In this paper, we describe a mechanism for the transfer of nutrients from symbiotic microbes (bacteria and fungi) to host plant roots that we term the ‘rhizophagy cycle.’ In the rhizophagy cycle, microbes alternate between a root intracellular endophytic phase and a free-living soil phase. Microbes acquire soil nutrients in the free-living soil phase; nutrients are extracted through exposure to host-produced reactive oxygen in the intracellular endophytic phase. We conducted experiments on several seed-vectored microbes in several host species. We found that initially the symbiotic microbes grow on the rhizoplane in the exudate zone adjacent the root meristem. Microbes enter root tip meristem cells—locating within the periplasmic spaces between cell wall and plasma membrane. In the periplasmic spaces of root cells, microbes convert to wall-less protoplast forms. As root cells mature, microbes continue to be subjected to reactive oxygen (superoxide) produced by NADPH oxidases (NOX) on the root cell plasma membranes. Reactive oxygen degrades some of the intracellular microbes, also likely inducing electrolyte leakage from microbes—effectively extracting nutrients from microbes. Surviving bacteria in root epidermal cells trigger root hair elongation and as hairs elongate bacteria exit at the hair tips, reforming cell walls and cell shapes as microbes emerge into the rhizosphere where they may obtain additional nutrients. Precisely what nutrients are transferred through rhizophagy or how important this process is for nutrient acquisition is still unknown. Fagales, Rosales and Cucurbitales) where roots may become inter-cellularly and intra-cellularly colonized by diazotrophic actinomycetes of the genus Frankia that inhabit nodules in roots [Betulaceae, Elaeagnaceae, Fagaceae, Myricaceae, Rosaceae and so forth [Fabaceae) [Gunnera that possess specialized glands that secrete polysaccharides to attract cyanobacteria, which enter into tissues of the stem and become intracellular within host cell vesicles where they fix nitrogen that is subsequently transferred to the host plant [It is widely known and accepted that most plants obtain nutrients generally through absorption of dissolved inorganic nutrients from soils . Howeverin roots . Familieso forth . Other nso forth ,6. Rhizoabaceae) . In someabaceae) . Among tst plant .In all the previously discussed nitrogen-transfer symbioses, hosts evolved ways to internalize and engage in prolonged symbiosis with diazotrophic prokaryotes using specialized plant symbiosis organs or tissues. All of these associations are restricted to specific families that evolved specialized symbiosis organs (nodules or glands); however, all species of plants internalize microbial endophytes into plant tissues that do not involve specialized organs ,16,17,18Lycopersicum esculentum and Arabidopsis thaliana) internalized bacteria and yeasts into root cells where microbes appeared to be degraded in time. Paungfoo-Lonhienne et al. [Evidence for a mechanism for direct transference of nutrients from symbiotic microbes to plant roots was provided by Paungfoo-Lonhienne et al. . Throughe et al.  later dee et al.  and Paune et al.  demonstre et al.  documente et al.  later doe et al. ,46.Over the past several years, we have conducted experiments using various host species and endophytic bacteria to elucidate how the plant microbivory process works in plant roots ,53,54,55Bosea, Methylobacterium; Beta-proteobacteria Achromobacter and Burkholderia; Gamma-proteobacteria Acinetobacter, Klebsiella, Pantoea, Pseudomonas and Micrococcus; and Gram-positive Bacilli Bacillus and Paenibacillus; and Actinobacteria Curtobacterium and Microbacterium. Bacteria that function in the rhizophagy cycle belong to diverse classes of bacteria and it seems likely that any bacterium that colonizes roots and can be induced to enter root cells may be involved in the process [In our experiments, we employed microbes that were transmitted within or on the surfaces of seeds of several species of plants ,48,56,57 process ,48,50,55Plants produce a root exudate zone adjacent to and just behind root tip meristems . Plants Burkholderia and Klebsiella) involved in the rhizophagy cycle have been frequently found to fix atmospheric nitrogen, while others are sometimes or rarely reported as nitrogen fixing [Burkholderia only showed evidence of active nitrogen fixation in roots of switchgrass (Panicum virgatum) after root tissues senesced. On the other hand, it seems logical that bacteria could deliver nutrients to plants via the rhizophagy cycle without nitrogen fixation occurring; instead bacteria need only be capable of obtaining nutrients in the rhizosphere by scavenging of nutrients from soils, bacteria, fungi or plants. The capacity of some bacteria (including species of Bacillus) to produce and secrete resistant proteases may enable these bacteria to scavenge nitrogen from other microbes by degrading and absorbing their proteins in the rhizosphere [Bacillus amyloliquefaciens to cotton seedlings growing in soil resulted in increased growth and an increased expression of nitrate transporter genes. In isotope tracking experiments using grass seedlings both with and without bacteria, grown in agarose amended with 15N-labeled proteins, White et al. [Some of the bacteria  iron was acquired directly from bacteria at the root tip meristems. In the process of rhizophagy, it is at the root tip meristems that bacteria enter plant cells and are exposed to reactive oxygen that likely extracts nutrients from the intracellular bacteria—inducing electrolyte leakage of bacteria and making nutrients available for absorption into plant root cells. Further work is needed to determine precisely which nutrients may be obtained by plants from the direct consumption/oxidation of microbes.Through the activities of some symbiotic microbes, nutrients  may be solubilized in the rhizosphere . SolubilPseudomonas spp.  and Micrococcus luteus (from seeds of Lycopersicum esculentum) that differed in resistance to reactive oxygen degradation due to their relative capacities to produce antioxidants  have suggested that bacteria that participate in the rhizophagy cycle may be matched to their particular host plants. More specifically, rhizophagy bacteria must be degradable by levels of reactive oxygen produced by their hosts. Pseudomonas spp. that stimulated growth of several grasses [Taraxacum officionale) [Micrococcus luteus, a tomato endophyte, was shown to stimulate growth of seedlings of tomato , carrot (Daucus carota), curly dock (Rumex crispus) and Japanese knotweed  resulted in internal colonization of root cells and suppression of root growth. Examination of roots of these species bearing Micrococcus luteus showed that bacteria colonized the interior of root cells, and elicited reactive oxygen production, but did not degrade within root cells, instead copiously replicating in seedling roots  within roots. The degradable pseudomonads may thus provide a source of nutrients to their grass host plants, resulting in growth stimulation. Micrococcus was resistant to degradation by several hosts and likely did not provide nutrients. Further, because Micrococcus luteus was resistant to degradation in the root several hosts, the numbers of bacteria in cells could not be regulated and likely overwhelmed the seedlings due to bacterial overload. This emphasizes that plants and their symbiotic microbes may be adapted optimally to maximize benefits of the symbiosis. In an optimal rhizophagy cycle relationship, microbes would enter plant roots and some would be degraded oxidatively, keeping intracellular microbe numbers in check, while survivors would exit roots to rejoin soil populations.Experiments using bacterial endophytes  grasses ,49 were cionale) ,49. Micrf tomato . Howeverng roots  to exit  Abishek  showed ta et al.  showed tArabidopsis thaliana and Lycopersicon esculentum inoculated with Escherichia coli showed that plants expressed higher levels of cellulases than un-inoculated controls. These authors also found that bacteria on surfaces of root cells were sometimes covered by a matrix composed of cellulose fibers as evidenced by immuno-histochemical experiments. It was proposed that coating microbes in a matrix could be an early stage of endocytosis into root cells. Further, Paungfoo-Lonhienne et al. [Paungfoo-Lonhienne et al.  proposede et al.  reasonedSalmonella spp. and Candida spp.) remain in the biofilm phase but when butyric and propionic acid concentrations fall, virulence genes in microbes are up-regulated and microbes parasitize epithelial tissues in the walls of the animal’s gut [Allium cepa root tip meristem cells. Why plant cell division in the meristem slows is unknown, but it shows that the plant may respond to presence of the microbes around the meristem. The active absorption of these SCFAs by root meristem cells, may stimulate bacteria in the biofilm around the root meristem to infect the thin-walled meristem cells in a comparable way microbes in intestines infect intestinal epithelial tissues. Thus, absorption of SCFAs by the root meristem is a possible ‘rhizophagy trigger,’ essentially inducing microbes to parasitize the meristem cells. In this sense, the removal of organic acids from the microbial biofilm around root meristems may represent part of the symbiotic ‘cross talk’ between plant and microbe, comparable to interactions that occur in establishment of rhizobial symbioses [Bacteria growing in biofilms on the root surface and in the rhizosphere anaerobically ferment carbohydrates present in root exudates to form short-chain fatty acids (SCFAs). Butyric and propionic acids are among the SCFAs fermented by bacteria associated with roots . In otheal’s gut ,76. Butyal’s gut ,78. In ial’s gut  showed tal’s gut  found thymbioses .Poa annua (inoculated with Pseudomonas fluorescens) on agarose  that contained 0, 2, 4, 6, 8, or 10 mM of butyric or propionic acids . After approximately 10 days, seedlings were stained for 15 h in a solution of 3,3-diaminobenzidine (DAB)  and counterstained using aniline blue , then examined