tana. Much of the district has low relief with expansive floodplains providing year‐round pasture and dwellings and crops occupying adjacent slopes. Cultivated land occupies 72% of the district where rainfed agriculture predominates, the main crops being The climate of the region is moist subtropical with little annual temperature variation though high‐diurnal variation. The median annual total rainfall is 1541 mm, as measured 1987‐2017) at the National Meteorological Agency (NMA) weather station in Dangila town, 91% of which falls during May to October  recharge dominates across the study site Figure  with qua2) and Brante (65.5 km2) are sub‐catchments of the Kilti (631.7 km2). The catchment‐scale recharge assessment methods were applied to all three catchments. Thirty‐one shallow groundwater samples were collected for chloride analysis, from locations distributed throughout the study site, in March/April 2015 and October/November 2015. Rain could only be sampled during the second field visit because it did not rain during the 4 weeks of the earlier dry season visit, nor during a third visit in January 2017. Three samples were collected from two sites and occurred whenever rainfall was sufficient to enable direct sampling. All samples were filtered upon collection and, to prevent evaporation, the nalgene bottles were completely filled and kept in a refrigerator prior to laboratory analysis by Dionex ion chromatography. Additional data used in development of the conceptual model and required to parameterize models resulted from three periods of fieldwork, which included pumping tests on hand‐dug wells , AET is actual evapotranspiration (from the unsaturated and saturated zones and from surface water), R is recharge, A is abstraction, Qout is groundwater flow out of the basin, and ΔS is the change in storage. The assumptions are that ΔS is balanced over long time‐periods , Qin and Qout are negligible as these are headwater catchments with thin aquifers and rivers founded on bedrock (hence no groundwater flow beneath the gauge), and abstraction is negligible due to sparse wells with manual‐lifting technology. AET is not straightforward to estimate and was calculated with three methods for comparison: (1) The T‐M method; (2) Application of Turc's formula .Recycling of chloride does not occur within the basin area.The chloride concentration in runoff is equal to that in precipitation.Evaporation of groundwater does not occur upgradient of groundwater sampling points.The CMB method requires mean annual precipitation, chloride concentration of that precipitation and chloride concentration of the groundwater, is independent of whether recharge is diffuse or focussed, and integrates recharge rates both spatially across a region and temporally over long time‐periods. The method has several assumptions , Clwap is the weight‐average chloride concentration in precipitation including dry deposition, and Clgw is the average chloride concentration in groundwater. Clgw averaged 2.10 mg/L with a standard deviation of 1.33 mg/L and Clwap averaged 0.68 mg/L (standard deviation = 0.32 mg/L). Details of the parameterisation can be found in the Appendix The basic equation applicable for evaluation of recharge using the CMB isR is calculated for a particular well by multiplying the change in water level of two successive groundwater level readings by the specific yield Sy of the aquifer:h is water level and t is time. To correctly estimate Δh, it is necessary to extrapolate the antecedent recession curve to the point below the peak, that is, the point that the groundwater level curve would have reached without precipitation  is a physically‐based spatially distributed finite difference modeling system for coupled surface and subsurface water flow in river basins and is openly available at Recharge estimates from the various methods show high variability: 45‐814 mm/a or 3‐53%MAP  for the median annual recharge . Therefore, some assumptions must have been unsatisfied, which, rather than invalidating a method altogether, meant that the method was computing potential or minimum recharge or change in aquifer storage. Insights gained on the conceptual model mostly concern the amount and type of evapotranspiration, and the spatial variability of groundwater availability. High‐recharge values from the SMB methods indicate that all infiltration, which unsaturated zone methods are actually measuring, does not form recharge and there is likely to be interflow followed by discharge and/or evapotranspiration. The streamflow hydrograph methods' lowest recharge estimates indicate that groundwater is depleted prior to contributing to baseflow. Evapotranspirative losses from the saturated zone must be significant, which was thought likely given the shallow wet season water tables and spring/seepage‐fed inundated floodplains. High‐recharge values from the water balance methods are also suggestive that evapotranspiration may have been underestimated. Further evidence for this is the lower recharge estimate from SHETRAN that is due to its comprehensive simulation of canopy and open water evaporation and transpiration from the unsaturated and saturated zones resulting in greater total evapotranspiration losses. The high‐recharge values from the water table fluctuation methods, and high variability between wells, demonstrate the spatial variability in groundwater availability. The results show that groundwater flow, interflow and storage in certain areas can provide high potential for abstraction. Examples of other studies where fewer methods were applied and useful insights were gained are included in the Appendix The obvious insights gained from the multi‐method