Novel poly(tetrafluoroethylene) (PTFE) hollow fiber membranes were successfully fabricated by electrospinning, with ultrafine fibrous PTFE membranes as separation layers, while a porous glassfiber braided tube served as the supporting matrix. During this process, PTFE/poly (PVA) ultrafine fibrous membranes were electrospun while covering the porous glassfiber braided tube; then, the nascent PTFE/PVA hollow fiber membrane was obtained. In the following sintering process, the spinning carrier PVA decomposed; meanwhile, the ultrafine fibrous PTFE membrane shrank inward so as to further integrate with the supporting matrix. Therefore, the ultrafine fibrous PTFE membranes had excellent interface bonding strength with the supporting matrix. Moreover, the obtained ultrafine fibrous PTFE hollow fiber membrane exhibited superior performances in terms of strong hydrophobicity (CA > 140°), high porosity (>70%), and sharp pore size distribution. The comprehensive properties indicated that the ultrafine fibrous PTFE hollow fiber membranes could have potentially useful applications in membrane contactors (MC), especially membrane distillation (MD) in harsh water environments. Poly(tetrafluoroethylene) (PTFE) is a type of high-performance perfluoropolymer with a unique combination of outstanding chemical resistance, excellent thermal stability, strong hydrophobicity, exceedingly good antifouling property, and so on . These eHowever, owing to the ultrahigh melt viscosity and its insolubility, PTFE membranes or fibers are difficult to produce by conventional solution-spinning or melt-spinning methods ,6. NowadElectrospinning has proven to be an effective, straightforward, convenient, and versatile technology to synthesize continuous submicron structures from a huge range of materials ,17. In aIn our previous work, we fabricated ultrafine fibrous PTFE flat-sheet membranes by sintering electrospun PTFE/poly (PVA) ultrafine fibrous membranes, and their performance in the application of vacuum membrane distillation (VMD) was investigated ,21. BaseN,N-dimethylformamide (DMF) was purchased from Tianjin Kermel Chemical Reagents Co., Ltd., Tianjin, China. All the reagents were used as purchased without further purification. A glassfiber braided tube was provided by Dongguan Goddess Co., Ltd., Guangdong, China, and the basic properties of the glassfiber braided tube are shown in PTFE aqueous dispersion (FR301B) was purchased from 3F New Materials Co., Ltd., Shanghai, China. The specifications of the dispersion are shown in PVA aqueous solution (10 wt%) and PAN solution (10 wt%) were prepared by dissolving the materials in distilled water and DMF respectively. Then, PTFE emulsion and a certain amount of boric acid (0 wt% and 0.025 wt% of the solution) were added into PVA solution to obtain a homogeneous spinning solution. The composition of the spinning solution was tabulated in The schematic of electrospinning PTFE hollow fiber membranes was shown in The membrane morphologies were investigated by scanning electron microscopy  after coating the membrane with gold. The membrane surface roughness was investigated by confocal laser scanning microscope . The diameter distribution of the ultrafine fibers was calculated from the SEM images using Image Proplus software.Fourier-transform infrared spectroscopy  was used to identify the surface chemistry composition of the ultrafine fibrous membranes before and after sintering. The crystalline phases of the samples were confirmed by X-ray diffraction , with the diffraction angle 2θ having a value from 10° to 45°. The standard thermogravimetric (TG) analysis was completed using a thermogravimetric analysis instrument  . Water contact angle (WCA) measurements were performed using an optical contact angle meter  by the sessile drop method of water drops, and more than fifteen readings were obtained for each sample. The mean pore size and porosity were tested using an automatic mercury porosimeter . The membranes’ liquid entrance pressure (LEP) was determined using a self-made setup which is illustrated in −1 (C–F) and 1150 cm−1 (C–C), which corresponded to the stretching vibrations of PTFE. For the spectrum of nascent PTFE membranes (−1 (O–H), 2910 cm−1 (C–H), 1460 cm−1 (CH2), and 952 cm−1 (C–O) were assigned to PVA [−1.The FTIR spectra of the ultrafine fibrous PTFE hollow fiber membranes before and after sintering are shown in embranes b, the abd to PVA . These pOwing to the non-self-supporting property of flat-sheet micro-nano fibrous mats, they must be coupled with additional porous backing materials. The porous glassfiber braided tubes are mainly utilized as the backing material of electrical insulation, owing to their excellent mechanical strength, flexibility, and thermal stability .In this study, the porous glassfiber braided tube was used as the supporting matrix of ultrafine fibrous PTFE hollow fiber membranes, which provided not only high mechanical strength but also pressure resistance. As shown in The morphology of the membranes can provide an insight into the fiber stacking and the fiber’s microstructure as well as its pore network. As shown in After the sintering process, as shown in The XRD curves of PTFE hollow fiber membranes are shown in In general, the permeability increased with an increase in membrane pore size and porosity, while it decreased with the membrane thickness and pore tortuosity porosity. However, it is well known that for a good MD process, the porous membrane should exhibit a high LEPw, and applying a transmembrane hydrostatic pressure that exceeds the LEPw value may lead to pore wetting and a lower quality of the permeate water. The LEPw achieved by a membrane with high hydrophobicity (high water contact angles) and small pore size (ranging from 100 nm to 1 μm) was an essential condition for the process to successfully reduce pore wetting.The nascent PTFE/PVA membrane was completely wetted by water, which was attributed to the highly hydrophilic PVA. However, the obtained ultrafine fibrous PTFE hollow fiber membrane exhibited strong hydrophobicity (higher than 140°), which were not only owing to the natural hydrophobicity of PTFE, but also the micro-nano structure on the fiber’s surface, which further resulted in a higher membrane roughness. The membrane showed a good hollow fiber structure, and the ultrafine fibrous PTFE layer covered the supporting tube. The hollow porous glassfiber braided tubes increased the resistance of the membrane and provided a high specific surface area. The interaction between nanofibers and rope and the formation of nanofiber layers on the