microscopically for evidence of intracellular bacteria using a compound light microscope  [To test whether absorption of propionic and butyric acids from the bacterial biofilm by the root tip meristem is the trigger to initiate entry of bacteria into root meristem cells, we conducted experiments in which we grew seedlings of the grass NY, USA) . In thesNY, USA) ,51,53. TNY, USA) , or actiCynodon dactylon and Poa annua) and dicots  infected by Pseudomonas spp. or Micrococcus luteus shows that bacteria initially enter root meristem cells as walled cells—pseudomonads are rod-shaped and Micrococcus luteus cells are spherical and in tetrads. In the periplasmic space of root cells, bacteria lose cell walls and form spherical L-forms  from seeds and seedlings of the plant Froelichia gracilis (Amaranthaceae). We also isolated the endophytic yeast Rhodotorula sp. from Abrus precatora (Fabaceae). Experiments using A. pullulans and Rhodotorula sp. demonstrated that these yeasts internally colonized seedling roots of several plant species, entering into root cells and locating in the periplasmic spaces of root parenchyma [In a recent study, we isolated the fungus renchyma . Initialrenchyma . In our renchyma  observedExperiments using seedlings of grasses and other plant species have demonstrated that some, or perhaps all, microbes that internalize in plant cells modulate development of the seedlings. Seedlings from rigorously surface-disinfected seeds using sodium hypochlorite to remove all surface microbes invariably are diminished developmentally, often showing loss of root gravitropic response (roots fail to grow downward) and reduced or no root hair formation. Re-inoculation of the symbiotic bacteria onto axenic seeds restores normal root growth in seedlings ,48,49,50Entry of microbes into root cells is generally accompanied by increased production of defensive reactive oxygen by the root cells . IncreasBacillus endophyte of English ivy (Hedera helix) produced antifungal lipopeptides that directly inhibited growth of the pathogenic fungus Alternaria tenuisima and protected the host from disease. Pseudomonad endophytes of plants also venture into the soil and colonize potential fungal pathogens, repressing their growth and reducing virulence [Some of the microbes that become intracellular in plant roots have been shown to inhibit pathogenic fungi in the soil or in the plant ,50,93,94irulence . Pseudomirulence .Phragmites australis showed that the bacteria stimulated growth of several grasses but suppressed growth and increased mortality in seedlings of dandelion  and curly dock (Rumex crispus) [Rhodotorula sp. and Aureobasidium pullulans and bacteria Micrococcus luteus and Paenibacillus sp. from seedlings of plant species Abrus precatorius, Froelichia gracilis, Lycopersicum esculentum and Poa annua, respectively, suppressed root growth and increased mortality of seedlings of dandelion , curly dock (Rumex crispus) and clover (Trifolium repens). Here, microbes that were most inhibitory to seedling growth were those that were resistant to reactive oxygen due to production of antioxidants—and likely internal replication of microbes could not be controlled by the inhibited seedlings, resulting in replication of endophytes at the expense of host growth. We do not know the mechanisms by which endophytic colonization of non-hosts led to increased seedling mortality. Endobiome interference could be a factor that affects competition between plant species in natural plant communities—with endophytes serving as a means to suppress the growth of competitor plant species [Microbes involved in the rhizophagy cycle intimately associate with plant root tissues and cells, growing around the root tip meristems and passing through cell walls where they come into direct contact with root cell plasma membranes. We hypothesize that this intimate association requires that the microbe must be subject to control by the plant cells. If the intracellular microbes are resistant to degradation by host-produced reactive oxygen due to production of antioxidants the microbe may replicate itself excessively at the expense of the host cell and host growth may be reduced. If microbes produce metabolites such as hydrogen cyanide or other compounds that restrict respiration or other functions of the host cell, plant growth may again be compromised. Thus, it seems reasonable that hosts and rhizophagy cycle endophytes are adapted to maximize benefits to both hosts and microbes. Colonization of rhizophagy cycle endophytes into hosts to which they are not adapted may result in ‘endobiome interference,’ where endophytes reduce growth and competitiveness of hosts and perhaps interfere with the growth promotional activities of native endophytes . In somecrispus) . In othecrispus) , we show species . AdditioThe ‘rhizophagy cycle’ is a process where microbes alternate between an endophytic phase and a free-living soil phase. Rhizophagy microbes become intracellular in root cells by infecting cells at the root tip meristem. Microbes exist primarily as wall-less protoplasts (‘L-forms’ for bacteria and ‘mycosomes’ for fungi) in close association with host cell plasma membranes in roots. Root cell plasma membranes secrete reactive oxygen onto microbes and this may reduce replication of intracellular microbes and cause leakage of nutrients from microbes. Intracellular microbes trigger formation of root hairs on roots and they exit cells at the elongating root tips, reforming cell walls as they exit from root hairs. Plants appear to manipulate microbes in the rhizophagy cycle by: (1) stimulating bacterial growth around root tip meristems of seedlings by secretion of root exudates around the root tip; (2) triggering bacteria to enter into periplasmic spaces in root cells at the root-tip meristem by absorbing bacterial fermentation products including butyric acid, causing bacteria to up-regulate virulence/endoparasitism genes; (3) subjecting bacteria in periplasmic spaces to superoxide formed on root cell plasma membranes to extract nutrients from bacteria, and (4) depositing surviving intracellular bacteria back into the rhizosphere from the tips of elongating root hairs to maximize new nutrient acquisition by bacteria. Microbes engage in the rhizophagy symbiosis likely because they also benefit from the nutrients provided by host plants in terms of root exudates and nutrients that leak from root cells during the endophytic/intracellular phase. The rhizophagy symbiosis may be viewed as a mutualism involving an exchange of nutrients between the plant and microbe participants. Much of our research to date on the rhizophagy cycle has been done on seedlings, however future research is needed to confirm that the rhizophagy cycle also occurs at root tips of more mature plants. While ‘rhizophagy’ has been shown to increase movement of nitrogen into plants, we hypothesize that the real benefit of the rhizophagy cycle may be in acquisition of iron and other micronutrients from symbiotic microbes that sequester these soil nutrients using siderophores, although this must be proven. The biological implications of the rhizophagy cycle and microbes that are involved in it in terms of plant growth promotion and plant-plant interactions via symbiotic microbes are topics that require much additional research.


They received detailed information regarding how to correctly complete these forms several months before the go-live date in July 2013 and reminded them afterwards at regular graduate medical trainee, faculty and staff meetings to ensure their engagement and that institutional-wide participation would happen in these assessment strategies.The promotion for launching the new professionalism and mistreatment tools was advertised as "Press the Button", as the person accessing these forms would press a button-shaped desktop icon that opens the graduate medical education forms' website on any institutional-computer to start reporting an event. This website is also accessible from other computers and connected mobile devices where the "Button" shortcut can be added to personal devices. Any hospital graduate medical trainee, faculty, or staff person in the institution  who witnessed or personally experienced a reportable event was encouraged to activate the online forms by describing the incident in as much detail as possible. These electronic forms can be reported anonymously or by self-identification. Graduate medical education leadership immediately reviewed a completed form, then forwarded to the programme director , to the department chair (for faculty activations), or to the director of medical staff affairs . A written summary of actions and remediation is required to be sent back to graduate medical education within ten business days. The event remains open until a written summary of actions is received by the graduate medical education office and subsequently closed.As a way to perform quality improvement and assurance on the button activations, we conducted an observational analysis of these activations from July 2013 (button inception) through July 2017.  Over this four-year-period there were 315 button activations, most were anonymous (69.0%) with graduate medical trainees being the primary offender (74%) in their first and second year of graduate medical education . Having this readily available and easily accessible web-based tool allowed for a high rate of reporting and participation, and early intervention of unprofessional behavior. We believe continuing this process will encourage professionalism within the institution and subsequently lead to decreased incidents in an improved professional training environment.,,,,,Competence to practice medicine includes the ability of physicians to demonstrate professionalism in all relationships, including honesty to patients, patient confidentiality, maintaining appropriate patient relations, improving the quality of patient care and access to care, having the ability to work with finite resources, with applicable scientific knowledge and principals.The authors declare that they have no conflict of interest.