comparison were that not all methods were computing actual recharge at multiple catchment‐scales are therefore of primary interest. Although spatial assessments of aquifer storage change for small‐scale shallow aquifers, particularly at the seasonal‐scale, are also of significant interest to identify areas with the greatest potential for groundwater abstraction.Considering which types of recharge and spatiotemporal scales are relevant to this study, we restate the purpose as being to determine the resilience of shallow groundwater resources used for irrigation by rural communities in the Dangila area of Ethiopia; estimates of long‐term annual actual recharge averaged over the general study area lies somewhere between the minimum and potential values of 176 and 547 mm/a, given by the lowest streamflow hydrograph and highest SMB methods, respectively. The range of median values given by all actual recharge methods is 279‐535 mm/a.Considering the different types of recharge Table , while tWith regard to spatial scales, the methods based on groundwater level time series are highly localized and dependent on lateral inflows and other local factors, with values of recharge for individual wells from the RIB and the WTF methods ranging from under 100 to over 1600 mm/a. At the catchment‐scale, recharge values for the three catchments for each method used were generally consistent Appendix , indicatactual recharge estimates for the general area around Dangila requires consideration of the confidence given to each relevant method. This can be based on factors discussed earlier, including: temporal representativeness of time‐series data; spatial representativeness of data; errors and uncertainties in input data; sensitivity of models to parameter values and input data; whether assumptions of methods are met. We have greatest confidence in the water balance method using the higher AET rate, the CMB method, and the SHETRAN modeling. Thus, we identify a reliable recharge range for the Dangila area of 280‐430 mm/a, which is consistent with the range from the national map  the saturated zone, and (2) the unsaturated zone following infiltration past the root zone due to interflow and seepage. Second, by revealing the spatial variation of the change in aquifer storage, which locally can be significantly higher than This study has demonstrated for an extensive range of commonly used recharge methods applied at a single site that, in addition to quantifying uncertainty of recharge estimations, results from multi‐method comparisons should be clearly interpreted in relation to the types of recharge and spatiotemporal scale they represent, but can also provide additional benefits through improved hydrogeological understanding.The author(s) does not have any conflicts of interest to report.Appendix S1. Supporting Information.Table S1. Methods of recharge estimation grouped by hydrological zone. Methods applied in this study are marked with *.Table S2. Comparison of recharge estimates from large‐scale mapping/modeling studies.Table S3. Details of the recharge estimation studies used to develop a new empirical recharge method for Ethiopia based on the rainfall‐recharge relationship. Note that multiple recharge results from the same study relate to different recharge estimation methods applied and/or to different catchments or areas of the study site. CMB = chloride mass balance method, SMB = soil moisture balance method, SNNPR = Southern Nations, Nationalities and Peoples' Region, WTF = water table fluctuation method.Table S4a. Calculation of actual evapotranspiration (AET), soil moisture deficit and soil moisture surplus (from which 50% forms recharge) using the Thornthwaite and Mather  method. The year 2000 has been selected and grassland LULC category (MC = 200 mm) as an example. All values are in mm.Table S4b. Calculation of actual evapotranspiration (AET) with the application of a runoff factor, soil moisture deficit and soil moisture surplus (which is equated to recharge) using the Thornthwaite and Mather  method. The year 2000 has been selected and grassland LULC category (MC = 200 mm) as an example. All values are in mm.Table S5. Representative MC values and proportional coverage of LULC classes for Dangila woreda.Table S6. Comparison of rainfall chloride concentrations with other studies.Table S7. Details and statistics of the calibration and validation periods for the SHETRAN catchment models.Figure S1. Location map of the study area with other recharge study sites identified in the literature review shown on the right .Figure S2. The components of a streamflow hydrograph. Total flow is the sum of the three components, or the entire area below the Overland flow curve. The plot is a snapshot of the WETSPRO analysis of Kilti river flow.Figure S3. Snapshot of the Brante and Kilti hydrographs showing uncertainties encountered with the Meyboom method.Figure S4. Location map of the AMGRAF and ILSSI monitoring wells (a kebele is similar to a parish).Figure S5. Groundwater hydrograph through the wet season and determination of water table rise for the WTF method. MW1 refers to the groundwater level in monitoring well 1 from where this snapshot is taken. (mbgl = metres below ground level).Figure S6. Graphical output of the RIB model showing observed rainfall, observed groundwater level fluctuation (WLF), simulated groundwater level fluctuation (dh [rib]) and computed recharge. This plot shows the simulation of monitoring well MW3.Figure S7. Graphical comparison of annual recharge estimates from the catchment‐scale techniques separated into catchments. T‐M = Thornthwaite‐Mather method of AET estimation.Click here for additional data file.


