GDF15 has recently gained scientific and translational prominence with the discovery that its receptor is a GFRAL-RET heterodimer of which GFRAL is expressed solely in the hindbrain. Activation of this receptor results in reduced food intake and loss of body weight and is perceived and recalled by animals as aversive. This information encourages a revised interpretation of the large body of previous research on the protein. GDF15 can be secreted by a wide variety of cell types in response to a broad range of stressors. We propose that central sensing of GDF15 via GFRAL-RET activation results in behaviors that facilitate the reduction of exposure to a noxious stimulus. The human trophoblast appears to have hijacked this signal, producing large amounts of GDF15 from early pregnancy. We speculate that this encourages avoidance of potential teratogens in pregnancy. Circulating GDF15 levels are elevated in a range of human disease states, including various forms of cachexia, and GDF15-GFRAL antagonism is emerging as a therapeutic strategy for anorexia/cachexia syndromes. Metformin elevates circulating GDF15 chronically in humans and the weight loss caused by this drug appears to be dependent on the rise in GDF15. This supports the concept that chronic activation of the GDF15-GFRAL axis has efficacy as an antiobesity agent. In this review, we examine the science of GDF15 since its identification in 1997 with our interpretation of this body of work now being assisted by a clear understanding of its highly selective central site of action.  GDF15 is a stress-regulated hormone that signals exclusively via the GFRAL-RET heterodimer.GFRAL expression is restricted to the area postrema and nucleus tractus solitarius.GDF15-GFRAL-RET signaling suppresses appetite in mice and nonhuman primates and is perceived as aversive by mice.GDF15 is implicated in a wide range of human disease states associated with weight loss and nausea, including cancer cachexia, chemotherapy-related nausea and vomiting, and hyperemesis gravidarum.Metformin increases circulating GDF15 in humans and an intact GDF15-GFRAL-RET axis is required for the weight loss effects of metformin in mice.Growth differentiation factor-15 (GDF15) is a member of the transforming growth factor- β (TGF-β) superfamily, which is secreted by cells exposed to a broad range of stressors. Its role in appetite and weight regulation was first postulated when it was observed that tumors overexpressing this hormone could induce cachexia in mice, which was ameliorated by an anti-GDF15 antibody. In spite of this, its primary physiological function and the molecular basis of its actions remained enigmatic until recently, when its receptor, the GDNF family receptor alpha-like proto-oncogene tyrosine-protein kinase receptor Ret (GFRAL-RET) heterodimer, was simultaneously identified by 4 groups working independently. GFRAL is expressed exclusively in the hindbrain and activation of the GFRAL-RET heterodimer suppresses food intake. These findings firmly establish the GDF15-GFRAL axis as a novel hormonal system regulating ingestive behavior and have invigorated interest in the translational relevance of this system. Here we review the cellular and molecular biology of GDF15-GFRAL and consider the emerging roles of this hormonal axis.GDF15 (then termed macrophage inhibitory cytokine-1 [MIC-1]) was discovered by the Breit group in 1997, who used subtraction cloning to identify genes that were relatively enriched in monocytoid cells treated with phorbol 12 myristate 13-acetate (PMA) to model activated macrophages . In 2001The human GDF15 gene  is locatIn humans and old-world primates, a 73 base pair transcript encoding a microRNA (miRNA) miR-3189 is found in the GDF15 intron . Existinth cysteine in the 9 cysteine domain forms a disulphide bond with a free 6th cysteine from another pro-GDF15 monomer to form a pro-GDF15 homodimer . Be. Beth cyomodimer  and pairomodimer  26). Pr. Prth cyomodimer .Unlike other members of the TGF-β superfamily, GDF15 does not require an intact propeptide domain for processing and secretion, as when a GDF15 mutant lacking the propeptide is overexpressed the proteasomal inhibition has no effect on secretion of mature GDF15 and GDF15 does not accumulate intracellularly . This isIn the processing of canonical TGF- β superfamily members, the prodomain remains noncovalently associated with the mature peptide and confers latency. In the case of GDF15 there is no evidence to suggest that it remains associated with its prodomain when processed and secreted. Instead, it is thought to circulate exclusively as an active homodimer. However, a number of transformed cell lines, including the choriocarcinoma cell line BeWo, the monocytoid cell line from which GDF15 was initially cloned, U937, and the prostate cancer cell lines, PC-3 and LNCaP, all secrete an unprocessed proGDF15 dimer where the propeptide domain has not been proteolytically cleaved . InteresAs noted above, GDF15 exhibits important structural differences, which set it apart from its counterparts in the TGF-β superfamily. Examining the evolutionary history of this divergence is instructive. GDF15 likely evolved in the common ancestor of jawed vertebrates, as there is no clear orthologue observed in the genomes of the other 2 lineages of craniata, hagfish, lampleys, or lower vertebrates. Orthologues have been annotated in mammals, reptiles, amphibians, bony fish, and birds with a high level of conservation observed in the C-terminal region of the protein that represents the mature peptide. In contrast, the amino acid propeptide conservation is considerably lower, indicating significant remodeling and simplification during evolution (note progressive accumulation of deletions in vertebrate evolution) ).Unfortunately, the structure of pro-GDF15 is not yet known; however, the structure of the homologous TGF-β can be used to draw inferences . The amiThe significance of these changes in terms of evolutionary fitness is unclear at present.The high expression of GDF15 in placenta and the high circulating levels observed in pregnant women suggest it is important in pregnancy . It may Circulating levels of GDF15 are raised in a variety of disease states, including cancer, atherosclerotic cardiovascular disease, and obesity. However, the specific molecular mechanisms underlying this upregulation are less well known and the data that do exist are largely from in vitro studies using transformed cell lines. One of the first transcription factors identified as a regulator of GDF15 was p53. Overexpression or pharmacological induction of p53 in lung, osteosarcoma, and breast cancer cell lines was shown to markedly upregulate GDF15, findings that have been confirmed in cancer cell lines from other tissues . This tuAnother putative tumor suppressor protein that has been implicated in regulation of GDF15 is EGR1, a zinc-finger protein induced in response to growth factor signaling and DNA damage. A pharmacological agent that coordinately upregulated GDF15 and EGR1 in a colon cancer cell line also activated transcription at a region in the GDF15 promoter containing 2 experimentally validated EGR1 binding sites, suggesting that EGR1 is a direct transcriptional regulator of GDF15  13)..13).The integrated stress response (ISR) is an adaptive cellular response to a diverse array of cellular stressors. Cellular stress results in phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). This remodels cellular translation, suppressing global protein synthesis while simultaneously activating transcription of an adaptive gene program via the ISR-effector, activating transcription factor 4 (ATF4). The net effect is the repartitioning of nutrients and the biosynthetic machinery to specific pathways that adapt cells to stress. Recently, our group have shown that this pathway is a potent regulator of GDF15 in a variety of cell types . The celThe initial report describing GDF15 noted that it was induced by an array of proinflammatory stimuli . There iThe GDF15 promoter also contains a number of Kruppel-like factor (KLF) response elements. Recent work has demonstrated that the complement component C5a can upregulate GDF15 in a nonsmall cell lung cancer cell line via KLF5 . In the The transcription factors implicated above are all potent inducers of GDF15 in response to cell stress. However, what regulates basal GDF15 expression is unclear. Do the above stressors occurring at physiological levels as part of tissue and organ homeostasis contribute to circulating GDF15 levels observed in health or is there constitutive expression of GDF15 in unstressed cells that can be suppressed? The transcription factors SP1 and SP3 have been suggested to be regulators of basal GDF15 transcription . GDF15 pGDF15 mRNA and ChIP demonstrated enrichment of GATA4 at the GDF15 promoter.A range of other transcriptional regulators of GDF15 have been proposed