Mechanical ventilation [MV] is a life-saving technique delivered to critically ill patients incapable of adequately ventilating and/or oxygenating due to respiratory or other disease processes. This necessarily invasive support however could potentially result in important iatrogenic complications. Even brief periods of MV may result in diaphragm weakness ], which may be associated with difficulty weaning from the ventilator as well as mortality. This suggests that VIDD could potentially have a major impact on clinical practice through worse clinical outcomes and healthcare resource use. Recent translational investigations have identified that VIDD is mainly characterized by alterations resulting in a major decline of diaphragmatic contractile force together with atrophy of diaphragm muscle fibers. However, the signaling mechanisms responsible for VIDD have not been fully established. In this paper, we summarize the current understanding of the pathophysiological pathways underlying VIDD and highlight the diagnostic approach, as well as novel and experimental therapeutic options. Invasive mechanical ventilation (MV) is a life-saving procedure applied to critically ill patients to achieve adequate pulmonary gas exchange and unload excessive respiratory muscle work. Observational studies report that the use of mechanical ventilation has led to a decrease in the mortality rate of critically ill patients –3.Nevertheless, and based on the advances in the understanding that mechanical ventilation itself can cause and potentiate lung injury , 5, tranRecently, a similar concern has emerged with regard to the potential adverse effects of invasive mechanical ventilation on the respiratory muscles. This entity was originally termed ventilator-induced diaphragmatic dysfunction (VIDD) , althougIt is well known that muscle dysfunction in critically ill patients defined as ICU-acquired weakness is associated with weaning failure and unfavorable outcomes in critically ill patients , 14. HowCurrently, VIDD represents one of the most challenging fields within translational research applied to critically ill patients. This review provides the knowledge necessary for intensivists to better understand the signaling mechanisms responsible for VIDD, as well as summarizes the development of diagnostic bedside tools. Based on these findings, future therapeutical approaches may have the ability to prevent VIDD and to improve weaning success as well as other relevant clinical outcomes.Multiple recent studies have shown that VIDD is reported in up to 53% of mechanically ventilated patients within 24 h of intubation. An additional 26% may develop VIDD while on mechanical ventilation during their stay in the intensive care unit (ICU).In the literature, the variations observed in the incidence of this complication are based fundamentally on the diagnostic tool used. The assessment of the transdiaphragmatic twitch pressure (PdiTw) generated in response to bilateral anterior magnetic phrenic nerve stimulation (BAMPS) is an objective, nonvolitional measurement which represents the gold standard technique used to specifically assess diaphragm strength. According to several observational studies, mechanically ventilated patients in the ICU, on average, generate a PdiTw that is only 20% of normal . The useThe potential improvement in the outcome that might be obtained by implementing prevention or therapeutic strategies for diaphragm atrophy caused by ventilation is therefore also uncertain.Most studies show that VIDD occurs in a progressive, time-dependent manner , althougThe molecular mechanisms underlying VIDD currently represents one of the more attractive fields of translational research in critically ill patients. We summarize some of the key discoveries that may provide a unified mechanistic framework for understanding the pathogenesis of VIDD Fig. .Fig. 1SuThe mechanical properties of diaphragm muscle fibers are characterized by the relationship between cytosolic-free calcium, cross-bridge attachment/cross-bridge cycling rate, and sarcomere length . Any facNonetheless, it seems likely that the cause of MV-induced diaphragm contractile dysfunction is multiplicative and includes oxidative modifications to contractile proteins resulting in depressed fiber sensitivity to calcium, protease activation leading to sarcomere disruption, and a loss of myosin heavy chain protein.Overall, we can consider that VIDD is characterized by a disbalance in protein homeostasis defined as a decrease in diaphragm protein synthesis and an increase in diaphragm protein degradation. The activation of catabolic pathways, induced in the diaphragm under mechanical ventilation, occurs in the short-term under controlled modes of mechanical ventilation . This apTherefore, mitochondrial oxidative stress (MOS) may play an integral role in MV-induced atrophy of the diaphragm by inducing catabolism , 28.The force component of VIDD can also be affected by MOS in the following two ways: firstly, by producing free radicals that can induce a post-translational modification of muscle proteins  and, hence, alter their structure and function, specifically reducing the calcium sensitivity of myofilaments , and secAutophagy is a catabolic process that degrades cytosolic proteins and organelles using lysosomal proteases. More specifically, lysosomal proteases  have the biological function of removing both organelles and nonmyofibril cytosolic protein aggregates . During A recent study found that prolonged mechanical ventilation increases the expression of key autophagy proteins  and the number of autophagosomes in the human diaphragm . FurtherHowever, it is not clear whether autophagy is an effector of VIDD, or a protective mechanism that may actually offset VIDD , 37. CriCalpains are cysteine proteases that promote muscle atrophy by cleaving over 100 different cellular proteins, and the two ubiquitous calpains located in skeletal muscle are calpain I and II . NumerouImportantly, it has been established that prolonged mechanical ventilation activates calpain in the diaphragms of both humans and animals . In factSpecific diaphragm muscle proteins have been identified that are degraded during mechanical ventilation-induced sarcomere damage, concretely both titin and myosin. A recent work found a significant reduction in rat diaphragm single muscle fiber force production after 18 h of mechanical ventilation, which was accompanied by a proportional loss of myosin heavy chain concentration together with a titin dysfunction in diaphragm fibers . InteresAnother possible link between mechanical ventilation-induced oxidative stress and diaphragm contractile dysfunction is the connection between reactive oxygen species (ROS) and calpain activation. MV-induced ROS production in diaphragm muscle promotes calpain activation , 48; in Finally, recent studies have shown that regulatory cross-talk exists between calpain and caspase-3 in the diaphragm during controlled MV in rats. Caspase-3 belongs to a large family of cysteine proteases and plays an important role in apoptosis. This protease may also contribute to muscle protein degradation during a variety of muscle wasting conditions.There is a body of research demonstrating that full support MV activates caspase-3 in both the rodent and human diaphragm and induces myonuclear apoptosis in the diaphragm , 49, 50.The ubiquitin-proteasome system of proteolysis plays an important role in muscle protein catabolic pathways during a variety of wasting conditions . Recent Both experimental and human studies have shown that prolonged mechanical ventilation induces diaphragm contractile dysfunction . In an aIn critically ill patients, some factors have been identified that along with prolonged mechanical ventilation adversely impact diaphragm contractile function including senescence , 66, intThese studies however present divergent findings, and further research is needed to address the effect of treatments on diaphragm function in animals using both partial and controlled mechanical ventilation.2O per year from the maximal inspiratory pressure (MIP) between the ages of 65 and 85 years  showed preserved diaphragmatic force production after 72 h of mechanical ventilation  can also be measured by simultaneously recording the esophagus and the stomach pressures, but the interpretation of the results is also limited and may be dependent on the level of patient co-operation, the depth of sedation, or the use of neuromuscular blockers.Other non-invasive beside volitional tests such as the sniff maneuvers can be used in combination to enhance the performance of certain tests.sniff nasal pressure  may result as precise as when more invasive tests  are added to the evaluation /thickness at end-expiration]for TFdi .Both DE and TFdi have been shown to correlate with functional measurements of diaphragmatic function in spontaneously breathing patients, but the use of ultrasound in the process of weaning remains controversial , 97.Several clinical studies suggest that these ultrasound parameters can be reliable predictors of weaning and extubation outcomes. Indeed, a recent study  found a On the other hand, a study  reportedRecently, a systematic review that included 13 studies on 742 adult critically ill patients found that ultrasound had excellent intra- and inter-observer reproducibility, as well as higher accuracy in investigating diaphragm dysfunction compared with other weaning indexes or fluoroscopy . FinallyClinicalTrials.gov Identifier: NCT03295344).A