cylindrical rope enhanced the hydrodynamic conditions. Electrospun ultrafine fibrous PTFE fibers served as the outer layer which offered very high porosity, controllable pore size, and strong hydrophobicity. This was undoubtedly beneficial to the MD process.The porosity and pore size distribution in the results of In regards to the LEPw valve, the M-3 membrane held an LEPw value more than 0.2 MPa, which was higher than that of the M-2 membrane. Again, this is due to the larger pore size of the M-2 membrane, although the advancing WCA of the M-2 membrane was higher than that of the M-3 membrane. Moreover, the MD membranes must have good thermal stability and excellent chemical resistance to feed streams. The TG and DTG curves of M-3 membrane shown in These results suggested that the ultrafine fibrous PTFE hollow fiber membranes may provide a wide range of potential applications for the wastewater treatment, especially in harsh water environments.In summary, we reported a novel method of preparing ultrafine fibrous PTFE hollow fiber membranes by the electrospinning method, with PTFE ultrafine fibrous membranes acting as separation layers while the porous glassfiber braided tube acted as the supporting matrix. The obtained ultrafine fibrous PTFE membranes have excellent interface bonding strength with the supporting matrix. Moreover, ultrafine fibrous PTFE hollow fiber membranes exhibited superior properties such as strong hydrophobicity, high porosity, and sharp pore size distribution. It is expected that the ultrafine fibrous PTFE hollow fiber membrane will attract more attention not only in applications of membrane contactors but also in wastewater treatment.


RAS genes result in the elevation of cellular active RAS protein levels and increased signal propagation through downstream pathways that drive tumor cell proliferation and survival. These gain-of-function mutations drive over 30% of all human cancers, presenting promising therapeutic potential for RAS inhibitors. However, many have deemed RAS “undruggable” after nearly 40 years of failed drug discovery campaigns aimed at identifying a RAS inhibitor with clinical activity. Here we review RAS nucleotide cycling and the opportunities that RAS biochemistry presents for developing novel RAS inhibitory compounds. Additionally, compounds that have been identified to inhibit RAS by exploiting various aspects of RAS biology and biochemistry will be covered. Our current understanding of the biochemical properties of RAS, along with reports of direct-binding inhibitors, both provide insight on viable strategies for the discovery of novel clinical candidates with RAS inhibitory activity.Oncogenic mutations in  RAS mutations are among the most common mutations in cancer, driving aggressive, metastatic malignancies with poor patient prognoses. Notably, pancreatic (95%), colorectal (45%), and lung (35%) cancers harbor KRAS mutations at remarkably high frequencies. Mutations in RAS genes are also known to cause developmental disorders of the heart and nervous system, known as RASopathies ..KRAS G12Targeting the nucleotide-free state of RAS is a relatively unexplored strategy for inhibiting nucleotide loading of RAS. This state of RAS only seems to exist transiently in cells prior to GTP loading. However, a recent report has shown that nucleotide-free RAS exists in a large enough fraction in cells to participate in signal transduction, as it can indeed bind to and negatively regulate the activity of class II PI3Ks. This interaction in turn negatively regulates RAS activation by blocking GTP binding to RAS and locking RAS in a nucleotide-free conformation . The negKRAS mutant tumor models [Another promising approach for directly targeting RAS is disrupting the interaction between active, GTP-bound RAS and its effectors . Yeast tr models . Howeverr models . Currentr models . Additior models .RAS cell-based assays, which suggest that the mechanism is unrelated to COX inhibition [RAS mutant tumors in a chemical-induced rat model of breast cancer [50 values for cell growth inhibition and inhibition of RAS-RAF binding.Additionally, they may have other binding partners that contribute to their anticancer activity at micromolar concentrations. However, these studies suggest that chemical modification of sulindac to further improve RAS inhibitory activity and eliminate COX inhibitory activity represents a promising approach to target RAS for anti-tumor activity with reduced potential for toxicities.The cyclooxygenase inhibitor, sulindac sulfide E, has behibition ,73. Whilt cancer . Attemptt cancer ,76. ThesWhile most discovery strategies target one specific aspect of RAS nucleotide cycling, a novel inhibitor was recently discovered that inhibits several protein-protein interactions that are critical for RAS functionality. This compound binds to a shallow pocket between the switch I and switch II domains of RAS that was previously thought to be “undruggable.” The novel compound, BI-2852, binds to both the RAS-GDP and RAS-GTP states to inhibit GEFs, GAPs, and effectors all from interacting with RAS . This deRAS mutations other than G12C. Taken together, significant progress has been made towards drugging the “undruggable” RAS, and the hunt continues for a promising clinical candidate that could be the first FDA-approved RAS inhibitor.Despite extensive efforts to identify small molecule inhibitors of RAS, there are currently no FDA-approved drugs that directly bind to RAS and selectively inhibit RAS-driven tumor growth. The cycling of guanine nucleotides to shift RAS between inactive and active conformations provides new opportunities to target RAS. The most successful approaches, thus far, have been covalently trapping RAS in its inactive GDP-bound state, along with inhibiting RAS-effector binding. Selective covalent inhibitors of the KRAS G12C mutant, such as AMG 510, elicited promising results in clinical trials for the treatment of lung and colorectal cancers. Additionally, the RAS mimetic rigosertib is currently being evaluated in clinical trials for the treatment of hematologic cancers. While the progress towards developing clinically effective RAS inhibitors is promising, the therapeutic potential of compounds targeting specific mutants is limited to subsets of RAS-driven cancers. The current understanding of the mechanism of RAS nucleotide exchange presents numerous opportunities for reversible inhibitors, with several recent discovery campaigns producing novel reversible RAS inhibitors. A reversible RAS inhibitor would be highly significant to the oncology field at large and extremely valuable for the treatment of patients harboring