based on the correlation of transcription factor expression with GDF15 and the presence of putative transcription factor binding motifs in the GDF15 promoter but without definitive experimental evidence. For example, single cell RNA sequencing has been used to identify transcription factors that correlate with GDF15 expression in single cells from heart tissue, and a cell-specific gene regulatory network of GDF15 has been proposed based on this data . This woGDF15 expression , a negative regulator of RNA III polymerase activity, binds to the GDF15 promoter where it suppresses basal pression . Traditipression . The autAn additional emerging negative regulator of GDF15 transcription is the transcription elongation factor SPT5. Canonically, SPT5 facilitates the transcription of stress and inflammation-related genes. However, this does not seem to be the case for GDF15, as SPT5 inhibition using a small molecule inhibitor potently induced GDF15 mRNA in vitro . The mecGDF15 mRNA by binding to AU-rich elements in its UTR. This process was partly dependent on CHOP-mediated suppression of peroxisome proliferator-activated receptor γ (PPARγ) expression. When CHOP was suppressed during ER stress using a ShRNA, PPARγ expression was enhanced and GDF15 induction was impaired. The authors found that PPARγ binds to PKCα and prevents its nuclear translocation, therefore preventing it from phosphorylating ELAV1  has also been suggested to regulate GDF15 via a post-transcriptional mechanism. Overexpression of RNPC1 in various cancer cell lines upregulated GDF15 mRNA and protein and prolonged the alf-life . MechaniCrif1 gene, which results in mitochondrial dysfunction , suggesting that the elevated GDF15 may be elaborated from macrophages infiltrating adipose tissue , and Gdf15 mRNA and a 2-fold increase in circulating GDF15, which was observed as soon as 1 hour after exposure to the lysine deficient diet and persisted for at least 4 hours   . More dr0 pg/ml) .It is important to note that not all studies have found an acute effect of exercise on GDF15. In 1 study investigating the effects of exercise on heart transplant recipients, neither high intensity interval training, nor sustained moderate intensity exercise, altered GDF15 levels . The reaRepeated exercise training may also influence GDF15 levels. Volunteers with obesity underwent an exercise intervention consisting of 1 hour of aerobic exercise a day, 5 days a week, for 12 weeks . Serum GThe elevation in GDF15 with intense physical activity is notable as a transient suppression in appetite can be observed following intense exercise in some settings , 92. WhiGDF15 expression in relation to exercise  arm of the ISR is activated in skeletal muscle in response to exercise in mice and may upregulate GDF15 in this context . Howeverexercise .Living at higher altitudes is inversely associated with the prevalence of obesity and exposure to moderate, high, or extreme altitudes is associated with a reduction in fat mass and fat-free mass . The ascCellular sensing of hypoxia occurs via accumulation and transactivation of the hypoxia-inducible factors hypoxia inducible factor-1 (HIF-1) and HIF-2. Hypoxia and CoCl2, which stabilizes HIF, can upregulate GDF15 expression in vitro . In addiGDF15 expression, primarily in the kidney  . In a prAgeing is associated with the development of a frailty syndrome defined as “a state of vulnerability to poor resolution of homoeostasis after a stressor event, as a consequence of cumulative decline in many physiological systems” . PhysicaA cellular correlate of organismal ageing is senescence, a programmed cellular response to various stressors resulting in exit from the cell cycle and the acquisition of a secretory phenotype characterized by the secretion of a diverse array of typically proinflammatory mediators and growth factors—the so-called senescent-associated secretory phenotype (SASP). A range of studies have identified GDF15 as upregulated in senescent cells, but a recent proteomics study has identified GDF15 as a “core” SASP protein, upregulated in senescence in 2 different cell types by all of the stimuli tested . Thus, lAbnormal levels of circulating GDF15 compared to control and changes of GDF15 within a cohort have been associated with alterations in childhood growth. In one cross-sectional study of patients with congenital heart disease, children with congenital heart disease and a failure to thrive had significantly higher levels of GDF15 compared to their normal weight controls . In a loGiven that GDF15 is potently regulated by cellular stress, it is not surprising that its circulating levels are markedly elevated in a number of disease states. Even modest elevations in GDF15 above the defined upper limit of normal (1200 pg/ml) have been associated with increases in all-cause mortality . As suchCirculating levels of GDF15 are raised in a range of human malignancies, including malignant glioma , pancreaA subset of patients with cancer develop an anorexia/cachexia syndrome characterized by involuntary weight loss . Cancer In esophageal and gastric cancer, prechemotherapy circulating GDF15 was found to be elevated in patients who had lost weight at the cessation of chemotherapy , 127. InClinical studies have demonstrated that GDF15 is elevated in patients with subclinical vascular dysfunction, atherosclerosis, and in those who subsequently develop the complications of atherosclerosis . WhetherGDF15 is upregulated in infarcted human myocardium relative to remote, noninjured myocardium and is elevated in the blood of individuals after myocardial infarction . In addiGDF15 is elevated in chronic heart failure, which correlates with severity and predicts its development . GDF15 iGdf15 is upregulated almost 20-fold at the mRNA level in the myocardium versus noninjured hearts, but plasma levels are not significantly different   and declGdf15 mRNA is increased in a mouse ischemia reperfusion model of acute kidney injury   . Thus, i = 0.01) .Like heart failure and cancer cachexia, it is likely that GDF15 is a pathogenic factor in the anorexia/cachexia syndrome observed in advanced CKD. The association of GDF15 with the renal cachexia syndrome has been studied previously and GDF15 was found to be inversely associated with BMI in ESRD . The effTK2) gene. GDF15 was upregulated 150-fold . TGdf15Csf1R), Breit and colleagues demonstrated that Gdf15Csf1R mice ate less food and had reduced weight compared to their wild-type controls , and the dorsal motor nucleus of vagus (DMX) relatively rapidly (60 minutes), consistent with a direct effect on these neurons. Ablation of the NTS and AP using microaspiration completely prevented the anorectic actions of GDF15 treatment .It should be noted that work from the Eling group suggests that mice with transgenic overexpression of GDF15 may have a reduced weight independent of food intake. Similar to the Breit group, Eling et al found that mice that overexpressed human GDF15 were protected from diet-induced obesity and dysglycemia . AdiposeIt is worth commenting that food intake in this study was normalized to body weight, and the validity of this approach has been challenged . MoreoveApcmin/+ model of intestinal neoplasia, where mice develop spontaneous intestinal adenomas, mice harboring a copy of the GDF15 transgene exhibited a reduction in the number of adenomas (Gdf15Csf1R mice described above) that exhibit a reported elevation of circulating GDF15 between 10 and 90-fold, reduced local tumor burden as expected, although the rate of metastasis was unexpectedly increased  and 2 slightly more distant homologs—GFRAL and growth arrest specific-1 (GAS1). The GFRAs are glycosylphosphatidylinositol (GPI)-linked proteins with a variable number of common GDF-receptor (GFR) domains, which classically have 10 conserved cysteines, conserved cysteine–cysteine pairing, and 5 α-helices that form a rigid core between the first and last cysteines . In contGFRAL gene, which in humans is found on the short arm of chromosome 6. It consists of 9 exons and encodes a 394 amino acid protein  and 1 cysteine-rich domain (CRD) and GFRAL of 3 GFR-like “D” domains . GDF15 bo and 125o. The dimer formation brings 2 RET monomers in close association and triggers mutual phosphorylation of their intracellular domains , a finding that has been validated in vivo using GDF15 treatment of mice and coimmunostaining of GFRAL+ neurons with pERK , 37, 38.Gfral in the AP and NTS and demonstrate that this expression distribution is conserved in rodents, monkeys, and humans  could only find evidence of GFRAL expression in the AP and NTS, where it was coexpressed with RET. These findings were confirmed by laser capture dissection and subsequent Nanostring mRNA analysis and immunohistochemistry . qPCR and humans , 36–38, se brain . No evidt groups , 36, 37.hemistry . An addiGfral-/- mice or in animals pretreated with a GFRAL-blocking antibody  