novel field of research is the implementation of tissue Doppler as a diagnostic tool in the analysis of diaphragm function. Tissue Doppler imaging  is an echocardiographic technique and promises a number of applications to further extend the unique diagnostic role in echocardiography. In particular relation to the diaphragm, it might allow the assessment of regional diaphragmatic contractile function at rest and with stress. It should also provide a reliable identification of viable respiratory contractile muscle. There is currently a French clinical trial which aims to look for correlations between the time movement mode and tissue Doppler in the assessment of a threshold value of diaphragm mobility in patients in the postoperative period following scheduled cardiac surgery (Diaphragm electromyography (EMGdi) reflects the muscle electrical activity, and it is considered the gold standard in the assessment of neural respiratory drive.Electromyography (EMG) comprises the temporal and spatial summation of neural impulses from the brain that are translated into muscle fiber action potentials. There are different approaches to obtaining a diaphragm EMG, and at the bedside, it can be recorded using an esophageal catheter with multiple electrodes .The processed signal can be obtained easily and continuously in ICU patients and is referred to as the amplitude of the electrical activity of the diaphragm (EAdi). EAdi records action potentials from the diaphragm and can therefore assess whether the phrenic nerve is intact. This may be helpful in monitoring respiratory muscle loading, patient–ventilator synchrony, and efficiency of breathing in critically ill patients . EAdi isThis index reflects the ability of the diaphragm to convert the respiratory drive into ventilation. A low VT/EAdi suggests severe impairment of neuromechanical coupling. EAdi-derived indices have been used to identify patients with weaning failure . InteresSimilarly, the electrical activity of the diaphragm (EAdi) records action potentials from the diaphragm and therefore assesses whether the phrenic nerve is intact .A clinical study  reportedThe second tool is the diaphragm electromyography (EMGdi), which measures the activation of action potentials along the diaphragm muscle and can be used to assess muscle contractility. Intramuscular electrodes can be used to record, relatively selectively, from the diaphragm and intercostal muscles .However, the techniques are invasive and technically difficult.There is a body of research showing that EMGdi is feasible in routine clinical care for monitoring respiratory muscle function. However, there are some limitations in the interpretation of EAdi: it is still necessary to identify the threshold level of EAdi that should be achieved in the daily management of mechanically ventilated patients ; respiraNevertheless, there are nasogastric tubes for enteral feeding, which are already commercially available, with EMG electrodes.Taken together, EAdi is a well-evaluated parameter to monitor respiratory muscle unloading and patient–ventilator synchrony from an early phase of critical illness. The implementation of EAdi would be clinically relevant as an important tool for respiratory muscle monitoring during mechanical ventilation and weaning.One of the challenges in translational research is the development of a management strategy in order to avoid VIDD in the critically ill patient, with the aim of improving clinical outcomes.No preventive or therapeutic interventions have been tested in clinical trials.Potential treatments arising from translational or from small clinical studies should be considered as initial steps towards large clinical trials. These larger trials are necessary in order to establish a definitive approach to VIDD in the critically ill patient. The current strategies which target preventive, experimental, and/or therapeutic approaches might support the development of well-designed clinical trials. Any VIDD management strategies should firstly consider the optimal medical treatment of concurrent conditions, such as malnutrition , in addiA better understanding of the pathophysiological mechanisms involved in VIDD at the cellular, transcriptional, and molecular levels is leading the search for new therapeutic approaches. This includes experimental pharmacological agents such as the use of mitochondria-targeted antioxidants , JAK-STATranscription factors of the FOXO and signal transducer and activator of transcription (STAT) families are upregulated and act as important mediators of VIDD .Data from both animal , 117 andAn experimental study found in a rat model of mechanical ventilation that the intravenous infusion of an antioxidant  prevented VIDD by attenuating the oxidative stress and subsequent proteolysis and contractile dysfunction during mechanical ventilation .In summary, antioxidant agents acting on more than one molecular target may mediate VIDD produced by oxidative stress, and therefore, this represents an important clinical issue that warrants further investigation.Since VIDD has been identified as a cause of weaning failure, improving respiratory muscle function is an important strategic option. In the ICU, the most promising therapy seems to be inspiratory muscle training (IMT); however, only a few studies have been conducted. When looking at studies on inspiratory muscle training, two factors must be considered. First, the modalities of the “control” arm to which inspiratory muscle training is compared should be carefully examined because there is considerable heterogeneity in practice. Second, the impact of the intervention should have clinical relevance. There is no real benefit of improving inspiratory muscle force itself; attention has to be paid to clinical outcomes, such as shortened duration of mechanical ventilation, successful extubation, or greater survival. The largest study enrolled 69 long-term ventilated patients and randomized them to receive either IMT (defined as a 5-day-a-week program with four sets breathing through a threshold inspiratory muscle trainer of 6 to 10 breaths per day) or control training . PatientIn this study, IMT significantly increased maximum inspiratory pressure but did not affect clinical outcomes such as weaning time. In another study, it was recently reported that IMT performed after successful extubation improved respiratory muscle function, but without any clinical benefit . In concTherefore, IMT is feasible and appears safe in critically ill patients with respiratory muscle weakness and weaning failure . Studiesp value 0.01 and 0.02, respectively).When choosing ventilatory modes, maintaining spontaneous breathing efforts during invasive mechanical ventilation seems protective and seems to alleviate VIDD in healthy animals , 123. EaThe preferred ventilation mode, the optimal level of support (“unloading”), or the rate of support reduction for patients is currently unknown. Spontaneous breathing is well maintained with so-called effort-adapted modes , for exaMethylxanthines, especially aminophylline and theophylline, have been widely prescribed in poorly controlled asthma or COPD based on some of their pathophysiological mechanisms of action. These drugs mainly relax airway smooth muscle by inhibiting phosphodiesterase-3 activity, leading to bronchodilation . They anThe increased histone deacetylase-2 activity reduces formation of peroxynitrite radicals, stimulates the respiratory neuronal network, and increases the activity of respiratory muscles, including the intercostal and transversus abdominis muscles, as well as the diaphragm –142.These findings suggest that methylxanthines may have a role as therapeutic agents in ventilated patients with weaning difficulties associated with VIDD –146.A study using phrenic nerve conduction showed that theophylline infusion rapidly reversed the reduction of transdiaphragmatic pressure resulting from resistive loaded breathing in normal human subjects .In subjects with severe COPD, theophylline significantly increased the maximal transdiaphragmatic pressure and suppressed diaphragmatic fatigue when compared with placebo . Several2O vs. 26.9 ± 9.1 cm H2O; p value = 0.034), but no statistically significant differences in clinical outcomes were found , and with no significant adverse reactions among the study patients.The investigators found that the primary outcome (PImax) was significantly better in the theophylline group than in the nontheophylline group  Ca [2+] release channel/ryanodine receptor (RyR1) in the diaphragm has been found . CalciumExperimental studies have shown that levosimendan improves calcium sensitivity of diaphragm muscle fibers in patients with COPD . In healp = 0.03].However, in a recent trial  in whichFurther studies are therefore needed to elucidate the potential indication of levosimendan in patients with diaphragm dysfunction.ClinicalTrials.gov identifier NCT01721434] is currently investigating whether levosimendan facilitates liberation from the ventilator in patients difficult to wean from the ventilator.Finally, a randomized clinical trial  partially restored calcium sensitivity in diaphragm fibers isolated from piglets after 5 days of mechanical ventilation . Taken tPharmacological interventions aimed at restoring protein balance during muscle dysfunction in the critically ill patient seem a consistent pathophysiological approach. The most important endogenous anabolic hormones are growth hormone, insulin-like growth factor-1, insulin, and the anabolic