IntroductionBrain metastases are common in patients with advanced systemic cancer and often recur despite treatment with surgical resection and radiotherapy. Whole brain radiation therapy (WBRT) and stereotactic radiosurgery (SRS) have significantly improved local control rates but are limited by complications including neurocognitive deficits and radiation necrosis. These risks can be higher in the re-irradiation setting. Brachytherapy may be an alternative method of additional targeted adjuvant radiotherapy with acceptable rates of toxicity.MethodsA retrospective chart review of all patients undergoing resection for metastatic brain lesions and permanent low-dose rate Cs-131 brachytherapy was performed for one institution over a 10-year period. All patients had previous radiation therapy already and, after surgery, were followed with imaging every three months. Patient demographics, disease characteristics, intracranial disease, peri- and post-operative complications, and outcomes were recorded. The primary outcome of interest was local tumor recurrence at the site of brachytherapy while secondary outcomes included distant disease progression (within the brain) and complications such as radiation necrosis.ResultsDuring the study period, nine cases of individual patients met inclusion criteria. The median preoperative lesion diameter was 3 cm (0.8–4.1). The median overall survival after surgery and brachytherapy was 10.3 months, after excluding two patients who were lost to follow-up. Six of nine patients had no local recurrence, while three patients had development or progression of distant lesions. No patients experienced acute or delayed complications.ConclusionCs-131 brachytherapy is a promising alternative method for controlling brain metastases after previous radiation interventions and surgical resection. In this case series, there were no incidences of local tumor recurrence or complications such as radiation necrosis. Brain metastases are common and can occur in 25% of patients with systemic cancer . AlthougTreatments for brain metastases often involve a combination of surgical resection and/or radiotherapy ,3. HowevSince brain metastases often recur locally and WBRT and SRS are limited in certain patient scenarios, there has been growing interest in alternative methods of focused re-irradiation. Permanent low-dose rate (LDR) brachytherapy seeds are one such option that can deliver targeted radiation to a specific site . HoweverInstitutional review board approval from the senior author’s institution (IRB00092610) was obtained for this retrospective series. All patients with brain metastases treated by surgical resection and permanent Cs-131 LDR brachytherapy from 2007 to 2017 by the senior author were reviewed. Patient consent was not obtained due to the retrospective nature of this study. Inclusion criteria for relevant cases were patients over 18 years of age with a history of established metastatic cancer. The intent at the time of surgery was gross total resection of their brain metastases with placement of radioactive Cs-131 seeds lining the operative bed. Patient follow-up was obtained with imaging every three months after surgery. Patient demographics, disease characteristics, intracranial disease, peri- and post-operative complications, and outcomes were recorded and de-identified appropriately. The primary endpoint was local tumor recurrence, while secondary outcomes of interest included development or progression of distant (within CNS) metastatic disease and complications related to brachytherapy implantation. Local recurrence was defined as a progressively expanding lesion at the site of resection and brachytherapy seen on multiple scans. Distant progression was evaluated similarly for other sites within the CNS. Early complications include acute hemorrhage or infections; delayed complications include worsening headache, progressive neurological deficits, volume loss from atrophy or gliosis, and radiation necrosis . Follow-During the study period, nine cases of individual patients met inclusion criteria. Their average age at the date of surgery was 53.8 years. Patient demographic information is summarized in Table The operative parameters are reported in Table Table As the incidence of brain metastases become more frequent, new methods of delivering focused radiation are warranted to address the variety of tumors that may metastasize to the brain, especially in the setting of previous irradiation . CurrentSRS has become the modality of choice for delivering targeted intracranial radiotherapy after surgical resection. Surgical resection alone has local control rates ranging from 45 to 60% ,16. HoweBrachytherapy is capable of delivering a low amount of radiation over a longer period of time, potentially addressing the risk of endothelial damage and subsequent radiation necrosis caused by high dose SRS . InterstLong-term local control is a key aim of adjuvant focal irradiation techniques after surgical resection. Though WBRT and SRS have significantly improved rates of local control, the results of this case series and several other reports suggest that brachytherapy is also effective in delivering robust levels of local tumor control. Wernicke et al.’s prospective trial on Cs-131 therapy after surgical resection in lesions ≥2 cm showed 100% local freedom from progression for all treated tumors regardless of size or primary cancer type . FurtherThis case series sought to report the outcomes of Cs-131 brachytherapy for a variety of metastatic brain lesions. However, this study of nine cases over 10 years is limited by the small sample size and single center experience. Additional multicenter studies incorporating larger sample sizes would be able to better define rates of local and distant control and provide a broader overview of complication rates. Further, the inherent nature of retrospective studies includes a risk of unexpected effects from unmeasured variables. Nonetheless, this case series clearly demonstrates high local control and low complications from brachytherapy in a variety of metastatic brain lesions.Brain metastases are common and account for most intracranial tumor resections. Standard of care radiotherapy often employs SRS but is limited by radiation necrosis and tumor size. Cs-131 brachytherapy is a potential alternative method for focal irradiation, especially for previously irradiated lesions. In this series, there was a remarkably high rate of local control and no reported complications including radiation necrosis.