Four outbreaks of leukoencephalomyelopathy in colonies of SPF cats on a long‐term diet of irradiated dry cat food were observed in the Netherlands between 1989 and 2001. As a primary defect in myelin formation was suspected to be the cause of the disease and myelin consists mainly of lipids and their fatty acids, we investigated the fatty acid composition of the white matter of the spinal cord of affected and control cats and of irradiated and non‐irradiated food. The irradiated food had low levels of alpha‐linolenic acid compared to linoleic acid as well as a high total omega‐6:omega‐3 ratio of 7:1 in the irradiated and of 2:1 in the non‐irradiated food. The white matter of the spinal cord showed low levels of linoleic acid and absence of alpha‐linolenic acid in affected cats as well as absence of lignoceric and nervonic acid in both affected and control cats. These abnormalities in fatty acid composition of the white matter of the spinal cord may reflect an increased need for alpha‐linolenic acid as a substrate for longer chain omega‐3 fatty acids to compose myelin and thus indicate a particular species sensitivity to dietary deficiency in omega‐3 polyunsaturated fatty acids, particularly alpha‐linolenic acid in cats. Our findings indicate that abnormalities in fatty acid metabolism in myelin play an essential role in the pathogenesis of this acquired form of leukoencephalomyelopathy in cats. These cats originated from specific pathogen‐free (SPF) colonies and the neurological condition affected up to 40% of litters in a colony thyroid gland, thymus, heart, lung, liver, spleen, kidney, adrenal gland, stomach (fundus), pancreas, duodenum, colon, ovary and uterus respectively testicle and skeletal muscle (M. semitendinosus) and in the cats from the 2001 outbreak also from subcutaneous  and perirenal fat tissue. Samples were fixed in 10% neutral, phosphate‐buffered, formalin. For neuropathological examination, brain, spinal cord including spinal ganglia, right brachial and sciatic nerves, coeliac ganglion, trigeminal ganglion and pituitary gland were fixed by immersion in 10% neutral, phosphate‐buffered, formalin. After routine dehydration and embedding in paraffin, 4 µm sections were made and stained with haematoxylin and eosin (HE). Specific staining methods used in selected slides from the nervous system were toluidine blue and immune‐histochemical stains for glial fibrillary acidic protein (GFAP), ionized calcium binding adaptor molecule 1  and alpha‐smooth muscle actin (αSMA) using standard procedures.2.3Spinal white matter was collected from three affected cats from the 1997 outbreak and six affected cats from the outbreak in 2001 as well as from the three control cats. Spinal cord material was frozen in liquid nitrogen and stored at −70°C until use. Immediately prior to analysis the material was thawed and ground. Also, a sample of the irradiated food associated with the 2000 outbreak  and of a standard dry cat food from the same manufacturer were obtained and ground prior to analysis.Cholesteryl esters (CE) were isolated by a modified “Bligh & Dyer” extraction according to Retra et al. , followe33.1All animals were in good bodily condition, and no gross abnormalities were seen.Histologically, lesions were consistently present in the central nervous system and comparable in distribution and severity in all severely affected cats. In both the brain and the spinal cord, lesions were restricted to the white matter, and consisted of bilateral symmetric vacuolation, loss of myelin and variously associated astrocytosis, microgliosis and capillary hyperplasia. The vacuoles often contained intact and sometimes swollen axons but also loss of axons associated with cellular debris and/or myelophages was observed Figure . BesidesThe lesions in the white matter had a particular and consistent distribution. In the cerebrum, marked lesions were seen in the white matter of the corona radiata, the outer cortex, the capsula interna and in the optic tract. The corpus callosum and the thalamic white matter showed no or only minimal lesions. In the mesencephalon, lesions were almost restricted to the corticospinal tracts and the rostral colliculi. The