steroid testosterone and its analogues. Although several trials have been conducted in patients with chronic disease, no clear clinical benefits have been identified and they are not indicated in ICU patients , 160.It is well known that invasive mechanical ventilation may suppress the electrical activity of disused diaphragm muscle , 161. InSimilar findings have been consistently demonstrated in different studies in rat , piglet Diaphragm pacing can be applied through a transvenous phrenic nerve pacing system designed for percutaneous placement into the left subclavian vein. In an experimental study, pigs subjected to mechanical ventilation that received transvenous phrenic nerve pacing in synchrony with ventilation exhibited less diaphragm atrophy . IdentifBrief transcutaneous stimulation of phrenic nerves is often used for diagnostic purposes, but is not suitable for therapeutic purposes. Critically ill patients requiring prolonged mechanical ventilation are not ideal candidates for surgically implanted systems, but may benefit from short-term phrenic nerve pacing to diminish VIDD.Transvenous unilateral phrenic nerve stimulation has been shown to be safe in the treatment of central sleep apnea . AnotherRecent investigations based on a semiblinded, nonrandomized preclinical interventional study test the hypothesis that early phrenic nerve pacing stimulation is a means of increasing diaphragmatic activity during mechanical ventilation . FurtherDespite technical improvements in phrenic-pacing systems, activation of the diaphragm does not provide sustained, full ventilatory support , and evaAs described above, diaphragm pacing is a novel method that can stimulate the diaphragm. This method could be viewed as an effort to support, maintain, and strengthen the diaphragm in patients with weaning failure. It is in these situations that diaphragm pacing (DP) could be proposed in order to remove positive pressure ventilation and restore a more “physiological” breathing obtained through diaphragm contraction .The pathophysiological basis underlying electrical intramuscular DP is seen in experimental studies that have demonstrated that DP increases type 1 slow-twitch fatigue-resistant fibers, thereby reinforcing the resistance of the muscle to fatigue . The proThe use of DP has been proven in canine animal models to provoke the conversion of diaphragm muscle fibers into slow-twitch muscle fibers .These studies portray a clear model of muscle atrophy from disuse and how electrical stimulation  can have effects in its own right, independently of any superior voluntary control. The inactivation of a skeletal muscle, due to disuse or direct trauma, seems to lead to the transformation of type I muscles  into type II muscles (fast twitch glycolytic metabolism) , 188.The two techniques for the implantation of the intramuscular DP system are the thoracic approach, which has been regularly performed with success in a few centers around the world , and a lCE Mark Approval). Currently, there are two ongoing randomized clinical trials  to investigate the safety, effectiveness, and performance of a temporary percutaneous phrenic nerve pacing device. The Lungpacer Diaphragm Pacing Therapy System™  is a central venous catheter that incorporates pacing electrodes.Recently, this technique has also been tested as a temporary tool to avoid diaphragmatic amyotrophy in patients requiring MV for whom spontaneous breathing recovery seemed possible. Thus, these two approaches have led to broadened indications not all validated to date. In fact, some controversy exists as the DP system has been accepted in the USA under compassionate use for some non-pulmonary diseases , and recClinicalTrials.gov Identifier NCT03096639) to investigate the safety and effectiveness of a temporary transvenous diaphragm pacing (TTVDP). The aim is to improve weaning from MV in up to 88 mechanically ventilated adult patients who have failed at least two spontaneous breathing trials over at least 7 days.The current clinical trials include patients undergoing invasive mechanical ventilation ≥ 7 days and/or have failed two or more spontaneous breathing trials [SBT]. The pacing was performed by stimulating the diaphragm through daily therapy sessions, with the intention of exercising and rehabilitating the diaphragm muscle and with the objective of accelerating the process of liberation from mechanical ventilation. A multicenter, randomized, controlled, and open-label interventional study  will carPatients will be randomized to TTVDP as the intervention group or standard of care as the control group. The primary endpoint is time to successful extubation with no reintubation within 48 h. Secondary endpoints include physiological parameters such as the maximal inspiratory pressure and ultrasound-measured changes in diaphragm thickness and diaphragm thickening fraction over time. It is expected that enrollment will be completed by September 2019, and this will be the first clinical trial of TTVDP in critically ill patients with difficultly weaning.Considering the current evidence, there are limitations regarding the effectiveness and safety on the use of DP in critically ill patients with VIDD.Until more results are available from forthcoming clinical and translational studies in this patient population, this therapy should for the moment be considered as a rescue intervention and always performed in specialized centers with experience.The majority of critically ill patients admitted to the ICU require mechanical ventilation, and a substantial amount of time spent in the ICU is dedicated to withdrawal from the ventilator –3. It isThe novel concept of myotrauma may have several implications for research and practice . First, The concept of “bundling” adjunctive therapies is well established in the ICU, and one could even envision the concurrent use of several interventions such as diaphragm pacing with pharmacological interventions to prevent or treat VIDD in the future Fig. . Until fWhile using interventions that might affect patient inspiratory effort or patient–ventilator synchrony—i.e., invasive or non-invasive ventilation strategies, sedation strategies, high-flow oxygen therapy, and extracorporeal life support techniques— the possibility of myotrauma should be considered as a mechanism which may impact on outcomes. The benefits of early mobilization and sedation avoidance strategies might, in part, result from improved recruitment of respiratory muscle effort and avoidance of over-assistance myotrauma.Ultrasound is a powerful tool used to diagnose VIDD in the clinical setting—it enables assessment of both respiratory effort and detection of structural changes in the muscle and the development of muscle weakness. Future studies should consider incorporating simple diaphragm ultrasound measurements to explore the role of the various forms of myotrauma and diaphragm weakness in the determination of outcomes and hence integrate ultrasound in algorithms for decision-making process.Concerns about myotrauma are further increased by the possibility that it might contribute to long-term functional disability in ICU survivors.The major implication of the myotrauma paradigm might be to provide a conceptual framework for how to titrate ventilator support to prevent diaphragm injury.2O [Evidence suggests that the optimal level of respiratory muscle effort might be that of healthy individuals breathing at rest, equivalent to a respiratory muscle pressure swing of 5–8 cm H2O . The afoMonitoring of respiratory effort should become a routine aspect of clinical practice in the ICU. In view of the current evidence, clinicians should attend to patient inspiratory effort and minimize the duration of diaphragm inactivity during invasive mechanical ventilation. If there is no clear indication for neuromuscular blockade, clinicians should aim to maintain a normal level of spontaneous inspiratory effort as well as minimize the volume and transpulmonary pressure applied to the lung in accordance with the paradigm of protective ventilatory strategy. Indeed, the implementation of a diaphragm-protective ventilation strategy aimed in avoiding changes in diaphragm structure, function and myotrauma should be examined in further clinical translational studies.Ventilator-induced diaphragmatic dysfunction is a common deleterious effect of invasive mechanical ventilation and a current relevant clinical challenge. The pathophysiological pathways include several alterations mainly produced by a protein imbalance and oxidative stress. VIDD is often associated with poor clinical outcomes. A thorough evaluation is required to identify its origin and properly manage its effects on symptoms, sleep homeostasis, and exercise capacity.The increasing availability of diaphragmatic ultrasound has provided a simple and effective means of routinely evaluating diaphragm function at the bedside that should help clinicians identify the optimal treatment for the correct patient.Referral to a center with experience in this disease and access to diaphragmatic ultrasound, phrenic stimulation or pacing and surgical expertise in diaphragm stimulation should be considered, where applicable.The integration of evidence from a large body of research based on preclinical and experimental studies of animal models and critically ill patients should allow us to develop a conceptual framework for understanding VIDD. Implementing this knowledge to patient management will eventually result in improved clinical outcomes.