Lung adenocarcinoma (LUAD) patients experiencing lymph node metastasis (LNM) always exhibit poor clinical outcomes. A biomarker or gene signature that could predict survival in these patients would have a substantial clinical impact, allowing for earlier detection of mortality risk and for individualized therapy.With the aim to identify a novel mRNA signature associated with overall survival, we analysed LUAD patients with LNM extracted from The Cancer Genome Atlas (TCGA). LASSO Cox regression was applied to build the prediction model. An external cohort was applied to validate the prediction model.We identified a 4-gene signature that could effectively stratify a high-risk subset of these patients, and time-dependent receiver operating characteristic (tROC) analysis indicated that the signature had a powerful predictive ability. Gene Set Enrichment Analysis (GSEA) showed that the high-risk subset was mainly associated with important cancer-related hallmarks. Moreover, a predictive nomogram was established based on the signature integrated with clinicopathological features. Lastly, the signature was validated by an external cohort from Gene Expression Omnibus (GEO).In summary, we developed a robust mRNA signature as an independent factor to effectively classify LUAD patients with LNM into low- and high-risk groups, which might provide a basis for personalized treatments for these patients. Lung cancer is the leading cause of cancer-related death worldwide, with adenocarcinoma being the most common histological type . DespiteIn recent years, the development of gene expression profile technologies, such as microarray and next generation sequencing (NGS), have provided further opportunities to comprehensively characterize the molecular features of cancer , 8. ConsIn this study, based on The Cancer Genome Atlas (TCGA) LUAD mRNA-seq and clinical datasets, we sought to develop a gene expression signature to predict overall survival for LUAD patients with LNM, and then the proposed gene signature was validated in an external cohort from the Gene Expression Omnibus (GEO) database.https://xenabrowser.net/). GSE68465 was download from the GEO database. The LUAD patients with LNM were filtered by the criteria that N stage of patients was I–IV.TCGA RNA-seq datasets and clinical data for LUAD were downloaded by UCSC Xena browser  was used to construct the gene co-expression network . The co-A weighed network adjacency was defined by raising the co-expression similarity to a power nt}β ≥ 1 . We selecument}q . The modThe topological overlap measure (TOM) plots visualized inter-connectivity patterns and suggests the presence of large modules. This property of dense connections among the genes of module DEG analysis was performed by the Limma package . The tisCox regression, also called Proportional Hazards Regression, is a survival analysis model . It can LASSO (Least Absolute Shrinkage and Selection Operator) is an important regularization in many regression analysis methods  . The ide package . The GSE package .The flowchart of our study is shown in Fig. The whole transcriptome of LNM-positive samples was included to perform DEG analysis, and the volcano plot showed 974 differentially expressed genes (DEGs) in 173 LNM-positive (LNM+) tumour samples compared to 335 LNM-negative (LNM-) tumour samples Fig. a. To conThen, LASSO Cox regression analysis was performed to identify robust markers among the 575 candidates. By forcing the sum of the absolute value of the regression coefficients to be less than a fixed value, certain coefficients were shrunk to exactly zero, and the most powerful prognostic markers were identified with relative regression coefficients. Cross-validation was applied to prevent the over-fitting of the LASSO Cox model Fig. e. FigureTo comprehensively investigate the association between the 4 identified genes and prognosis in these patients, we developed a 4-gene signature-based prognostic model according to their individual coefficients. Then, we calculated the risk score for each LNM+ patient in the training set and ranked them. Thus, LNM+ patients with follow-up information were divided into two groups: a low-risk group (n = 78) and a high-risk group (n = 78) based on median cut-off value Fig. a. FigureWe investigated the 4 genes’ expression profiles in different AJCC-TNM stages in the TCGA cohort. As shown in Fig. Then, we constructed a nomogram that integrated clinicopathological features with the 4-gene signature to predict survival probability of LNM + patients Fig. a. CalibrTo confirm our findings in the training set, we validated the prognostic function of the 4-gene signature in an independent GEO cohort GSE68465). After extracting the microarray data of 140 LNM+ patients with follow-up information, we calculated the risk score for each patient by using the same formula in the training set. Figure 8465. AftAccumulating evidence shows that lung adenocarcinoma patients with local invasion or lymph node metastasis always exhibit poor responses to standard treatments and thus tend to have poor clinical outcomes –5, 25. IIn this study, we identified 4 coding genes associated with overall survival in LUAD patients with lymph node metastasis, namely, LDHA, ABAT, FAM117A and INPP5J, in the training set. Among the 4 coding genes, LDHA was widely reported to promote malignant progress and predict poor survival in various cancer types –30. In aBased on multivariate Cox coefficients derived from LASSO analysis, we developed a 4-gene signature-based risk score model. Moreover, we investigated the prognostic value of the signature in different subgroups. In detail, the signature still exhibited powerful prediction for overall survival in LNM+ patients with same TNM stage and genomic alteration (including EGFR and KRAS mutation status), confirming that the signature is a promising marker independent of different clinicopathological features. In addition to survival prediction, GSEA showed that the signature-identified high-risk group was significantly correlated with certain hallmarks of cancer, such as EMT and hypoxia, indicating the potential molecular mechanisms underlying the lethal tendency of these LNM+ patients. By integrating established clinicopathological features with the signature, we developed a nomogram to predict the survival probability of LNM+ patients. The predictive power was measured by the time-dependent area under the ROC curve (AUC(t)), and the result showed that the integrated nomogram model had higher predictive power than individual markers. Lastly, an external GEO cohort was used to validate the prognostic value of the 4-gene signature, and the survival analysis showed the same tendency in the validation cohort.The limitations of our study should be acknowledged. This is a retrospectively designed study, and the sample size of the training and validation sets is relatively small.In summary, the novel 4-gene signature proved to be a robust model with high predictive power in LUAD patients with LNM+. The predictive power was stable over time and showed promising survival prediction in combination with established markers. The use of the signature integrated with clinicopathological features can help to further stratify LNM+ patients into risk groups, thus serving as a predictive tool for clinical outcome, guiding personalized treatment, and resulting in more aggressive therapy for high-risk patients or less aggressive therapy for low-risk patients. Further research is needed to reveal the interplay between these genes, and thereby, we will be able to develop better treatment alternatives for high-risk LUAD patients with lymph node metastasis.In conclusion, based on publicly available data, we constructed a robust mRNA signature that could serve as a reliable marker to stratify a high-risk group among LUAD LNM+ patients. Subgroup analysis indicated that the signature works effectively independent of other clinical features. Validation in an external cohort from GEO further confirmed the prognostic value of the signature. We hope the identified signature could help to improve the strategies for personalized treatment of LUAD patients with LNM.