targeting Gfral into the AP and NTS was injected to achieve post-natal knockdown of Gfral in mice that were then subjected to HFD-feeding. On a HFD, Gfral-knockdown mice had increased bodyweight and adiposity compared to animals treated with a control ShRNA.The Breit group hypothesized that compensatory changes occurring during the early development of proaches . FirstlyConsidering the totality of the evidence, it seems unlikely that GDF15 is a physiological regulator of appetite in the normal state. It is not acutely regulated by over or underfeeding in humans and loss of GFRAL does not have any effect on food intake or adiposity in chow-fed mice , 36–38. Whether or not GDF15 provides a regulatory brake on weight gain in chronic overfeeding, it seems very unlikely that this is its primary evolutionary purpose. GFRAL expression is restricted to the hind brain in rodents, monkeys, and humans, and it is highly expressed in the AP, a sensory circumventricular organ with a highly permeable blood brain barrier, meaning it is exquisitely positioned to integrate endocrine signals from the peripheral blood and regulate behavior. Coupled with the fact that GDF15 is potently regulated by cellular stress, it seems likely that the primary role of GDF15 is to act as an endocrine message conveying somatic distress to the brain . What reHsu and colleagues  found thGfral-/-animals  with an annotated pQTL (rs45543339), determined using aptamer based technology, with the minor variant apparently associating with elevated plasma GDF15  denotes a histidine to aspartic acid substitution at the sixth amino acid of mature GDF15 , Au Yeung et al were unable to provide any evidence that GDF15 was related to the development of type 2 diabetes, HbA1c, blood pressure, or BMI . The genThe above studies suffer from some limitations but most notably the differences in GDF15 are modest compared to the alterations seen in human disease, and there is uncertainty regarding the validity of GDF15 measurements by current methods in the presence of the common rs1058587 variant.An alternative approach to uncovering the functional importance of human GDF15 in human health and disease is to identify coding variants that may alter GDF15 structure and examine their association with human phenotypes. rs1058587 (H202D) has been discussed above. Three further common (MAF > 1%) polymorphisms exist: V9L rs1059519 27%, S48T rs1059369 27%, and S164SS  rs199673307 2.2%. Our molecular modeling does not indicate that the amino acid replacements introduce significant functional changes in the three-dimensional structure. V9L is a very conservative replacement in the segment preceding the transmembrane helix in the signal peptide, and S48 lies in a mobile loop on the surface. Moreover, none of them are conserved in evolution ).The first indication that GDF15 may play a specific role in pregnancy came from the findings of high levels of expression in the placentas of PTGF-β  and PLABGDF15 is expressed at high levels in human syncytiotrophoblast and maternal GDF15 levels rise markedly in the first trimester of pregnancy and continue to rise, albeit more slowly, through the rest of pregnancy . This faNausea and vomiting in pregnancy, colloquially  placental GDF15 is the key determinant of plasma GDF15 in pregnancy and the effect size observed in this study is diluted by pregnancies in which the fetus does not inherit the risk allele from the mother, and (2) extra-placental GDF15 makes a meaningful contribution to plasma GDF15 in pregnancy and the risk alleles modulate GDF15 risk even when the genotype of the mother and her pregnancy are discordant with respect to the causative SNPs. Further work delineating the risk of HG by maternal and fetal genotype and the effects of these SNPs on circulating GDF15 in pregnancy will be necessary to resolve these issues. It should be reiterated that the rs1058587 variant which is protective for HG  is assocIf, as we believe, GDF15 acts solely through GFRAL in the hindbrain, why are circulating levels so high in pregnant women from the very early stages of pregnancy? The robust association of SNPs close to GDF15 with severe NVP, a phenomenon which is present to a milder degree in the majority of pregnant women, and the almost universal reports of changes in food preferences in pregnancy provides support for the idea that high levels of maternal GDF15 may influence maternal appetite, food preference, and food intake. Why would this have been selected for during our evolution? One hypothesis might be as follows. The human fetus is highly susceptible to teratogens, especially in the first trimester. During most of human evolution, as hunter–gatherers, daily life involved the ingestion of calories from a wide variety of sources, many of which contained chemicals potentially damaging to fetal development. Might high GDF15 levels sensitize the CNS and encourage avoidance of certain foodstuffs? For example, pregnant women frequently report an intolerance of tea and coffee, both of which contain multiple alkaloids , 240. SeAside from NVP and HG, altered food perception and eating behavior is relatively common in pregnancy. Pica, voluntary consumption of non-nutritive substances, occurs in almost 30% of pregnancies, according to one meta-analysis . Why andIt is also possible that GDF15 has other central effects through, for example, the autonomic or neuroendocrine axes, that might facilitate maternal adaptation to the pregnant state, but this has not been explored.Antagonism of the GDF15 hormonal axis has been achieved in preclinical settings with blocking antibodies to both GDF15 and GFRAL. The potential therapeutic utility of GDF15-GRFAL antagonism has been demonstrated most clearly in the setting of cancer cachexia, where a blocking antibody to GDF15 prevented the cancer cachexia associated with xenograft tumors in mice . The assMice lacking GFRAL are relatively resistant to the suppressive effects of cisplatin on food intake , with obGFRAL in a normal human fetus appears extremely low, bordering on undetectable , so the risk of mechanism-based toxicity to the fetus, even if some antibody did cross the placenta, would be very low.As discussed above, both genetic and biochemical evidence support the notion that GDF15 is causally related to severe NVP and HG. Could a therapy based on antagonism of the GDF15–GFRAL axis be both effective and safe in the treatment of these conditions? The considerable overlap in GDF15 concentrations between asymptomatic women and those with severe NVP suggest that factors other than the high GDF15 levels themselves must be involved. Indeed, the seminal genetic study in the area did highlight several other genes . HoweverThe therapeutic potential of GFRAL agonism has been demonstrated in preclinical studies in mice, rats, and monkeys and modified; long acting molecules have been developed with impressive preclinical effects , 84. GDFGdf15 mRNA at these sites but not in adipose tissue, liver, or skeletal muscle. Importantly, these findings have been independently replicated and published almost simultaneously by the Steinberg group. Using Gdf15-/- mice they showed that endogenous GDF15 was required for the weight-lowering actions of metformin in diet-induced obesity; however, they did not explore the source of GDF15 in vivo , or the specific method used to ablate GDF15-GFRAL-RET signaling. Interestingly, the elevation of circulating GDF15 observed with metformin treatment in both studies was much more modest in chow-fed than HFD-fed mice, suggesting that obesity somehow potentiates GDF15 induction in response to metformin. This study also demonstrated a small effect of metformin on food intake in chow-fed mice in both In addition to the obvious mechanistic interest of these studies, they are of immediate translational relevance, as the fact that metformin chronically elevates GDF15 is reassuring for the development of GDF15-GFRAL agonists as antiobesity agents given the safety profile of metformin over 60 years of widespread use in man.There is an unhelpful tendency in medical science to eulogize or demonize biological pathways. Such an oversimplistic view seems particularly inappropriate for GDF15, where context is all important.On the one hand, it is highly likely that elevated levels of GDF15 actively participate in driving nausea, emesis, and cachexia syndromes in the context of cytotoxic chemotherapy, cancer, and other severe systemic diseases.However, it is unlikely that GDF15 evolved to make us ill. It surely must have served some adaptive functions during our evolutionary history. Its response to ingested toxins and its perception as aversive may well have alerted us to potentially harmful chemicals in plants or animals encountered while we were hunter–gatherers. Emerging evidence of actions in the context of acute inflammation suggest that it may have adaptive functions that remain relevant