Nonetheless, recent advances in our understanding on the mechanisms of human Tfh differentiation and identification of multiple risk loci in genome-wide association studies have revealed several pathways potentially associated with exaggerated Tfh response in human autoimmune diseases. In this review, we will first briefly summarize the differentiation mechanisms of Tfh cells in humans. We describe the features of “Tfh-like” cells recently identified in inflamed tissues of human autoimmune diseases. Then we will discuss how risk loci identified in GWAS are potentially involved in exaggerated Tfh response in human autoimmune diseases.Convincing lines of evidence in both mice and humans show that exaggerated T follicular helper (Tfh) responses is pathogenic in autoimmune diseases. However, the cause of exaggerated Tfh response in humans is still much less clear than in mouse models where genetic factors can be manipulated for  Although this encouraging outcome remains to be confirmed, it is of great interest to determine whether the treatment with ustekinumab reduced Tfh responses, and more importantly whether the reduction of Tfh response correlated with the improvement of clinical parameters. Alternatively, treatment with ustekinumab might target other immune cells including Th1 and Th17 cells, which are also proposed to play pathogenic roles in SLE , 63. DetLOF mutations as well as patients with IL-21 receptor deficiency display significantly less cTfh cells  mutations display normal frequency of cTfh cells  are not among the risk loci identified in GWAS on autoantibody mediated autoimmune diseases. ICOSLG was instead found to be highly associated with Crohn’s disease and ulcerative colitis  on lupus nephritis. Although the trial was prematurely terminated due to the development of thrombotic events and associated fatality in some patients, the early results were encouraging and the treatment reduced serum anti-dsDNA titers, increased serum complement levels, and reduced nephritis score . Thus, tHU and SH, have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Foodborne disease surveillance in the United States is at a critical point. Clinical and diagnostic laboratories are using culture-independent diagnostic tests (CIDTs) to identify the pathogen causing foodborne illness from patient specimens. CIDTs are molecular tests that allow doctors to rapidly identify the bacteria causing illness within hours. CIDTs, unlike previous gold standard methods such as bacterial culture, do not produce an isolate that can be subtyped as part of the national molecular subtyping network for foodborne disease surveillance, PulseNet. Without subtype information, cases can no longer be linked using molecular data to identify potentially related cases that are part of an outbreak. In this review, we discuss the public health needs for a molecular subtyping approach directly from patient specimen and highlight different approaches, including amplicon and shotgun metagenomic sequencing. Clinical diagnostic laboratories are increasingly moving away from traditional culture-based methods to culture-independent diagnostic tests (CIDTs) which are molecular tests that identify pathogens directly in the specimen without producing an isolate  infections were caused by non-O157 serotypes not easily recognized in culture. However, CIDTs for enteric pathogens gained popularity during the past decade when polymerase chain reaction (PCR)-based “syndromic” panels were commercialized, permitting detection of up to 22 bacterial, viral, and parasitic pathogens in a stool sample in a matter of hours.Among the first CIDTs were enzyme immunoassays (EIAs) detecting single or multiple pathogens. Among foodborne enteric bacterial pathogens, the most notable EIA was developed for the detection of Shiga toxins in Salmonella enterica, Listeria monocytogenes, and STEC, cultured isolates are forwarded to local and state public health laboratories to perform subtyping for public health surveillance. Although these subtyping results rarely contribute to direct patient care, subtype information is critical for public health surveillance and enteric disease outbreak detection.These tests are highly accurate and are performed on minimally processed patient specimens. Thus, these tests can provide clinically actionable information more rapidly than gold standard culture-based identification techniques. However, unlike gold standard methods, the CIDTs do not yield a pure culture of the pathogen. For reportable pathogens such as Subtypes of foodborne pathogens are compared at the local, state, and national levels through PulseNet, the national foodborne molecular surveillance system. Outbreaks caused by nationally or regionally distributed products or from local food handling problems can be identified by using subtype information to group together case patients likely to have a common exposure, such as a common food source. Annually, PulseNet processes subtype data from about 70,000 isolates of clinical foodborne pathogens from public health partners, and include data streams from human and animal diagnostics as well as regulatory testing of food and food production environments.et al., PulseNet's routine, systematic use of subtype data from isolates at state and local public health laboratories has a tremendous public health impact, preventing an estimated 270,000 illnesses annually and saving the U.S. economy more than 500 million U.S. dollars , which costs 95–300 U.S. dollars in materials per isolate, depending on the workflow in the laboratory. In addition, the laboratory and analytical workflow needs to be completed in 3–4 working days and be compatible with a standard 8-h workday and minimal hands-on time.For the laboratory workflow, all sequencing should use existing next-generation sequencers in public health laboratories; new equipment should entail minimal equipment costs and service contract expenses. Analytical tools must be user-friendly and push-button because a majority of laboratories have limited information technology (IT) and bioinformatic support and limited, if any, access to high-performance computing. Critically, the final subtyping output must be able to distinguish outbreak from background sporadic isolates at both a local and national level and link specimens from epidemiologically related cases.Ideally, the output should also be compatible with isolate-based analysis workflows used to identify and/or confirm potential outbreaks and outbreak sources, and it should ensure historical compatibility with isolate-based WGS data. Given these requirements and limitations, the ideal solution for public health surveillance for foodborne infections would be a user-friendly laboratory procedure generating sequence data directly from the primary specimen that could be analyzed using push-button bioinformatic tools that are easily deployable in a public health laboratory.Unfortunately, there is no single existing specimen-based subtyping approach that can meet all these requirements . Here weet al., A classic example of the amplicon sequencing approach is the 16s rDNA sequencing, typically used to assess microbial diversity in a specimen. However, this approach only provides resolution at the genus or species level, limiting its utility for diagnostic and public health purposes  scheme. The target regions for the HMAS panel must be flanked by pathogen-specific primer sites and be of the correct length to be compatible with the intended sequencing platform.The design of HMAS panels requires a well-curated pathogen-specific database to identify the genomic regions needed to distinguish an outbreak from sporadic cases. A database has been developed for in silico studies of amplicons generated from isolate genome assemblies have shown that it is possible to subtype Salmonella at a resolution similar to that of the PulseNet cgMLST scheme using ∼1500 loci (unpublished data).The flexibility of HMAS panels allows additional informative targets, such as antimicrobial resistance (AMR), virulence, and serotyping genes, to be added to the subtyping amplicon panel. Existing HMAS panel laboratory methods are also amenable to sample multiplexing, allowing simultaneous amplification of several specimens and loading of the products onto a sequencing platform with minimal processing. Analysis will require the development of new or repurposed existing open-source bioinformatic pipelines. Our Salmonella, in extremely complex and bacteria-rich clinical specimen backgrounds such as stool. In addition, some enteric pathogens are closely related to the commensal gut microflora, making it difficult to identify enough pathogen-specific targets for the high-resolution subtyping required for foodborne outbreak surveillance. For example, commensal E. coli frequently co-occurs in stool specimens with STEC, but virulence genes are the only markers known to distinguish STEC from other strains of E. coli. Unfortunately, virulence genes alone do not contain enough information to identify outbreak-related strains.While amplicon sequencing is a mature technology, there are several obstacles to implementing HMAS panels at scale. One challenge is the need for large numbers of primers specific to the pathogen of interest, for example, E. coli-specific single-copy core genes. The different alleles for each gene will then appear in the sequencing data in the same proportion as the strain of origin in the E. coli population of the patient sample. These proportions can be used to group alleles from different genes by their strain of origin. This concept is illustrated in et al.  . This requires input of enrichment targets, which can include AMR genes or other known subtyping targets. The initial study demonstrated enrichment of AMR targets on respiratory metagenomic samples. Both Cas9 approaches have low hands-on time in the laboratory and a low cost per sample after the initial guide RNA generation costs. The methods are briefly outlined in Another Cas9-based approach, FLASH (Finding Low Abundance Sequences by Hybridization), targets DNA to enrich for sequencing (Quan et al., et al., et al., One challenge for FLASH is that the high specificity of the approach may require individual guides to be generated for each targeted sequence variant to enrich for all of the possible subtypes. This contrasts with PCR primers, which have some template mismatch tolerance. Other target enrichment approaches rely on the generation of a biotinylated bait library to target specific pathogen library DNA to enrich those libraries for subsequent sequencing workflows (Briese et al., a priori knowledge about the target microbe, which is helpful if the specimen contains multiple or novel pathogens. Even if the agent is not detected by the initial CIDT panel, unculturable, or unknown, shotgun metagenomics can detect and identify such pathogens.The other specimen-based subtyping approach, shotgun metagenomics, is used to sequence all DNA in the specimen in an unbiased manner, from which pathogen signals can be isolated and characterized. Although this approach is more costly and with limited throughput, advances in information and sequencing technology may bring the cost down to meet the requirements of a public health molecular surveillance system (Scholz et al., et al., A few proof-of-principle studies have been published proving the utility of this approach (Loman In addition, there are few bioinformatic pipelines that produce the resolution needed for strain-level characterization from metagenomic reads. Most shotgun metagenomic analysis pipelines provide resolution to the species level, but not to the subtype level (Wood and Salzberg, et al., et al., et al., et al., Emerging approaches for binning metagenomic reads and contigs using reference-based and reference-free approaches are making it possible to sort sequences into species-based bins for further analysis (Sangwan et al., et al., Moreover, both binning approaches are able to take advantage of whole sets of samples as might be found in presumptive clusters during an outbreak investigation. By focusing on genomic material in common among samples, the output from these programs can be used to generate a phylogenetic tree showing the relatedness of samples using only the common sequence bins, which may correspond to shared pathogens (Nayfach et al., There are some laboratory-based approaches to link together sequences generated from the same organism found in a specimen. One approach includes crosslinking DNA from the same cell and then using the markers left by crosslinking to bioinformatically link reads generated by the same cell. Phase Genomics and others have commercialized this approach, and it has been successful in human gut metagenomic samples (Press Another promising laboratory approach is low-cost long-read metagenomic shotgun sequencing. For example, Oxford Nanopore's technology can generate 15–100 kB reads, which are long enough to physically link pathogen marker genes, such as the Shiga toxin phage region of STEC, with other core genes useful in subtyping from the surrounding regions.et al., et al., et al., et al., There are several proof-of-principle studies that have shown the utility of long-read sequencing from metagenomic samples, although these approaches focus on pathogen identification or identification of resistance genes, rarely strain level subtyping (Lemon et al., Although real-time shotgun metagenomics for public health surveillance may not currently be feasible, clinical laboratories are also moving to metagenomic approaches as a way to identify pathogens from clinical specimens (Gu The advantages and disadvantages of the specimen-based subtyping approaches outlined in this review are highlighted in Such a solution has the potential to reduce turnaround time by weeks compared with current outbreak detection methods, thereby enabling a faster and more efficient public health response. If public health and food and regulatory databases contain comparable sequence data, then signals could be quickly detected for potential outbreak sources. In addition, if molecular data were linked to patient and population-based demographic and eating habit data and other food consumption information, such as shopper cards, investigators could more quickly combine the epidemiologic information and use it to confirm or disprove signals in the molecular data. Finally, as the food industry moves to higher fidelity approaches to track food from farm to fork, such as block chain, the powerful combination of these different data sources can speed up outbreak investigations and prevent more foodborne illness.This vision cannot be achieved with the current state of sequencing, informatics, and bioinformatic technology. However, what may sound like science fiction today could become reality within the next 10–20 years.


Worldwide 2–8% of pregnancies are complicated by pre-eclampsia (PE), with an estimated maternal mortality of 50 000 cases annually . PE is aDelivery is the only definitive treatment. In preterm stages of pregnancy treatment of the disease occurs mainly symptomatically, aiming for analgesia and control of hypertension. In high-risk groups, a slight risk reduction of developing PE is achieved by administration of acetylsalycic acid in early pregnancy . A centuFor successful implantation of the placenta, maternal immune system has to develop local tolerance for the non-self-embryo . FailureDown-regulation of local immunity in the decidua is likely to be shaped by an ‘evolutionary tradeoff’. Pregnancy yields higher susceptibility to infection and tolerance for fetal tissue as the trophoblast has shown to be disrupted by infection during pregnancy. Immunological tolerance is subject to regulation, reflected in the reduced risk to develop PE in a subsequent pregnancy from the same partner .PE seems to occur in pregnancies with a reduced immunological tolerance. This yields a placenta with lower capacity, more vulnerable to a mismatch between fetal demands and maternal carrying capacity.‘Parental antagonism’ hypothesizes that maternal and paternal interests are not entirely aligned in (partly) promiscuous species. The resulting conflict is (among other stages of the life cycle) fought using endocrinological influence of the trophoblast  on maternal metabolism and circulation. By mechanisms like imprinting, genes are competing: paternally derived genes will manipulate maternal metabolism as to allocate more resources, while maternally derived genes aim to reduce expenses and risks of gestation and labor . Both paPE is likely to be too fundamentally rooted in evolutionary tradeoffs and parental antagonism to allow full prevention. Improvements in clinical practice could be achieved by a combination of first trimester screening and more causal treatment of the dysfunctional placenta. Early diagnostics will benefit from knowledge of the evolutionary background. Signs of dominant paternally derived genes could be found in (epi)genetics itself, but so far no candidate genes could be selected to reliably predict PE . ScreeniConflict of interest: None declared.