Athalia rosae (Hymenoptera: Tenthredinidae). We then assessed whether offspring of starved and non-starved parents differed in their own life-history if reared in conditions that either matched that of their parents or were a mismatch. Early life starvation effects were more pronounced within than across generations in A. rosae, with negative impacts on adult body mass and increases in developmental time, but no effects on adult longevity in either generation. We found some evidence of higher growth rates in larvae having experienced starvation, although this did not ameliorate the overall negative effect of larval starvation on adult size. However, further work is necessary to disentangle the effects of larval size and instar from those of starvation treatment. Finally, we found weak evidence for transgenerational effects on larval growth, with intra-generational larval starvation experience being more decisive for life-history traits. Our study demonstrates that intra-generational effects of starvation are stronger than transgenerational effects on life-history traits and consumption measures in A. rosae.Resource availability during development shapes not only adult phenotype but also the phenotype of subsequent offspring. When resources are absent and periods of starvation occur in early life, such developmental stress often influences key life-history traits in a way that benefits individuals and their offspring when facing further bouts of starvation. Here we investigated the impacts of different starvation regimes during larval development on life-history traits and measures of consumption in the turnip sawfly,  As a major determinant of growth and developmental trajectories, resource abundance in early life plays a crucial role in shaping an individual’s phenotype , not leaApis mellifera) starved in early life live longer than individuals raised in an environment of plenty when both are exposed to starvation in adulthood  as the response variable and initial larval body mass, larval starvation treatment, sex, and two two-way interactions between initial larval body mass and larval starvation treatment or sex as the predictors. The RCR was estimated via the analysis of the log of total fresh mass of consumed leaf material (mg) as the response variable and the same predictors as for the RGR analysis. The ECI was estimated via the analysis of the change in larval body mass as the response variable and the total fresh mass of consumed leaf material, larval starvation treatment, sex, and two two-way interactions between mass of consumed leaf material and larval starvation treatment or sex as the predictors.We estimated relative growth rate (RGR), relative consumption rate (RCR) and food conversion efficiency (ECI) of larvae using general linear models , to avoivia the analysis of the change in larval body mass [final mass (mg)—initial mass (mg)] as the response variable and initial larval body mass, parental (F0) larval starvation treatment (NS or LS), offspring (F1) starvation treatment (NS or LS) and their interactions (three-way interaction) as predictors. The RCR was estimated via the analysis of the log of total fresh mass of consumed leaf material (mg) as the response variable and initial larval body mass, parental (F0) larval starvation treatment (NS or LS), offspring (F1) starvation treatment (NS or LS) and their interactions (three-way interaction) as predictors. The ECI was estimated via the analysis of the change in larval body mass as the response variable and the total fresh mass of consumed leaf material, parental (F0) larval starvation treatment (NS or LS), offspring (F1) starvation treatment (NS or LS) and their interactions (three-way interaction) as predictors.Offspring (F1) generation: Due to the low number of females (n = 6) compared to males (n = 64) reaching adult eclosion in the F1 generation, the full analysis was carried out only on males . The influence of starvation treatment in both the parental and offspring generation on larval developmental time was assessed using a GLM , where larval development time (days) was the response variable, and parental (F0) larval starvation treatment (NS or LS), offspring (F1) starvation treatment (NS or LS) and their interaction were the predictors. A survival analysis  was use23,96 = 52.08, p < 0.001). Larval development time was significantly longer when larvae had periods of starvation  than when they were fed ad libitum (NS)  or sex alone on larval developmental time . In contrast, sex influenced adult longevity, with females living significantly longer than males , independent of larval starvation treatment . Adult females were heavier than adult males and adult body mass decreased with increasing bouts of starvation . The total number of hatched larvae per female varied with female body mass in dependence of the larval starvation treatment . Females of the MS or LS treatment showed a decreasing number of hatched larvae with increasing female body mass, whereas females of the NS treatment showed the reverse  had a significantly lower larval survival than larvae of the three other starvation treatment levels . Across all treatments there was a significant positive relationship between the amount of leaf material consumed by larvae and their initial body mass, i.e. the RCR , with increases in the initial body mass of larvae that had previously been starved  resulting in larger increases in the amount of consumed leaf material than equivalent increases in initial mass in larvae of the NS treatment  or on the relationship between leaf consumption and initial larval body mass .The relative growth rate (RGR) differed significantly between F0 larvae of the different starvation treatments . In both starvation treatments  larvae that had a higher increase in body mass consumed more food over the course of the consumption assay, while in larvae of the NS treatment there was no apparent relationship between consumption and body mass change  or on the relationship between leaf consumption and larval change in body mass .The ECI differed significantly between F0 larvae of the different treatments . There was no