today.To complicate matters further, our increasingly obesogenic environment may yet restyle this aversive, potentially emetogenic substance as a therapy for overnutrition-related diseasesMany puzzles remain to be solved. Are the elevated GDF15 concentrations found in conditions such as ageing and frailty, with chronic renal and cardiovascular diseases playing an active role in the pathogenesis of these disorders? If not, are they part of a helpful adaptive response to disease or are they simply a biomarker?Disentangling GDF15 as a driver of disease to be inhibited or as a protective reactive change to be potentiated will continue to be difficult, but detailed mechanistic understanding afforded by the discovery of GFRAL will be invaluable while the therapeutic potential of targeting the GDF15-GFRAL-RET axis for the treatment of human disease is explored.GDF15 is a hormone that signals states of somatic distress to the brain via the receptor GFRAL-RET, which is highly anatomically restricted. It has clear anorectic actions and emerging data suggests that it may have aversive properties. We have proposed that a major adaptive consequence of stress-induced regulation of GDF15 is a learned aversion to toxic environmental stimuli and have extended this to the setting of pregnancy whereby placental GDF15 may facilitate hypervigilance against harmful environmental agents. The discovery of GFRAL as the GDF15 receptor has invigorated interest in this axis as a potential therapeutic target. The most obvious route to clinical utility is in the use of GDF15 antagonists in chemotherapy-induced nausea and vomiting and in various forms of cachexia associated with high circulating GDF15. Some support for the notion that chronic elevation of GDF15 action might be safe and effective in obesity is provided by studies demonstrating the chronic elevation of GDF15 by metformin and the importance of this action for metformin’s beneficial effects on weight.Our understanding of the physiological function of the GDF15-GFRAL-RET axis and its role in human disease remains in its infancy, and a number of key questions remain unanswered. While existing evidence has clearly defined a role for GDF15 as an anorectic peptide, it is likely that further physiological actions of GFRAL-RET signaling exist and remain to be defined. Identifying these actions and determining if they can account for some of the phenotypes previously attributed to a peripheral action of GDF15 will provide further mechanistic insight into the role of the GDF15-GFRAL-RET axis in human disease. Exploring the genetic epidemiology of circulating GDF15 may be a useful strategy to further understand the effects of GDF15 in humans, but the utility of this approach is likely to be limited until we can fully understand how the common H202D variant affects circulating GDF15 independent of antigenicity. The fact that metformin exerts its regulatory effect on weight via GDF15-GFRAL-RET provides proof of principle for GFRAL agonism as a therapeutic strategy in obesity. However, the efficacy and tolerability of more potent agonism aiming to achieve weight loss comparable to existing obesity therapies remains to be defined.


Music and language have long been considered two distinct cognitive faculties governed by domain-specific cognitive and neural mechanisms. Recent work into the domain-specificity of pitch processing in both domains appears to suggest pitch processing to be governed by shared neural mechanisms. The current study aimed to explore the domain-specificity of pitch processing by simultaneously presenting pitch contours in speech and music to speakers of a tonal language, and measuring behavioral response and event-related potentials (ERPs). Native speakers of Mandarin were exposed to concurrent pitch contours in melody and speech. Contours in melody emulated those in speech were either congruent or incongruent with the pitch contour of the lexical tone . Component magnitudes of the N2b and N400 were used as indices of lexical processing. We found that the N2b was modulated by melodic pitch; incongruent item evoked significantly stronger amplitude. There was a trend of N400 to be modulated in the same way. Interestingly, these effects were present only on rising tones. Amplitude and time-course of the N2b and N400 may suggest an interference of melodic pitch contours with both early and late stages of phonological and semantic processing. On incongruent items, the direction of pitch in speech diverged from pitch in melody .Speech stimuli were superimposed on the two last quarter notes positions of a melody  and merged in a single mono signal. Prior to this, both speech and melodic items were normalized in terms of loudness. The materials comprised of congruent and incongruent test items. On congruent items, the direction of the lexical tone matched the direction of the last melodic contour  and 72 were incongruent . In order to prime lexical processing, each trial was preceded by a visual cue on screen: a Mandarin character that corresponded to the word that followed. R and IR). Example audio stimuli can be found in The complete set of materials comprised a total of 144 items: 72 were congruent  would appear on screen and participants had to answer (yes/no) whether this character corresponded to the word they heard by pressing a key on a keyboard.The onset of each experimental trial was prompted with an asterisk on screen for 500 ms. A Mandarin character (the prime) then appeared on screen for 500 ms. The screen then went blank and an experimental item was presented through speakers . A blank screen for 1,000 ms then preceded a timed two-alternative forced choice lexical verification question for 2,000 ms—this time constraint was implemented to ensure rapid processing and control for attention as much as possible. The end of a trial was marked by a blank screen for 1,000 ms. Stimuli were pseudo-randomized with the sole restriction that an item could not be presented twice in a row. Stimuli were presented in 9 blocks of 48 items  at approximately 70 dB. Each experimental item was thus presented three times. Participants performed a practice session prior to the start of the experiment (with 12 items not used in the experiment) and were given feedback when appropriate. Behavioral response (percentages correct) and ERPs were recorded. The entire experiment lasted approximately 120 minutes (including self-paced breaks between blocks).The EEG was recorded continuously from 64 scalp locations using Ag–AgCl electrodes . Eye blinks and other ocular artifacts were recorded with a bipolar montage attached to the exterior canthi and the infraorbital and supraorbital regions of the right eye. Two additional electrodes were placed at the left and right mastoids. A Common Mode Sense (CMS) and Driven Right Leg (DRL) electrode were used as reference. The signal was amplified using a Biosemi ActiveTwo AD-box  with a band-pass of 0.1–100 Hz. Electrode impedance threshold was kept below 5 kΩ. The signal was digitized at a sample rate of 8 kHz with a 16-bit resolution.R, CF, IR, IF) were time locked to the last syllable  and segmented into separate epochs from -150 ms pre-stimulus onset to 1,000 post-stimulus (baseline corrected relative to stimulus onset). Epochs that contained amplitude variation exceeding 150 μV in a 500 ms sliding window (step size 250 ms) were rejected. The 5.61% of the trials were rejected on average. One participant with a rejection rate higher than 25% was excluded from analyses ) nor other interactions. Post hoc pairwise comparison indicated the difference between congruent and incongruent conditions to be significant at anterior  and central  sites. The analysis thus indicated significantly greater negative deflections at anterior and central sites for incongruent condition than for congruent condition.In the N2b latency, there were main effects of Congruency [F = 5.86, p = .029, F = 7.28, p = .003, F = 3.33, p = .049, F = 2.66, p = .041, R evoked overall stronger negative deflections than CR.In the N400 latency, there was a tendency of all three main effects to be significant  = 10.27, p < .001, The P3 analysis indicated strong main effect of Lateraity [In the N400 window, none of the main effects were significant.An extensive body of research has shown that experience with pitch in one domain can influence pitch perception in another. While the literature on transfer of pitch abilities between language and music is abundant, there are few studies that have explored simultaneous processing of pitch in both domains. The current study explored parallel processing of congruent and incongruent pitch contours in language and music. Native speakers of Mandarin were exposed to congruent and incongruent pitch contours in melody and speech. Results showed that processing two concurrent pitch contours did not affect lexical verification at a behavioral