Sleep has been classically described as an all-or-nothing global phenomenon. However, recent research strongly suggests that this view requires tempering. Invasive and non-invasive recordings in animals and humans show that neural activity typically associated with sleep can locally occur during wakefulness. Although local sleep is defined neuronally, it has been associated with impaired performance during cognitive tasks. Comparatively, the phenomenology of local sleep  has been less explored. Taking into account the literature on the neuronal and behavioral profile of local sleep intrusions in wakefulness, we propose that occurrences of local sleep could represent the neural mechanism underlying many attentional lapses. In particular, we argue that a unique physiological event such as local sleep could account for a diversity of behavioral outcomes from sluggish to impulsive responses. We further propose that local sleep intrusions could impact individuals’ subjective experience. Specifically, we propose that the timing and anatomical sources of local sleep intrusions could be responsible for both the behavioral consequences and subjective content of attentional lapses and may underlie the difference between subjective experiences such as mind wandering and mind blanking. Our framework aims to build a parallel between spontaneous experiences in sleep and wakefulness by integrating evidence across neuronal, behavioral and experiential levels. We use the example of attention deficit hyperactivity disorder (ADHD) to illustrate how local sleep could explain complex cognitive profiles which include inattention, impulsivity, mind-wandering and mind-blanking. Sleep and wakefulness have been traditionally considered as mutually exclusive states. At the behavioral level, sleep is characterized by a transient loss of responsiveness to the environment . This beRecent discoveries in animal and human sleep physiology have tempered the notion of slow waves as necessarily being a global physiological event . Within Glossary: Some of the terms used in this Perspective are widely used but not systematically defined and the same terms may be used by behavioral, phenomenological and/or neurophysiological approaches to refer to slightly different target phenomena. Below we define each term to the extent that is acceptable across different disciplines in order to facilitate further interdisciplinary work, both theoretical and empirical.Wakefulness: Wakefulness is a state in which individuals can rapidly and reliably react to environmental demands. At the physiological level, wakefulness is characterized by a pattern of brain activity dominated by fast, low-amplitude, desynchronized oscillations.Sleep: Sleep is a state behaviorally defined by a transient loss of responsiveness. To induce responses, stimuli have to be more intense than during wakefulness. The recovery of responsiveness is usually associated with a reversal to neural patterns of wake activity. Neuronal activity during sleep is dominated by slow, high-amplitude, synchronized oscillations in Non-Rapid Eye Movement (NREM) sleep and by low-amplitude, theta (4–7 Hz) and mixed-frequency oscillations in Rapid Eye Movement (REM) sleep.Local Sleep: A concept introduced by Microsleep: Microsleep is classically defined as a global shift in neuronal activity from wakefulness to light NREM sleep for a duration of 5 to 14 s. Above 14 s, the individual is considered asleep. Below 5 s, the individual is considered awake but could potentially show signs of local sleep (< 5 s).Attentional lapse: Attentional lapses refer to the redirection of an individual’s attention away from a specific task. These lapses are accompanied by a drop in objective performance as well as an increase in performance variability.Mind-wandering: Mind-wandering is defined as spontaneous, dynamic and often associative thought. In the context of laboratory experiments, this is often operationalized as task- and/or stimulus independent thought. Mind-wandering can be considered as the phenomenological dimension of a large group of attentional lapses.Mind-blanking: Mind-blanking refers to subjective reports of reduced awareness and a temporary absence of thought (empty mind) or lack of memory for immediately past thoughts. Mind-blanking can be considered as the phenomenological dimension of a distinct kind of attentional lapse compared to mind-wandering.ADHD: Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder with a worldwide prevalence of approximately 5%. The diagnosis is based on age-inappropriate levels of inattention, hyperactivity and/or impulsivity.Local sleep during wakefulness was originally defined based on the identification of a hallmark of NREM sleep within wakefulness: sleep slow waves . These sInvestigations in humans have therefore focused on the detection of local high-amplitude slow oscillations, which typically accompany OFF periods . HoweverIn summary, local sleep can be defined, at the level of neuronal networks, by the brief local appearance of events similar to sleep slow waves (delta and/or theta waves) which are in turn associated, at the level of single-neuron activity, with episodes of neuronal silencing . Local sInterestingly, the occurrence of local sleep intrusions varies as a function of both the time spent awake and the degree to which a particular brain region has been activated by environmental demands  . A recenAt the same time, it is unlikely that extensive usage of local neural circuitry is the sole predictor of local sleep occurrences. Rather, it is more likely that local sleep is modulated by a combination of use-dependent and circadian factors. Sleep in general is known to be influenced by both time spent awake and circadian processes . ConsistAlthough local sleep has been primarily defined at the physiological level, its occurrence during wake has been associated with impaired task performance: trials preceded by local slow waves typically show slower reaction times and increased error rates . These pLocal modulations of sleep-like activity not only impact behavioral performance during wakefulness, but might also affect the contents or structure of subjective experience during sleep. Recent research, relying on subjective reports and serial-awakening paradigms , has empLocal sleep in wakefulness could be associated with similar changes in subjective experience. Indeed, spontaneous experiences in wakefulness and sleep share similar properties at the phenomenological level. Wakeful cognition is a highly dynamic process and fluctuations in spontaneous experience forming the so-called “stream of thought”  are at tIt has been suggested that dreaming and mind wandering could be placed on a continuum . First, Other sleep-experiences lack the immersive character of dreaming and hence have been termed dreamless sleep experiences. A subgroup of these is characterized by an absence of reportable content; these seem to involve simple or perhaps even minimal forms of subjective experience in which specific forms of thought contents or imagery are lacking . This suIf sleep- and wake-related spontaneous experiences share similar properties at the phenomenological level, are they also related at the physiological level? More precisely, can local sleep explain the presence or absence (as well as content) of spontaneous experiences not just in sleep, but also in waking? This tantalizing question will need further investigation but initial results are promising. First of all, while the local decrease in slow-wave activity over parietal regions correlates with the occurrence of spontaneous experiences during sleep (dreams), a local increase during wakefulness over the same regions correlates with a reduction in spontaneous thoughts . These rWe propose a multi-level model of attentional lapses from neurophysiology to behavior and phenomenology. In our framework, local sleep intrusions represent a unifying physiological mechanism that could not only predict the occurrence of attentional lapses but also describe these lapses in terms of their behavioral consequences and phenomenological properties. Other markers of low arousal have been proposed to predict attentional lapses. In particular, pupil size can predict the occurrence of attentional lapses and mind-wandering . HoweverPrevious studies investigating local sleep intrusions during wake focused mostly on task and region-specific effects, after forcing participants to continuously perform a single task . The goIn particular, local sleep occurrences could explain the occurrence of seemingly opposed behavioral failures such as omission errors (failure to produce a response) and commission errors . The standard view is that commission errors are associated with executive control failure, whereas omission errors would rather mark the failure to maintain a more basic form of vigilance . This viNext, we hypothesize that local sleep can account not only for decreases in objective behavioral performance but also for changes in individuals’ subjective experience. The impact of local sleep on subjective experience has been less frequently studied  than itsImportantly, it has been previously suggested that these different types of subjective experiences correspond to different neural mechanisms and different levels of arousal. Mind-wandering has been proposed to be associated with a high level of arousal and fleeting or racing thoughts, whereas mind-blanking has been associated with decreased vigilance and low arousal . HoweverLocal sleep could also represent a powerful explanatory account of inter-individual differences in attentional lapses in conditions such as attention deficit hyperactivity disorder (ADHD). ADHD is a common neurodevelopmental disorder that affects 1–3% of adults and 3–7% of children . It is cKey behavioral attributes of ADHD, established across numerous tasks, are the increase in omission errors, greater intra-individual variability in response timing, as well as more infrequent extremely slow responses , b. At tThis behavioral and experiential fingerprint of ADHD involving sluggishness and mind-blanking portrays ADHD as hypo-vigilant. This could seem at odds with the popular association between ADHD and hyperactivity. However, the association between ADHD and hypo-vigilance fits well with the frequent observation of sleep disturbances in ADHD adults and children . In factA key question in the ADHD literature is therefore whether these sleep problems are mere correlates of the attentional disorder or whether they could be at the root of the behavioral features of ADHD. In favor of the latter, it has been hypothesized that ADHD symptoms arise from a dysregulation of arousal . AccordiThus, given that individuals with ADHD exhibit, when compared to healthy controls: (1) greater attentional lapses that affect task performance; (2) higher rates of mind-blanking; and (3) more sleep disturbances than healthy controls, local sleep could provide an interesting and largely overlooked neural mechanism for the behavioral phenotype in ADHD. This novel framework in ADHD research could also improve current therapeutic approaches, as current medications can improve daytime performance but can also perturb sleep . A more We outline here testable hypotheses regarding the relationship between the occurrence of local sleep and the behavioral and phenomenological profiles of attentional lapses. We hypothesize that local sleep occurrences could not only predict attentional lapses but also the type of lapse depending on the anatomical location of local sleep intrusions. Our model could lead to a better understanding of phenomena such as mind wandering and mind blanking. Indeed, mind wandering has been associated with the recruitment of the DMN . InteresWe further propose that local sleep could account for inter-individual differences in the frequency and type of attentional lapses. In particular, focusing on individuals with ADHD, we hypothesize that the reported increase in mind-blanking, at the expense of both mind-wandering and a focused state , may be Importantly, we do not argue here that all forms of attentional lapses can be explained by sleepiness and local sleep intrusions, nor that changes in subjective experience such as mind-wandering and mind-blanking are always associated with local sleep. It is of course possible that attentional lapses with similar behavioral consequences to those discussed here can occur in contexts of hyper-arousal rather than hypo-vigilance . NonetheAlthough promising, this framework needs to be further established. It has already been suggested that local sleep intrusions, depending on their location, can affect specific aspects of wake behavior . HoweverFinally, the conditions under which local sleep can be observed are yet unclear. Initial studies have shown local sleep intrusions following extensive sleep deprivation, with the first intrusions occurring only after a few hours of sleep restriction . A recenAll authors contributed to the review and discussion of the literature and to the proposition of future directions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diet quality is closely linked to child growth and development, especially among infants aged 6–23 months who need to complement breastmilk with the gradual introduction of nutrient-rich solid foods. This paper links Demographic and Health Survey data on infant feeding to household and environmental factors for 76,641 children in 42 low- and middle-income countries surveyed in 2006–2013, providing novel stylized facts about diets in early childhood. Multivariate regressions examine the associations of household socioeconomic characteristics and community level indicators of climate and infrastructure with dietary diversity scores (DDS). Results show strong support for an infant-feeding version of Bennett's Law, as wealthier households introduce more diverse foods at earlier ages, with additional positive effects of parental education, local infrastructure and more temperate agro-climatic conditions. Associations with consumption of specific nutrient-dense foods are less consistent. Our findings imply that while income growth is indeed an important driver of diversification, there are strong grounds to also invest heavily in women’s education and food environments to improve diet quality, while addressing the impacts of climate change on livelihoods and food systems. These results reveal