significant effect of either offspring larval starvation treatment , parental larval starvation treatment  or their interaction  on offspring adult longevity. Male adult body mass differed depending on starvation treatment; F1 adults were heavier when reared as larvae without starvation (NS) , offspring larval starvation treatment  nor their interaction  influenced the relationship between larval leaf consumption and larval initial body mass, i.e. RCR. Likewise, these two treatments and their interaction did not influence the relationship between larval leaf consumption and the change in larval body mass, i.e. ECI, in the consumption assay .There was a significant difference in the relationship between the initial larval body mass and the change in larval body mass (RGR) between F1 larvae of different treatments in the consumption assay  did not differ. These results suggest that there might be a limit to the prolongation of developmental periods in A. rosae in the face of harsh early life conditions, resulting in a trade-off with adult body mass. Several factors might select against extended larval development including increased risk of predation and parasitism as well as seasonal pressures such as peak adult food abundance, temperature or mate availability [A. rosae where only adults have high mobility, it may be advantageous to complete development at a smaller size to enable dispersal, whilst differentially allocating available resources during metamorphosis to improve dispersal abilities [Bicyclus anynana resource constraints in early life lead to a lower adult body mass, with these smaller adults having a higher thorax ratio  resulting in a better flight performance compared to larger adults [In the parental (F0) generation, periods of starvation during the larval phase prolonged larval developmental time. Increases in developmental time are often linked to starved individuals ‘catching up’ the overall growth achieved by non-starved individuals . Howeverult size . Adult mlability ,31,53–55bilities ,56,57. Fr adults .A. rosae, those individuals that were starved appeared to have a higher growth rate than those individuals that did not experience starvation. Likewise, previously starved larvae also showed more efficient consumption, despite all individuals consuming a larger amount of leaf material with increasing larval size. This might indicate ‘compensatory growth’, i.e. an increased growth rate, in starved larvae once food is available [A. rosae employs compensatory growth when recovering from larval starvation.In terms of treatment effects on the growth rate of vailable , howevervailable . The assvailable  and therA. mellifera [A. rosae based on early life experience, as the environment for all adults was benign, with a constant food supply.Starvation bouts during development had no effect on adult longevity (F0 generation). However, empirical work on the relationship between early life starvation and adult longevity provides a mixed bag of results, spanning the spectrum from positive to negative effects on adult lifespan –62. Arguellifera . In otheellifera . This maB. anynana that were starved in early life [A. rosae between maternal size and offspring number in the starvation treatments, although future measurements of offspring size would be necessary to test this assumption.Starvation also altered the relationship between female body mass and reproductive output in the parental generation, with a positive relationship in individuals experiencing no starvation and a negative relationship in individuals starved during development. Starvation and resource restriction in early life frequently reduce female fecundity ,29,68, irly life . This trA. rosae than the parental starvation treatment. Neither parental nor offspring larval starvation treatments influenced offspring adult lifespan, but offspring larval starvation increased developmental time and decreased male adult body mass independent of parental conditions. In contrast, in the grasshopper Chorthippus biguttulus high quality diet of the parental generation had positive effects on the offspring developmental time and body mass, when diet quality was kept high or low over the entire development of each generation [Drosophila melanogaster, offspring of mothers raised as larvae under poor environmental conditions had a larger size and shorter development time than the offspring of mothers that developed in a resource-abundant environment [versus transgenerational responses may be highly species- and dietary treatment-specific. Furthermore, both offspring and adult larval starvation treatments interactively affected offspring larval growth rate in A. rosae. The contrasting growth rates in particular between larvae of the LS-NS and the NS-NS treatment are most striking, as they indicate that the experience of poor larval conditions in the parental generation enhances the growth rate in offspring. However, as discussed above for the F0 generation, the different growth trajectories are probably mostly related to the body size and ontogeny of the larvae, with increasing growth rates up to a certain larval size and decreases once larvae approach pupation.The starvation regime of larvae in the offspring generation (F1) had a more pronounced effect on offspring life-history traits of neration . In Drosironment . Thus, iA. rosae, with starved individuals from parents that themselves were starved or not starved as larvae (LS-LS and NS-LS) having similar growth trajectories. One possible explanation is that the benefits of having high quality parents may mask the benefits of environmental matching, when offspring are developing in a poor environment. These types of transgenerational effects can be difficult to disentangle with a match-mismatch design, because the effects of both parental and offspring environments may not necessarily act in an additive manner [A. rosae, with much larger environmental effects of offspring diet.The environmental matching hypothesis predicts fitness benefits when the environment matches between parents and offspring ,24. Thuse manner . OverallA. rosae. Early life starvation negatively influenced adult body mass and increased individual developmental time, with growth trajectories in this experiment likely predominantly determined by current body size and ontogeny. Further work is necessary to disentangle the effects of larval size and instar from that of starvation treatment when studying consumption and growth rates in this species. Future studies may also consider the effects of single acute starvation bouts, simulating alternative field conditions where high numbers of later larval instars can decimate single host plants before finishing development. As the physiological effects of starvation are known to differ between different rates and duration of starvation bouts, there might also be differences in the intra-generational and transgenerational life-history responses of individuals to these different types of starvation.The effects of early life diet experience on key life-history traits were stronger within than across generations in S1 Table(XLSX)Click here for additional data file.