level. However, at a neurophysiological level, significant N2b response and a marginally significant N400 response were observed when incongruent pitch contours were presented. Although it was rathe unexpected that this effect only occurred in the rising contour conditions (see the discussion below), we propose that this can be taken as an indication that melodic pitch processing interacting with early and late stages of lexical processing: incongruent pitch contours would interfere with lexical processing and would elicit greater negative deflections for the N2b and N400.It is interesting that speech with only falling lexical tones evoked a positive component (P3b). Importantly, the amplitude of this component was not modulated by our Congruency manipulation, therefore, we think that this is caused by a more general feature of falling stimuli. In spoken Mandarin, a clear durational contrast exists between words with rising and falling tones. Previous behavioral studies have been able to demonstrate differences in the perception of both tones in language and music for speakers of Mandarin as a result of top-down interference from language experience –71. In tWhile we believe that our study opens up interesting future directions, because of the exploratory nature, our findings should be interpreted with care. One may argue that the domain-specific mechanism would still predict enhanced component amplitude because both domains would simultaneously activate similar neural mechanisms for semantic information processing. Here, we argue that, in our experiment, it is likely that top-down effects from language will prioritize the processing of lexical pitch over pitch in melody because, while our participants were primed to expect lexical tones to go up or down, such expectation was absent for concurrent musical pitch .Another important issue is the fact that the N2b and N400 are sensitive to other processes than the pure linguistic processing. For example, the level of attention required to perform a task is known to modulate the amplitude of both components . In thisFuture studies could also include a task focusing on pitch processing in both speech and melody. To further explore the interaction between both domains, it would be interesting to investigate the role of speech-specific modulation from language on tonal perception by exposing non-tonal speakers to concurrent pitch contours. As non-tonal speakers will have no knowledge of Mandarin semantics, bottom-up acoustic signal processing can be directly compared to the top-down listening strategies of the Mandarin speakers. By differentiating between bottom-up and top-down processing of pitch contours in both domains, we can significantly broaden our understanding of the domain-specificity of pitch cognition and pitch perception in language and music.To sum up, we used an explorative approach to address cross-domain interaction effects between melodic and lexical pitch processing mechanisms in speakers of Mandarin. Congruency between concurrent melodic and lexical pitch contours seemed to affect phonological and semantic processing at the neurophysiological but not the cognitive level. Since no observable behavioral effect of melodic pitch on lexical verification was found, interaction between domains might be restricted to lower level acoustic processing. This asymmetry between cognition and neurophysiology has been reported in other studies concerning pitch processing in speech and music see ,22. Our S1 FigRegions are divided from top to bottom by Caudality  and from left to right by Laterality .(PNG)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.S1 AudioA: Both speech and melodic contours rising (CR). B: IR Rising speech contour presented with falling melodic contour.(WAV)Click here for additional data file.


In 2015, new FIGO guidelines for CTG interpretation were presented (FIGO-15). In 2017, the previous Swedish guidelines (SWE-09) were replaced with guidelines adapted to FIGOs (SWE-17). The performance of these three templates had not been scientifically evaluated before its clinical implementation. The objective of this study was to compare the sensitivity and specificity to detect fetal acidosis at birth using these three templates during the second stage of labor.This case-control study included 295 neonates with cord blood pH < 7.05 and 591 controls with pH ≥ 7.15, born 2012−2017. Tracings from the last 30−80 min of labor were classified independently by three assessors , blinded to group and outcome.The classification pathological using FIGO-15 had a sensitivity of 50 % and specificity of 88 % in detecting fetuses with acidosis. For SWE-17, the sensitivity was 62 % and the specificity 85 %. For SWE-09 the sensitivity was 87 % and the specificity 56 %.By combining suspicious and pathological patterns the sensitivity for FIGO-15 increased to 97 %, and for SWE-17 to 83 %, whereas the specificity decreased to 23 % and 68 % respectively.The FIGO classification seemed to be insufficiently discriminative in the second stage of labor; most patterns in acidotic cases were classified as merely suspicious with this template, and the sensitivity of pathological patterns was low at 50 %. Combined pathological and suspicious patterns detected fetal acidosis at a specificity that was too low to be useful (23 %). SWE-09 showed the best ability to detect acidosis with pathological patterns (sensitivity 87 %). SWE-17 reached almost the same sensitivity (83 %) with the combination of suspicious and pathological patterns, and at a higher specificity (68 %). Randomised studies from 1976 to 1993 comparing CTG with intermittent auscultation indicated that CTG monitoring lowered the incidence of neonatal seizures  but onlyIn 1987 FIGO presented the first international guidelines of CTG , that waIn 2010 Ayres-De-Campos and Bernardes concluded that the FIGO guidelines from 1987 had limitations and called for a simpler and more objective guideline . Not havCTG patterns are often markedly different in the first and second stage of labor. The second stage is the period of highest risk of hypoxia, and the fetus is affected by the higher intrauterine pressure ,12.This study was undertaken to evaluate the sensitivity and specificity for the three templates in detecting hypoxia during the second stage of labor, as indicated by acidosis at birth after vaginal delivery or after cesarean section in the second stage of labor.The primary objective of this study was to compare the sensitivity and specificity of FIGO-15, SWE-09 and SWE-17, in identifying cases with acidosis at birth in the second stage of labor.2This is a retrospective case control study including neonates with acidemia (case group) defined as cord artery or cord vein pH < 7.05, and controls defined as neonates with a cord artery and cord vein pH ≥ 7.15 and Apgar scores of 9 or 10 at five and ten minutes, all with CTG monitoring during the second stage of labor.A power calculation estimated that 215 cases were needed to detect a difference in sensitivity between 80 % and 90 %, with 80 % power and a p-value of 0.05. To detect a difference in specificity between 60 % and 70 %, 386 controls were needed.d2013 – October 31st 2017, and Helsingborg Hospital March 13th2012 – December 31st 2016. Inclusion criteria for both groups were singleton pregnancy and an available CTG tracing of at least 30 min, ending at vaginal birth or within 30 min of second stage cesarean delivery. The case group consisted of newborns with cord artery or cord vein pH < 7.05 after vaginal birth or cesarean delivery in the second stage of labor. As controls, the first two neonates born consecutively after each case at the same hospital fulfilling the inclusion criteria above, except for acidosis, and who had both cord artery and cord vein pH ≥ 7.15 and at least 0.02 apart and Apgar scores 9 or 10 at five and ten minutes, were included.Cases and controls were collected from births at Skåne University Hospital in Malmö and Lund April 23In the international guidelines , pretermClinical data was gathered from patient files and computerized CTG tracings were evaluated. The last 30 min, and when available up to 80 min, of the tracings before birth were assessed. The tracings were anonymized and randomized.The interpretations of the CTG tracings were performed independently by three professionals, representing trained obstetric staff  with different levels in experience of electronic fetal monitoring from their daily work. All had performed educational programs including both the previous and the current classification templates. Each of the 886 traces were assessed by 3 of totally 21 interpreters. Each interpreter received a portfolio with tracings, information about the study and classification forms. The only additional clinical information provided was that it was a singleton pregnancy ending in vaginal birth or cesarean delivery in the