systematic patterns in how first foods vary across developing countries, pointing to new opportunities for research towards nutrition-smart policies to improve children’s diets. Growth In this study we explore why children 6–23 months are fed what they are fed in 42 low- and middle-income countries (LMICs), as measured through standard dietary diversity metrics and consumption of specific food groups in nationally-representative surveys. The scope of this study is novel, but also builds on existing literatures in nutrition and economics.Nutritionists have long recognized the importance of gradually introducing new foods in addition to continued breastfeeding after 6 months of age . ObservaEconomics also has a long tradition of research on consumer demand for variety in other kinds of goods, showing introduction of new goods and increased diversity in consumption as incomes rise . For fooRecent studies show how the diet diversification process is affected by differences in the relative cost of acquiring different foods, including the many situations in which certain foods are entirely missing from local markets . As showThis paper aims to provide a comprehensive exploration of the relative roles of wealth, parental knowledge/education, women’s empowerment, and geographical characteristics such as agroecology and infrastructure in shaping infant feeding patterns. We use recent Demographic and Health Surveys (DHS) from 42 countries, combined with geographic data about the locations of DHS survey clusters as described in 2Our work is motivated by household decisionmaking models such as those described in In this framework, if there were perfectly competitive markets for everything, child dietary intake would depend only on the household's wealth and full income , their preferences , and their food environment (including the relative prices and convenience of obtaining different foods). Missing or imperfect markets ensure that the household's own production possibilities and local conditions around each survey site also influence consumption, especially for bulky and highly perishable foods that have high transport and storage costs. These include many of the most important nutrient-dense foods needed for infant feeding, such as dairy products, eggs and many fruits and vegetables. Crop choice and productivity is very sensitive to temperature and climate , and tem2.1We use multi-country household survey data from Phases 5 and 6 of the DHS , which wOur primary measure of diet quality is the dietary diversity score (DDS), defined as the number of food groups  consumed by a child in the past 24 h. In addition to this diversity metric, we explore consumption patterns of four nutrient-rich vegetal foods and four nutrient-rich animal-sourced foods. The seven food groups included in the DDS measure are described in Overall, the dietary intake data we use are available for 76,641 children aged 6–23 months in 42 countries in five regions, thus providing substantial socioeconomic and geographic variation for our analysis (see Table A1 for a full list of countries): most (58%) of our sample is from sub-Saharan Africa, with 24% from Latin America and the Caribbean, 11% from Asia and 7% from the Middle East and North Africa).The theoretical framework described above emphasizes the importance of budget constraints in shaping food purchases. To capture purchasing power, we use DHS data on the household's ownership of durable consumption goods  and housing characteristics  that reflect permanent income. Time-varying purchasing power would be reflected in household expenditure, but it is measured with substantial error  and asseIn addition to wealth, we use several other socioeconomic indicators that we interpret primarily as proxies for knowledge and preferences. Formal education, in particular has been shown to be a strong predictor of nutritional knowledge . We meas2 lake or a major river) to reflect access to fisheries and large scale irrigation, and population density of the surrounding area to reflect the thickness of local markets.Our selection of community-level GIS indicators is motivated by the microeconomic theory around missing markets described above. Access to markets through improved infrastructure or inherent geographical advantages may be an important prerequisite for purchasing a greater variety of foods, especially perishable foods that are costly to trade long distances. The DHS records whether a cluster is urban or not, but definitions of urban vary substantially across countries and are somewhat arbitrary. Therefore, we use GIS estimates of travel time to cities to construct a “remote location” dummy variable that equals one if the DHS cluster (“village”) has more than a one-hour travel time to a town/city of 20,000 people or more, with that threshold suggested by nonparametric evidence from the association between DDS and travel times. We also use a satellite-based night lights intensity index to capture local economic development and electricity infrastructure , as wellIn addition to infrastructural and demographic conditions, agricultural conditions can substantially influence which foods are produced in a given community. We focus on three potentially relevant measures: average temperature, average rainfall, and cluster altitude, where temperature and rainfall are measured as 30-year annual averages (1980–2010). We expect that more rainfall increases the array of crops that can be grown, as well as fodder for livestock, while agronomic research shows that high temperatures (e.g. above 29 degrees Celsius) reduce the yields of many crops, and will therefore prevent farmers from even attempting to grow heat-sensitive crops . Tempera2.2i, j, and k respectively denote child, cluster and country-year identifiers and Our analysis begins with descriptive evidence on consumption patterns in the full sample and the five major developing regions, before turning to non-parametric local polynomial smoothing regressions to examine the relationships between dietary diversity and various explanatory variables of interest. As there are many non-linear relationships, particularly for the community-level GIS indicators described above, we measure continuous indicators with terciles to flexibly and parsimoniously capture these non-linearities. We then use ordinary least squares regression models and linear probability models to estimate a regression model with DDS, MDD or consumption of a nutrient-rich food group as a function of DHS child and household characteristics (H), community characteristics (C), child age in month (Z), and country-year (survey) fixed effects  of One important explanation of this pattern of results is that many of these GIS-level indicators are reasonably strong predictors of household wealth, parental education and access to health services. To examine this we estimated regressions with household wealth as the dependent variable and the various GIS indicators as explanatory variables. Coefficient plots with 95% confidence intervals are reported in H) or community-level variables (C) that the high diversity country have. Hence the predicted difference in DDS between high and low diversity countries due to any specific variable is just the product of the relevant regression coefficient and the difference in means across the two samples:Next, The results in Two important conclusions therefore stem from 4.2Next, we exploit the substantive geographical coverage in our data to explore heterogeneity of the DDS results across regions in For the GIS indicators there are limits in the number of clusters in most regions that likely restrict the ability of these regressions to accurately identify community-level effects, so we focus the discussion here on SSA, which has a large sample size and ample geographic variation in these indicators. In SSA we again observe positive associations between night lights intensity and DDS, but also that areas further from the coast have somewhat higher DDS. Given the region’s vulnerability to climate change, the sensitivity of DDS to rainfall and temperature is particularly striking. We also note that In 5Economic analysis of food choice and diet quality is usually confined to purchases by households, rather than individual intake, especially regarding infants from 6 to 24 months when growth faltering most often occurs. We overcome past data constraints on infant feeding by combining data from 42 countries covered by the DHS with a rich array of GIS-based community level data that capture economic and infrastructural development as well as exogenous agricultural conditions. This extensive dataset allows us to document diverse child feeding patterns across regions and economic strata, to estimate household and community level determinants with precision and flexibility, and to explore heterogeneity in these associations across developing regions. While the DHS dietary data are limited insofar as they do not provide quantities, their high degree of standardization allow us to analysed consumption patterns for a critically important age group.In this section we flag some of the most important findings from our results, and discuss their implications for new research and for nutrition-smart policies designed to improve child feeding.5.1We find a highly significant and approximately linear association between child diet diversity and household wealth, providing strong support for an alternative version of Bennett’s law defined by food group diversity . The marginal effect of household wealth is about the same size as the marginal effect associated with 10+ years of maternal education. Wealth and education account for similar fractions of the difference between low and high dietary diversity populations, which may partially account for the robust association between maternal education and stunting or other nutrition outcomes . It is p5.2We offer novel evidence that indicators of geographic, demographic and infrastructural characteristics – as well as night lights  – have significant and reasonably large associations with child dietary diversity scores. Night lights intensity is strongly associated with household wealth, suggesting that it can serve as a general indicator of economic prosperity  as well Of significant policy relevance in the context of a rapidly warming and more volatile climate, we find strong relationships between climate indicators and dietary diversity, as infants in the lowest tercile of temperatures and highest tercile of rainfall have the most diverse diets. This is consistent with a wide range of previous research suggesting that more temperate conditions facilitate agricultural growth and broader development e.g. , and tha5.3A subtle but important finding of our research is that while overall dietary diversity increases with wealth , different kinds of nutrient-rich foods vary differently with household wealth and aspects of the child's environment. At one extreme, dairy consumption rises sharply with household wealth and night lights intensity. The wealth gradient for meat is also relatively steep, while the gradient for eggs is more modest, and fish consumption does not rise with household wealth and is quite prevalent even among the lowest wealth tercile. In terms of vegetal foods, vitamin A-rich fruits and vegetables and other fruit/vegetables have modest wealth gradients similar to egg consumption, with children from the wealthiest tercile about 5.7 times more likely to consume these products than children from the poorest tercile of household wealth. However, legume/nut consumption is largely invariant to wealth, and DGL vegetable consumption actually declines with wealth. We note that these results are broadly consistent with economic studies of household level demand for these foods (see Supplement B).The fact that parental use of some healthy foods such as DGL vegetables declines with wealth, and that there is no income effect for fish, suggests a particularly important role for nutritional knowledge and preferences regarding these foods, and possibly a role for food preparation and time constraints as caregivers switch to other nutrient-dense foods notably eggs, meat or dairy. Since DGL vegetables and fish could provide valuable complements to other foods, parents might be encouraged to keep these healthy foods in child diets even as they add other food groups over time. Again, studying the evolution of infant diets in developing countries is an important area for future research.5.4All results in this study are purely observational, intended to establish novel stylized facts about the timing and degree of diversification in children’s diets in low- and middle-income countries. The DHS data we use provide nationally-representative samples of unprecedented size and scope, but are necessarily limited to a dichotomous indicator for intake of major food groups over the previous day or night. This single 24-hour food frequency questionnaire is less precise than more time-intensive forms of dietary assessment, and does not allow analysis of seasonal or other variation in feeding practices. Furthermore, our analysis focuses on the food groups used for dietary diversity scores, omitting premixed cereals or other packaged products. The potential correlates of feeding practices that we examine are limited to a relatively simple wealth index, and no direct measures of nutritional knowledge or exposure to programs designed to improve that knowledge. Finally, our analysis aims to establish global patterns, with only suggestive evidence about regional heterogeneity. Future work could aim to overcome many of these limitations, building on the steps taken in this study to understand and improve the diets of young children which are particularly important for physical and cognitive development . No prevOverall, our findings have important implications for future research and policy design, especially for countries with the highest burdens of malnutrition. Our results suggest that while wealth accumulation is indeed an important driver of diversification, women’s education and the food environment are likely to play an additional independent role as well. Access to maternal and child health care may also play an important role in how children are fed, highlighting opportunities to improve the nutritional messaging of conventional health services . All of


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