white matter of the cerebellar cortex showed no or minimal abnormalities, moderate lesions were seen in the white matter surrounding the cerebellar nuclei. In the pons and medulla oblongata, moderate to marked lesions existed in the vestibular, spinocerebellar and pyramidal tracts, the white matter of the formatio reticularis showed no abnormalities. In the spinal cord extensive lesions were found over the full length of the spinal cord. Dorsal, lateral and ventral funiculi were almost equally affected, although astrogliosis, microgliosis and perivascular cuffing with foamy lipid‐containing cells were most prominent in the lateral and ventral funiculi. Despite the extent of the lesions in the white matter of the spinal cord, the fasciculi proprii seemed spared. The spinal ganglia and the afferent and efferent nerves of the spinal cord, the peripheral nerves  as well as the coeliac and trigeminal ganglia showed no abnormalities.In the adipose tissue, only sampled in the animals from the January 2001 cases, a peculiar additional finding was the presence of single or groups of multi‐vacuolar adipocytes Figure  at varioImmunohistochemistry for IBA‐1 showed marked microglial proliferation in the affected areas, variable staining of myelophages in vacuolated myelin sheets and positive staining of the perivascular cuffs of lipid‐containing cells as well as solitary parenchymal lipid‐containing cells Figure . GFAP sh3.2The fatty acid composition of the spinal white matter of nine affected cats as well as of three control cats is given in Table 4The irradiated food‐induced leukoencephalomyelopathy in cats as demonstrated in our study has some special characteristics and thereby differs from many other forms of demyelination. It is an acquired disease that affects only cats and develops after feeding the irradiated food for four to six months. The disease, at least in the early clinical stage, is morphologically characterized by myelin vacuolation and demyelination with some myelophages but in general in the presence of intact axons, as well as by remyelinization with thin myelin sheaths around intact axons. When the cats at this stage are returned to a normal food, the animals show complete clinical recovery after 6–8 weeks although after 12 and 24 months these recovered cats still show thin myelin sheaths characteristic for remyelinated axons  and even absence of alpha‐linolenic acid (C18:3n‐3) and the high levels of docosadienoic acid (C22:2n‐6), docosatetraenoic acid (C22:4n‐6) and unknown fatty acids in the affected cats. As myelin composition is directly influenced by the composition of dietary fats . The alpha‐linolenic acid (C18:3n‐3) content of the irradiated food is low compared to the amount of linoleic acid. Although alpha‐linolenic acid is not regarded an essential nutrient for adult cats, it is regarded as such for growing kittens . The total omega‐6:omega‐3 ratio in the irradiated food is 7:1, whereas nutritionists typically recommend ratios between 2:1 and 1:1 for cat food. Such increased total omega‐6:omega‐3 ratio in the irradiated food might contribute to a relative deficiency of alpha‐linolenic acid (C18:3n‐3), as there is competition for metabolic enzymes with omega‐6 fatty acids such as linoleic acid (C18:2n‐6). Different omega‐6:omega‐3 ratios are reported for different indications (Simopoulos, Oligodendrocytes in the adult are thought to primarily rely on uptake to maintain their fatty acids pool Dimas, . OligodeIncreased peroxide levels in the spinal cord of affected cats as demonstrated by Caulfield et al.  in our oHowever, the myelin sheath has been regarded for a long time as an inert insulating structure, it has now become clear that myelin is metabolically active. Even if the turnover of lipids in myelin is slow with a half‐life of several weeks to months, there is a constant exchange of molecules (Schmitt, Castelvetri, & Simons, Apart from domestic cats also other wild felidae may be more sensitive to these alterations in dietary fatty acid pattern as a comparable form of leukoencephalomyelopathy has been described in captive cheetahs and other large felids (Brower et al.,  Click here for additional data file.