second stage of labor. The graphic of the tracings was 1 cm/min.Each interpreter completed a form including the assessment of all the variables relevant for classification. This was done twice; in 2017 with a protocol for SWE-09 when it was in clinical use, and in 2018 with a protocol for FIGO-15 and SWE-17 when the interpreters had been re-educated and used the SWE-17 in clinical practice. The description of all the included variables by each interpreter were then used to classify strictly according to each template to classification normal, suspicious or pathological.The final classification was composed of the three professionals’ assessments of the variables in each trace, and the classification for each interpreter for each template, representing the majority assessment of obstetric staff with different experience. If at least two out of three assessments agreed, that was the final classification. If all three classifications differed, a fourth assessor, an experienced obstetrician, also classified the trace, so that a final judgement was attained for all traces.2.1The main outcome was the sensitivity for the classification pathological to identify cases with acidosis at birth, and the specificity for the classification normal or suspicious together to rule out acidosis. The classification preterminal using SWE-09 was regarded as pathological in the final analyses. The interpretation suspicious mandates continued surveillance combined with additional active management to correct reversible causes and to evaluate the fetal condition in all three templates. Therefore, we also evaluated the sensitivity of suspicious and pathological patterns combined in identifying acidosis, and the specificity for the classification normal to rule out acidosis.2.2www.sample-size.net/confidence-interval-proportion provided by UCSF. The chi-square test was used to determine if there was a significant difference in sensitivity or specificity between classification systems, and a p-value < 0.05 was considered statistically significant.The information was gathered in Stat View® computer software. The sensitivity and specificity with 95 % CI for the final classification was calculated for the three classification systems, using 2.3Ethical approval was obtained from the Regional Ethical Review Board in Lund, Dnr 2016/371, 2016-05-24.3During the study period 57,582 neonates were born at the two hospitals. A total of 296 cases and 592 controls fulfilling the inclusion criteria were included. One case and one control were excluded due to birth before 34 gestational weeks, leaving 295 cases and 591 controls in the study . BackgroRates of agreement between the classifications determined by the assessment of the included variables of the three interpreters are shown for cases and controls in The result of the final classifications is summarized in When combining suspicious and pathological patterns the sensitivity for SWE-17 increased to 83.4 %, which was not significantly lower than the sensitivity for pathological patterns with SWE-09 (p = 0.26), whereas the specificity at 67.7 % was significantly higher than for pathological patterns with SWE-09 (p < 0.001). For the FIGO-15, combing suspicious and pathological patterns also lead to a high sensitivity (96.6 %), but the specificity declined to a very low level (22.5 %).44.1In this study we found that during the second stage of labor the FIGO-15 template had the lowest sensitivity, 50.2 %, to detect fetal acidosis when the cut-off was pathological patterns. When the cut-off was suspicious patterns, it had the lowest specificity, 22.5 %. The template lead to a high rate of patterns classified as suspicious in cases (46.4 %) as well as in controls (65.0 %).The SWE-09 template had the highest sensitivity to detect acidotic fetuses (87.1 %), whereas the specificity was low (55.5 %). Combining pathological and suspicious increased the sensitivity to 97.6 % with a concomitant decrease of specificity to 28.3 %.The SWE-17, modelled on the FIGO-15, had a sensitivity of 62.0 % for pathological patterns. When the cut-off was set at suspicious patterns the sensitivity was 83.4 %. This is similar as for pathological patterns with SWE-09, but the specificity was higher, 67.7 %.We consider that the safety of SWE-17 and SWE-09 was similar, if also suspicious patterns are acted upon with SWE-17. Acting does not always have to be to deliver, but may include diagnostic measures  as well as other therapeutic measures . The FIGO-15 classification results in too many suspicious tracings to be discriminative. The sensitivity of the classification pathological for FIGO-15 was low, and the specificity with a cut-off at suspicious was also low. This finding raises doubts concerning the validity in clinical practice.It is not clear to us why the interpretation using the SWE-17 template resulted in a higher rate of normal classification in acidotic fetuses (16.6 %) compared to both SWE-09 (2.4 %) and FIGO-15 (3.4 %). The main difference between the SWE-17 compared to the SWE-09 ad FIGO-15 is the definition of decelerations. This merits further analysis.4.2A few previous studies have compared different CTG interpretation templates ,14 and mBhatia et al. compared FIGO-15 with the NICE guidelines from 2007 and 2014 . They foOlofsson et al. compared the STAN classification system from 2007 (similar to SWE-09) with FIGO-15. They found that the two systems classified tracings differently  and that4.3The case group was defined as neonates born with a pH < 7.05. Jonsson et al. reported pH < 7.05 at birth as a useful variable for quality control of management of the second-stage of labor . A cord Each trace was interpreted by at least three different individuals, and the final classification was that of the majority (or two out of four). This might result in a higher sensitivity and specificity than if only one person assesses a trace. We chose the design with triple interpretation of each trace by clinicians with different experience both to reflect clinical practice and to achieve more accurate interpretations of the fetal heart rate parameters than from assessments of a single individual.The interpreters in the study were blinded to outcome, which is necessary to avoid ascertainment bias ,26. In tTo eliminate bias caused by different experience of the different classification systems, the classification of the different fetal heart rate parameters  were systematically transformed to classifications normal, suspicious or pathological according to each classification form by one of the authors.4.4The results of the study indicate that we are in dire need for improvement of interpretation protocols to ensure safe labor care. The purpose of intrapartum fetal monitoring is to detect fetal hypoxia in time to prevent asphyxia. With a low sensitivity of monitoring, fetal asphyxia may not be possible to avoid, whereas a low specificity may lead to unnecessary interventions. We consider a high sensitivity of CTG to be more important than a high specificity, since identification of fetuses at risk is indispensable if asphyxia should be avoidable, and since secondary diagnostic methods can be used to improve specificity.Further studies are needed to analyze how differences between the different parameters in the templates affect sensitivity and specificity. Improvements of SWE-09 to increase specificity or of SWE-17 to increase sensitivity might be possible, but before introducing new templates for fetal monitoring in clinical practice, studies of the validity of such templates should be performed.5We consider the FIGO-15 classification to be too restrictive in classifying tracings as pathological, and too liberal in the classification of merely suspicious tracings, to properly be able to discriminate between fetuses with and without acidosis. The sensitivity of the classification “pathological” to detect acidosis was low and insufficient for safe surveillance. The classification “suspicious” however, implied fetal acidosis with too low specificity to be clinically useful and to require clinical action in all cases of a suspicious trace. SWE-17 provided the best combination of sensitivity (83.4 %) and specificity (67.7 %), if the cut-off for indicating fetal acidosis was set at a suspicious pattern, whereas SWE-09 provided the best sensitivity (87.1 %) for pathological patterns and had the lowest false negative rate of tracings classified as normal in cases with acidosis (2.4 %).Region Skåne and funding from LÖF, the national Swedish patient insurance company. The funders played no role in planning or conducting the research or writing of the paper.This work was supported by research grants from The authors declare that they have no competing financial interest that have influenced the work reported in this paper. Andreas Herbst has contributed in the development of the Swedish classification systems from 2009 and 2017.