To assess the long-term effects of soft contact lenses (SCL) on the cornea and anterior chamber by topography.Thirty-nine eyes of 22 healthy patients were included in this prospective study. Changes in corneal and anterior chamber parameters before and after 12 months of daily SCL use  were evaluated with Pentacam .Best corrected visual acuity with toric SCL was significantly better compared to spectacles in the toric SCL group . None of the corneal  and anterior chamber  parameters showed a statistically significant change after long-term daily wear of SCLs.The results of this study revealed that long-term wear of current high oxygen permeable and relatively low modulus silicone hydrogel SCLs does not impact cornea and anterior chamber morphology or volumetric parameters. Furthermore, toric silicone hydrogel SCLs can provide better visual performance than spectacles. With the advances in contact lens technology, they have become more comfortable and safer; however, they still carry the risks associated with modification of corneal morphology. The primary goal in SCL design is to create a contact lens which provides biocompatibility with both the cornea and the surrounding ocular environment . The mechanical properties of the SCL  should not increase corneal and conjunctival epithelial irritation and should allow maximum tear circulation.6 Significant improvements in silicone hydrogel lenses have made them the first choice for new contact lens wearers.7 Silicone hydrogel lenses have eliminated corneal hypoxia.8 However, despite their high oxygen transmission levels, SCL-related central and peripheral corneal swelling which results in increased central corneal thickness and significant thinning of the central corneal epithelium has been reported.10Despite the developments in contact lens technology, the alterations in corneal metabolism and the mechanical forces associated with contact lens wear can affect the anterior shape of the cornea and result in central corneal steepening or flattening, loss of radial symmetry, and changes in astigmatism or optical higher order aberrations.11 Thus, monitoring changes in the anterior and posterior cornea is important in contact lens wearers who are candidates for keratorefractive surgery.Both the anterior corneal surface and corneal thickness are critical variables to consider before any clinical intervention, particularly refractive surgery. Furthermore, changes in the posterior corneal surface are useful in monitoring corneal pathologies and in the early detection of corneal ectatic disorders.The purpose of this study was to assess changes in corneal and anterior chamber parameters after 12 months of SCL wear.Thirty-nine eyes of 22 individuals who attended the contact lens clinic of Ankara Numune Training and Research Hospital between January 2014 and January 2016 were included in this prospective study. Patients with known history of ocular and/or systemic diseases, previous ocular surgery, prior contact lens use, ocular or systemic medication use, and more than 3.5 diopters of cylindrical and/or 6 diopters of spherical refractive error were not included. All subjects underwent complete ophthalmologic evaluation to ensure the presence of Snellen best corrected visual acuity of 20/25 or better, normal anterior and posterior segment biomicroscopy, normal tear film functions (Schirmer test >15 mm/5 minute and tear break-up time ≥10 second) and  normal intraocular pressure. Informed consent was obtained from all subjects and the study was carried out with approval from the ethics committee of Ankara Numune Training and Research Hospital.Refractive and topometric maps obtained with Pentacam  were used to evaluate anterior and posterior corneal axial curvature, corneal volume, central corneal thickness, corneal thickness at the thinnest point, anterior chamber depth, anterior chamber angle, and anterior chamber volume before contact lens fitting. All measurements were performed by a single author (C.C.) between 10:00 and 12:00 a.m. to avoid diurnal variations. Only reliable scans that marked “quality specification” of Pentacam were included in analysis.After the completion of Pentacam measurements, each patient was fitted with a suitable Lotrafilcon B  or Senofilcon A  SCL according to their manifest refraction. Subjects were instructed to wear these contact lenses in daily wear mode before returning for the final visit at 12 months. The cornea and anterior chamber were re-evaluated with Pentacam  at the final visit, at least 1 hour after contact lens removal to avoid lens-related mechanical changes. Subjects who wore their contact lenses regularly (>4 days/week) in daily wear mode were included in the final analysis.Statistical analyses were performed using SPSS version 13.0  software. Student’s paired t-test was used to compare changes in visual acuity, keratometry, corneal volume, central corneal thickness, corneal thickness at thinnest point, anterior chamber depth, anterior chamber angle, and anterior chamber volume over time. P value less than 0.05 was considered to be statistically significant.Thirty-nine eyes of 22 patients were initially included in the study, but 2 of the subjects were excluded due to irregular SCL wear. Thus, the study was completed with a total of 37 eyes of 20 patients . Mean age was 20.52±3.18 years (range 15-28 years). Nineteen eyes were fitted with spherical soft lenses and 18 eyes were fitted with toric soft lenses.Best corrected distance visual acuity (BCVA) for patients fitted with spherical lenses was 20/20 at baseline and final examination. There was a statistically significant increase in the distance BCVA of toric lens wearers  , and theThe corneal  and anterior chamber parameters  as evaluated by Pentacam are summarized in 