Kaposi's sarcoma involving the digestive tract in isolated form before the appearance of diffuse skin lesions is very rare in HIV‐negative patients and is a condition requiring watchful waiting. We describe a case of a 61‐year‐old woman undergoing an excision of a solitary nodule in the colon, with diagnosis of classic Kaposi's sarcoma. Cutaneous lesions appeared on the legs after three months from colon manifestation. Kaposi's sarcoma involving the digestive tract before skin lesions is very rare in HIV‐negative patients.1What is the percentage of Kaposi's sarcoma involving the digestive tract in HIV‐negative patients?22 every two weeks yielded partial response and was effectively continued until 12 courses.A 61‐year‐old woman was admitted to our hospital due to Stevens‐Johnson's reaction of uncertain etiology and mild anemia. Diffuse cutaneous macule‐papules were observed in thorax and lower limbs and empirically treated with antibiotics and steroids. Endoscopic examinations of stomach were negative, while colonoscopy allowed to evidence and remove a solitary purple nodule in the transverse colon, with diagnosis of classic Kaposi's sarcoma (Figure None declared.GC: participated in the management of patient care and wrote the paper. LT: analyzed the colon tissue specimens and made pathological diagnosis of tumor. SM: guided treatment decisions and contributed to the management of patient care.


Cell migration is a vital process in development and disease, and while the mechanisms that control motility are relatively well understood on two‐dimensional surfaces, the control of cell migration in three dimensions (3D) and in vivo has only recently begun to be understood. Vesicle trafficking pathways have emerged as a key regulatory element in migration and invasion, with the endocytosis and recycling of cell surface cargos, including growth factor and chemokine receptors, adhesion receptors and membrane‐associated proteases, being of major importance. We highlight recent advances in our understanding of how endocytic trafficking controls the availability and local activity of these cargoes to influence the movement of cells in 3D matrix and in developing organisms. In particular, we discuss how endocytic trafficking of different receptor classes spatially restricts signals and activity, usually to the leading edge of invasive cells. Abnormal cell migration is known to play a major role in a range of pathological conditions, including cancer metastasis, atherosclerosis and inflammation. As such, a complete understanding of the complex mechanisms that drive cell migratory processes should enable therapeutic manipulation.Accumulating evidence demonstrates a role for membrane trafficking in the regulation of cell migration in a variety of contexts. Many studies have shown that internalization and recycling of adhesion receptors to be particularly important, for example, integrins and syndecans, which regulate cell adhesion to the extracellular matrix (ECM),There is also extensive evidence that polarized recycling of receptor tyrosine kinases (RTKs) can generate localized signalling at the leading edge in response to extracellular signals or chemokine gradients, stimulating cell migration in a directional manner.Increasing evidence also implicates Golgi orientation to the leading edge, Golgi morphology and the polarization of post‐Golgi anterograde transport in the regulation of cell motility, particularly in two dimensions (2D).Study of cell migration in simple two‐dimensional (2D) environments has led to the detailed characterization of lamellipodial migration, which is driven by fan‐like actin‐rich membrane protrusions at the leading edge.BOX 12D assays: Scratch wound and random migration of cells plated on plastic/glass (and matrix‐coated surfaces), captured by time lapse imaging. Advantages include ease of imaging, ability to quantify numerous parameters . The major disadvantage is the lack of physiological relevance, as plastic and glass surfaces are more rigid than surfaces found in vivo, and often cells move through fibrillar 3D matrix of interstitial tissue.Cell‐derived matrices (CDMs): Fibroblasts lay down a collagen‐ and fibronectin‐rich fibrillar matrix  on tissue culture plastic before being removed leaving a 10‐20 µm thick layer of matrix behind.Hydrogels: Numerous hydrogel type systems exist, from artificially fabricated systems to purified matrix proteins . The clear advantage here is that cells move in a defined 3D environment, and matrix components can be “tuned” to resemble specific matrices found in vivo. The major disadvantage is the difficulty in imaging cells (particularly at high resolution) and tracking cell movement in xyx planes. Also, fibrillar