Training is a common and cost-effective way of trying to improve quality of care in low- and middle-income countries but studies of contextual factors for the successful translation of increased knowledge into clinical change are lacking, especially in primary care.The purpose of this study was to assess the impact of contextual factors on the effect of training rural healthcare workers in Kyrgyzstan and Vietnam on their knowledge and clinical performance in managing pediatric patients with respiratory symptoms.Primary care health workers in Kyrgyzstan and Vietnam underwent a one-day training session on asthma in children under five. The effect of training was measured on knowledge and clinical performance using a validated questionnaire, and by direct clinical observations.d (CI: 0.7 to 1.4) in Kyrgyzstan where baseline performance was lower and 1.5 Cohen’s d (CI: 0.5 to 2.5) in Vietnam. Consultations were performed by different types of health care workers in Kyrgyzstan and there was a 79.1% (CI 73.9 to 84.3%) increase in consultations where at least one core symptom of respiratory illness was asked. Only medical doctors participated in Vietnam, where the increase was 25.0% (CI 15.1 to 34.9%). Clinical examination improved significantly after training in Kyrgyzstan. In Vietnam, the number of actions performed generally declined. The most pronounced difference in contextual factors was consultation time, which was median 15 min in Kyrgyzstan and 2 min in Vietnam.Eighty-one healthcare workers participated in the training. Their knowledge increased by 1.1 Cohen’s The effects on knowledge of training primary care health workers in lower middle-income countries in diagnosis and management of asthma in children under five only translated into changes in clinical performance where consultation time allowed for changes to clinical practice, emphasizing the importance of considering contextual factors in order to succeed in behavioral change after training. Training is one of the most common and cost-effective methods of trying to improve quality of care in low- and middle-income countries but the effect of training does not always translate into clinical changes. Contextual factors that affect the translation of increased knowledge into clinical change have not been studied much and most available studies are not from the primary sector. Not much is known about which contextual factors impact the effect of training in primary care, where the most patients are seen globally, often by the least trained healthcare workers.As expected, the training increased knowledge in managing the disease in question (asthma in children under five) but only translated into clinical change in one of two settings where the most pronounced contextual difference was the time available for consultation (15 min vs. 2 min).Low quality of care in both the primary and secondary sector is a serious challenge in many low- and middle-income countries (LMICs) , 2. TherIt is generally unclear why training often does not translate into clinical changes in LMICs, which has prompted Rowe et al. to suggest studying contextual factors such as WHO regions, rural versus urban settings, types of health care workers being studied, and differences in baseline performance level for failure or success of educational interventions .We therefore used an opportunity that presented itself during a project we were involved in to explore if differences in context would affect clinical change after the same training program was conducted in two countries (Kyrgyzstan and Vietnam) that differ on all the contextual factors suggested by Rowe et al. .We studied how children with respiratory symptoms were managed in primary care in four low-resource settings during the FRESH AIR study , 5 and wThe purpose of this study was to assess if conducting the same training program in two contextually different countries would lead to differences in the translation of increased knowledge to clinical change.We developed a needs-based training program using interactive lectures and case discussions. The training consisted of an eight-hour program covering asthma in children under five targeted at primary care health care workers in LMICs. The curriculum contained major causes and clinical features of respiratory illness; asthma epidemiology; clinical features, diagnosis, and treatment of asthma; inhaler techniques; patient education; and case studies. The session was slightly adapted in each country in collaboration with the local research team and the content was guided to fit the local context based on previous qualitative studies  and the The pedagogical structure and learning methods of the training used plenaries, case discussions, practical assignments, and a focus on the importance of good communication with the family/caregivers. A participatory learning style was reinforced by having the participants complete a set of assignments and we engaged participants using cases with the aim of increasing self-confidence in management of asthma, and thus motivation, to implement the changes proposed by the program in daily practice.We collected quantitative observational data and questionnaires before and after the training to assess the effect of the training. The pre-training data were collected as part of a comprehensive baseline study that was reported in a separate paper  as well The Newcastle Asthma Knowledge Questionnaire (NAKQ)  was usedWe observed the healthcare workers who had participated in the training program while doing clinical consultations with children aged 2 to 59 months with cough and/or difficult breathing. We recorded history taking, clinical examination, diagnosis, and treatment to assess changes in clinical behavior 8 weeks after the training based on the methodology presented in Kjærgaard et al.  in Kyrgyzstan and 100% [11/11] in Vietnam), clinical officers (31.8% [7/22] in Kyrgyzstan), and nurses (40.9% [9/22] in Kyrgyzstan). Most of the participants were female (87.9%).. Just after the training session, 80 healthcare workers answered the questionnaire . At 8-week follow-up, only the 33 healthcare workers we originally invited were asked to answer and were observed during clinical practice. Thirty-three (100%) answered the questionnaire . We did follow-up observation of 20/22 (90.9%) healthcare workers in Kyrgyzstan and 5/11 (45.5%) healthcare workers in Vietnam, where follow-up was only possible in one of the two original districts d effect size of 1.1 (0.7 to 1.4). The participants had a higher pre-training baseline performance on the NAKQ score in Vietnam and the mean increase after training was 3.3 (CI: 1.4 to 5.2), corresponding to a Cohen’s d of 1.5 (CI: 0.5 to 2.5). NAKQ score at 8-week follow-up remained higher than the pre-training score in both countries (Table NAKQ scores showed increased knowledge in both countries. The mean NAKQ score increased by 3.7 points CI: 2.6 to 4.9) in Kyrgyzstan, corresponding to a Cohen’s .6 to 4.9 were diagnosed with asthma and 7.1 and 2.3%  were diagnosed with bronchiolitis. Although antibiotics were frequently prescribed (Table Table .Table 3Core respiratory symptoms and history: There was a 79.1% (CI 73.9 to 84.3%) increase in consultations where at least one core symptom of respiratory illness was asked in Kyrgyzstan (from 20.9 to 100%) and in Vietnam, the increase was 25.0% (CI 15.1 to 34.9%), from 20.1 to 45.1%. A family history of asthma or allergy was obtained in all consultations in Kyrgyzstan after the training, an increase of 56.6% (CI 49.3 to 61.9%). In Vietnam, family history of asthma or allergy was obtained in 5.3% of consultations, an increase of 3.6% (CI 0.5 to 7.7%).Generally, clinical examination improved significantly in Kyrgyzstan. There was an increase of 62.2% (CI 47.1 to 77.3%) in the number of children who had their respiratory rate counted. The other observed actions of clinical examination all increased to 100% after the training. In Vietnam, the number of clinical actions generally declined. There was a 25.1% (CI 19.6 to 30.6%) decrease in the number of children who had their respiratory rate counted, and the other actions remained low or decreased after the training. The changes in clinical behavior seemed evenly distributed across different health care worker types but there was a tendency towards higher increases in the rural setting in Kyrgyzstan, where baseline performance was lower than in the urban setting (data not shown). In Vietnam, only medical doctors participated, and we only had follow-up in urban areas.