structures and matrix ligands are randomly organized. An alternative approach is organotypic assays, in which fibroblasts pre‐strain a 3D collagen hydrogel and reorganize the matrix to facilitate invasion of other cell types.2Cell surface proteins are internalized via clathrin‐mediated endocytosis (CME) or clathrin‐independent endocytosis (CIE), and cargoes are delivered to early endosomes for recycling back to the cell surface or targeting for degradation.It is well established that receptor internalization is necessary to control the specificity, magnitude and duration of downstream signalling. Endosomes can function as signalling platforms where receptors elicit the same or different responses to those that occur at the plasma membrane. Endosomes have an appropriate pH to preclude ligand‐receptor dissociation and sequester these complexes from the proximity of phosphatases; both of these can lead to extended activation of the downstream signalling.Endocytic trafficking is particularly important in polarized cells, including epithelia  and PVR /VEGFR  orthologue) at the leading edge.In Endocytic recycling also plays a key role in endothelial cell function  drives invasive migration of cancer cells in 3D environments by forming a complex with α5β1 integrin, and subsequently recruiting RTKs for co‐recycling to the plasma membrane at the cell front4Directed cell migration is driven by cells sensing and responding to external gradients of chemotactic factors.Chemokine receptors can be internalized by clathrin‐dependent and ‐independent pathways, after which the fates of receptors and ligands may differ.Danio rerio (zebrafish) embryos revealed a specific role for CXCR4 internalization and subsequent downregulation. CXCR4 internalization is dispensable for initial detection and response to the ligand SDF‐1a, but crucial for the fine‐tuning of cell migration to ensure correct directionality of primordial germ cells to the gonad development region.Chemokine receptor trafficking is fundamental to directed cell migration in vivo. A study in 5Adhesion receptors, including receptors for ECM  and cell‐cell adhesion receptors (cadherins), play crucial roles in cell migration in physiological contexts. Trafficking of these receptor classes is important in controlling their localization at the cell surface, formation of cell‐matrix or cell‐cell adhesion complexes, and the signals generated downstream. Interestingly, crosstalk exists between receptor classes, for example, syndecan‐4 differentially regulates the trafficking of specific integrin heterodimers in migrating cells. Here, we will focus on the regulation and function of integrin trafficking in physiologically relevant 3D environments and in vivo, and highlight recent evidence for the involvement of cadherin trafficking in these processes.Integrins are the primary adhesion receptors for components of the ECM, composed of non‐covalently linked α and β subunits that heterodimerize to form 24 distinct integrins expressed in a cell‐ and tissue‐specific manner.It is now clear that focal adhesion complexes can form in cells migrating in 3D matrix5.1The rate of integrin degradation is low, and the majority of receptors are recycled back to the plasma membrane following endocytosis.There is now a wealth of evidence supporting a role for integrin recycling, particularly through Rab4 and Rab11 routes, in cell migrationRab11/RCP‐dependent recycling of α5β1 is of particular interest in the context of cancer and metastasis‐promoting gain‐of‐function mutant p53 expression. Mutant p53 acts to suppress the ribonuclease Dicer and miR expression, and this in turn relieves an inhibition of RCP‐α5β1 association and consequently leads to α5β1 recycling.α5β1 integrin recycling can also drive invasion into fibronectin‐rich environments by another mechanism. Rab25, a Rab11 family member with a more restricted expression profile, can directly bind the cytoplasmic tail of β1 integrin. This enables Rab25 to regulate the recycling of inactive α5β1 integrin back to the plasma membrane at the tips of migrating cells, for further interaction with the ECM to promote protrusion formation.5.2More recently, a more detailed picture of the integrin trafficking machinery has been uncovered in cells migrating in 2D, including a Vps3 and Vps8 route that controls β1 integrin recycling, and how GGA3, an Arf6 effector, controls SNX17 localization to control motility in 2D , from where integrins return to the plasma membrane at the leading edge of polarized cells. This promotes directional migration in cells in 2D and directional migration of the distal tip cell along the basement membrane to form the gonad in 5.3Leucocytes are fast‐moving cells that have to quickly transmigrate from blood vessels into tissues upon signals of inflammation and infection. The major integrin used by leucocytes to achieve this is LFA‐1 and it provides an excellent model as to how integrin recycling can regulate cell motility. The intracellular trafficking of the integrin LFA‐1 has been highly studied as this can regulate cell adhesion and motility. For instance, it has been shown that upon T‐cell stimulation with chemokines, the kinase Mst1 activates Rab13 through the Rab13 GEF (guanine nucleotide exchange factor), DENND1C. This activation facilitates the delivery of LFA‐1 to the leading edge, whereby this spatial distribution of LFA can drive lymphocyte migration and trafficking in vivo5.4Cadherins are the primary adhesion molecules that form cell‐to‐cell contacts called adherens junctions. Regulation of cadherins plays an essential role in physiological processes such as embryonic development, wound healing and cancer metastasis. Cadherins at the surface of adjacent cells connect via calcium‐dependent homophilic interactions between their extracellular domains. Intermediate proteins link the intracellular domain of cadherin to the actin cytoskeleton. Trafficking of cadherins has emerged as a fundamental mechanism by which these adhesive contacts can be regulated  is involved in cell migration during development and disease.Xenopus animal cap are regulated by internalization of C‐cadherin, controlled by dynamin and Rnd1.Cadherin trafficking also plays a role in the maintenance of cell‐cell junctions during morphogenesis and collective migration, and logically endocytosis could contribute to cell‐cell adhesion plasticity to allow morphological changes to take place. Movements in convergent extension of the 6Migration and invasion of individual cancer cells in 3D matrix and in vivo has been well studied, characterized and categorized into mesenchymal and amoeboid subtypes. Amoeboid migration is characterized by high levels of actomyosin contractility, which generates hydrostatic pressure to promote protrusion, and there is little protease activity required for cells to squeeze through gaps in the ECM.While numerous proteases, membrane tethered and secreted, contribute to cancer cell invasion and metastasis, MT1‐MMP  has become established as the major player in executing programmes of basement membrane and interstitial matrix invasion.Like other trafficking receptors, MT1‐MMP can follow several routes through the endocytic system. MT1‐MMP internalization is not well characterized but may occur via caveolae.C. elegans anchor cell is key to formation of invasive protrusions rich in the MMP ZMP‐1, a critical step in the programme of invasion across the basement membrane during development of the reproductive system.While most trafficking receptors that reach late endosomes/lysosomes are degraded, some can be recycled from these compartments, including MT1‐MMP.7Here, we have given an overview of the recent advances that have improved our understanding of how endosomal trafficking of a range of receptors regulates cell migration within 3D and in vivo contexts. Several recent studies have demonstrated the breadth of coordination between receptor families; from RTK and integrin co‐traffic to the synergistic relationship between integrins and syndecans, it is becoming increasingly clear that trafficking of different receptors cannot be considered in isolation. Indeed, gaining a complete understanding of the cooperation between signalling and receptors is essential to fully comprehend many aspects of cell behaviour. The discovery that many of these receptors actively signal from endosomal compartments has created another avenue of research that is not well established, especially in terms of in vivo cell migration. Studies that decipher the molecular pathways driving cell migration are a useful precursor for understanding and potentially targeting this process in specific diseases.Approaches to exploring endosomal trafficking in cell migration still rely heavily on biochemical and immunofluorescence experiments in 2D environments. Therefore, approaches in the future should endeavour to utilize the most physiologically relevant models Box . In vivoRecent studies that have used “omics” approaches to dissect receptor signalling have been very successful. For instance, an integrated multi‐layered proteomics approach was used successfully to decipher the selectivity of EGF and TGF‐α  on EGFR fate. It showed that phosphorylation of Rab7 and RCP recruitment were switches for the opposing fates of EGFR recycling and degradation, and that this controlled downstream signalling and subsequently cell migration.Editorial ProcessClick here for additional data file.