(Table 5).There were no significant changes in referrals in Kyrgyzstan  or Vietnam  A summary of effects of the intervention on knowledge and clinical behavior as well as contextual differences can be found in Table Self-reported changes to clinical practice, data on potential harm, and feasibility of performing the training session can be found in Additional file We studied the effect of training rural primary level healthcare workers in diagnosis and management of asthma in children under five on knowledge and clinical practice in two LMICs (Kyrgyzstan and Vietnam) where contextual factors differed widely. The training increased knowledge as expected but this only translated into behavioral change in Kyrgyzstan where the most prominent difference was duration of clinical consultations - but also the type of health worker, baseline performance level, and WHO region differed.It was striking that the biggest change in clinical practice was generally seen in Kyrgyzstan, where consultations lasted around 15 min (vs. Vietnam where consultations lasted 2 min) and most of the healthcare workers are not medical doctors. Other studies in LMICs have looked at the effect of training on knowledge and confidence in primary care but have not studied behavioral change or it’s relation to time available for consultations . From prChanges seemed most pronounced in Kyrgyzstan. This could be due to a different baseline level performance, as seen by the lower NAKQ score in the pre-training test, whereas the doctors in Vietnam generally had a higher baseline performance and thus perhaps did not feel compelled to change their behavior or did not have the possibility to do so with only limited time available for each consultation. There was a tendency towards bigger changes to clinical practice in the rural setting in Kyrgyzstan but there the baseline performance level was also lower, which might explain the finding. Another possibility is that healthcare worker motivation was higher in the rural setting at outset.n = 48, Fig. The literature on effects of training on clinical practice in LIMCs has been reviewed recently  and one Healthcare worker motivation can go down from training if there is a lack of medicine and equipment, or if the services in question cannot be provided . InhaledIt is a strength of our study that we evaluated the effect of training using two different approaches to assess whether our intervention was successful, making us able to evaluate if contextual factors affected whether increased knowledge was translated into changes in clinical performance. It is a limitation of our study that we were not able to follow each healthcare worker from observation at the baseline until completion of the follow-up, thus the population of healthcare workers was different at the baseline and at the intervention. This could potentially attenuate the effects that were found due to decreased possibilities to explain the variability in the data. Another limitation is the lack of follow-up in one of the Vietnamese provinces, which meant we were not able to study the effect of a rural versus an urban setting in Vietnam. It is another limitation that we did not have the possibility to do content validation of the NAKQ in the local languages but since we used the questionnaires to measure increases in knowledge, it would only introduce variability that would attenuate any effects. Also, the questionnaire has been thoroughly validated in the original language . In ordeOur training focused on pediatric asthma but there is, however, no reason to believe that our findings would have been different if we have trained in other subjects. Our study emphasizes that the context of training is important even if training results in increased knowledge and that the structure of the health system is crucial to assess if training should result in changes in clinical practice. Contextual factors, perhaps most importantly the time available for clinical consultations, should be taken into consideration prior to rolling out training interventions. Our study indicates that factors like baseline performance level, type of health care worker, rural versus urban setting, and WHO region may also influence translation of training into clinical change. Further studies of the effect of training programs on clinical behavior should assess a wider range of contextual factors in bigger cohorts, preferably with the possibility of adjusting for the varying contextual factors in order to narrow down which are the most important ones to take into account.Training primary care health workers in LMICs in diagnosis and management of asthma in children under five in different contextual settings increased knowledge but only led to clinical changes in Kyrgyzstan, where there was more time for consultations (15 min), and more mid-level providers among trainees as opposed to Vietnam where time for consultations was short (2 min) and trainees were all medical doctors. Several contextual factors seem to be important for training to lead to clinical change.Additional file 1.


Pitch perception is known to be difficult for individuals with cochlear implant (CI), and adding a hearing aid (HA) in the non-implanted ear is potentially beneficial. The current study aimed to investigate the bimodal benefit for lexical tone recognition in Mandarin-speaking preschoolers using a CI and an HA in opposite ears. The child participants were required to complete tone identification in quiet and in noise with CI + HA in comparison with CI alone. While the bimodal listeners showed confusion between Tone 2 and Tone 3 in recognition, the additional acoustic information from the contralateral HA alleviated confusion between these two tones in quiet. Moreover, significant improvement was demonstrated in the CI + HA condition over the CI alone condition in noise. The bimodal benefit for individual subjects could be predicted by the low-frequency hearing threshold of the non-implanted ear and the duration of bimodal use. The findings support the clinical practice to fit a contralateral HA in the non-implanted ear for the potential benefit in Mandarin tone recognition in CI children. The limitations call for further studies on auditory plasticity on an individual basis to gain insights on the contributing factors to the bimodal benefit or its absence. As a highlight of groundbreaking neural prosthesis in bioengineering, the cochlear implant (CI) provides accessibility to sound for individuals with profound hearing impairment ,2. The rThere has been a growing number of CI prescriptions not only for individuals with bilateral deafness but also for those with profound unilateral hearing loss. Some clinical practices point to compelling evidence in improving CI individuals’ speech perception by means of bimodal hearing for unilaterally implanted candidates with some residual acoustic hearing. Bimodal hearing refers to the combination of two different solutions for hearing loss, with fitting a CI in one ear while using a hearing aid (HA) in the opposite ear. Behaviorally, successful bimodal listeners obtained an improvement in speech understanding with CI + HA stimulation  relative to with a unilateral CI alone ,23,24. TThe great variability of the bimodal benefit leads to an initiative to pinpoint the contributing factors for this situation at the individual level. One important factor could be the degree of impairment in basic auditory function, which can lead to different amounts of residual hearing in the non-implanted ear . For exaIn recent years, there has been a surge in investigating the bimodal benefit on lexical tone perception in individuals from tonal languages ,34,35,36Several initiatives have been launched to investigate the lexical tone perception in Mandarin children with CIs. Based on differences in pitch height and pitch contour, Mandarin tones are traditionally classified into four categories: Tone 1 (T1) with a high-flat pitch, Tone 2 (T2) with a mid-rising pitch, Tone 3 (T3) with a falling-rising pitch, and Tone 4 (T4) with a high-falling pitch. Prior studies have demonstrated that T1 and T4 are significantly easier to perceive than T2 and T3 for native CI children, with more confusion between the identification of T2 and T3 ,37. HoweThe primary objective of this study was to investigate the nature of the bimodal benefit for Mandarin tone recognition at both group and individual levels in native kindergarten-aged children who use CI in one ear and HA in the other. Three specific hypotheses were tested: (1) the child participants would show confusion between T2 and T3, which can be alleviated by bimodal hearing; (2) the noisy listening condition would exhibit more robust bimodal benefit than the quiet condition; and (3) the audiometric thresholds and the demographic factors of the participants could account for the bimodal benefit in the preschool bimodal participants. The findings of this study would contribute to our understanding and potential development of age-appropriate intervention programs for aural rehabilitation and speech intervention for the pediatric CI population of tonal language speakers.Twenty native Mandarin-speaking preschoolers born with bilateral severe-to-profound hearing loss were recruited from the Shanghai Rehabilitation Center of the Deaf Children. They had no history of psychiatric disorders or brain injuries in their medical history. All the child participants were implanted unilaterally and fitted with a contralateral HA. They used the CI in conjunction with HA devices for at least 75% of waking hours c.f., ,28) acco acco28])Standard audiometric assessments for pure tones from 125 Hz to 8 kHz were performed to determine the hearing impairment of each child participant. Stimuli in the speech recognition task were the four Mandarin tones naturally produced with the /i/ syllable. They were recorded from four native Mandarin-speaking adults  in a double-walled, sound-attenuated booth. Each speaker was required to produce each tone 10 times. The speech samples were recorded at a sampling rate of 44.1 kHz (16 bit). An experienced phonetician selected five speech samples for each tone per speaker, resulting in 80 test stimuli in total. All stimuli were normalized with equal average root-mean-square (RMS) intensity level. The average duration of the T1, T2, T3, and T4 stimuli are 537, 532, 614, and 374 milliseconds (ms), respectively. The pitch contours of the four tones were extracted from the test stimuli using Praat )Findings of the present study confirmed a confusion pattern for T2 and T3 in Mandarin preschoolers using a CI and an HA in opposite ears. The additional low-frequency acoustic information from the HA in the non-implanted ear can alleviate the confusion between T2 and T3 in quiet, and contribute to an improvement in overall performance of lexical tone recognition in noise. Moreover, audiometric thresholds of PTA at low frequencies  and bimodal duration were two significant predictors for the varying degrees of bimodal benefit in lexical tone recognition. These results provide evidence for the practice of fitting an HA in the non-implanted ear to take advantage of the potential bimodal benefit to facilitate speech learning in kindergarten-aged children with a unilateral CI. Further research is needed to examine auditory plasticity on an individual basis to gain insights on the contributing factors to the bimodal benefit or its absence in quiet and in noise.