11 The purpose of our study was to investigate the long-term effects of SCL wear on corneal and anterior chamber parameters measured by Pentacam. To our knowledge, this is the first prospective study to investigate long-term SCL-associated changes in the cornea  and the anterior chamber  with Pentacam. This device has been found to be highly reliable and showed excellent repeatability in previous studies.13Contact lenses are a popular mode of refractive error correction due to advantages such as a larger field of vision, better optical quality, and cosmetic appeal. However, they still have the disadvantage of interfering with normal corneal physiology and curvature.15 We believe that with a good SCL-cornea adaptation process, the mechanical properties of contact lenses and the toric designs result in fewer aberrations with toric lenses compared to glasses.Snellen best corrected visual acuity of 20/20 was our targeted value for SCL wearers when prescribing contact lenses. Thus, some of the patients might have better visual acuity after SCL fitting. While we were unable to find a significant difference in the visual acuity of spherical lens wearers, a more detailed visual acuity study could also reveal significance in the spherical group. Visual performance in toric lens wearers was better with SCLs compared to spectacles. This result is in line with previous studies that have found improved visual quality and acuity with toric SCLs in low and moderate astigmatic eyes.16 evaluated corneal thickness and topography in long-term contact lens wearers and healthy controls. They found that corneal thickness was significantly reduced by 30-50 µm in contact lens wearers. Furthermore, corneal curvature and surface irregularity were increased in eyes wearing contact lenses. Yeniad et al.17 evaluated changes in corneal curvature and thickness after 1, 6, and 18 months of rigid gas-permeable and soft lens wear. SCL wearers in their study showed corneal thickening at 1 month, followed by corneal thinning at 6 and 18 months. An increase in radius of corneal curvature and central corneal thinning associated with continuous wear of first-generation high-Dk SCLs were previously reported in a small sample of patients.18Several studies have investigated the changes in corneal shape and thickness associated with contact lens wear. However, the follow-up for most of these studies was very short. Liu and Pflugfelder2 compared corneal topographical changes with 3 months daily wear of silicone hydrogel (SiH) lenses and found that corneal topographic indices were stable with daily wear of SiH lenses. The magnitude of corneal morphological changes associated with 8 hours wear of daily disposable lenses was also small.19 Radaie-Moghadam et al.20 investigated corneal hysteresis, corneal resistance factor, central corneal thickness, and keratometry on toric SCL wearers with negative history of SCL wear and concluded that corneal hysteresis and corneal resistance factor decreased at 1 month and returned to baseline after 3 months, while central corneal thickness and corneal curvature values did not change significantly.Contrary to these studies, Alba-Bueno et al.In the present study; Acuvue Oasys, Acuvue Oasys for Astigmatism, Air Optix Aqua, and Air Optix Aqua for Astigmatism were selected to evaluate the presumptive corneal alterations. These SCLs constitute the majority of SCL prescriptions in our clinic. The mean CCT was 553.64±23.22 at baseline and none of the corneal parameters showed significant difference after 12 months of SiH contact lens wear. Conflicting results in different studies are most likely due to differences in contact lens material, oxygen transmissibility, water content, and modulus. The results of our study show that low modulus, high oxygen transmissible lenses can prevent the corneal alterations associated with SCL use. Early corneal biomechanical and curvature changes which develop in the first few months of SCL wear regress in long-term follow-up after the cornea-contact lens adaptation process.This study also investigated changes in anterior chamber volume, depth, and angle with SCL wear. As expected, no significant changes were observed in anterior chamber parameters.This study has several limitations. First, our sample size was relatively small and did not allow for segregation of patients into subgroups depending on the modulus of lens materials.Another limitation is the lack of aberrometry measurements, which would be helpful to explain the improvement in visual acuity in the toric SCL group.In conclusion, the results of this study revealed that previously reported short-term SCL-associated corneal changes probably diminish in long-term follow-up. Long-term wear of current highly oxygen permeable and relatively low modulus SiH SCLs do not change corneal or anterior chamber morphology and volumetric parameters. Furthermore, toric SiH SCLs may provide better visual performance compared to spectacles. Further studies with a larger sample size and longitudinal follow-up are necessary to confirm these findings.


The hypocretins (Hcrts) are two alternatively spliced neuropeptides (Hcrt1/Ox-A and Hcrt2/Ox-B) that are synthesized exclusively in the hypothalamus. Data collected in the 20 years since their discovery have supported the view that the Hcrts play a broad role in the control of arousal with a particularly important role in the maintenance of wakefulness and sleep-to-wake transitions. While this latter point has received an overwhelming amount of research attention, a growing literature has begun to broaden our understanding of the many diverse roles that the Hcrts play in physiology and behavior. Here, we review recent advances in the neurobiology of Hcrt in three sections. We begin by surveying findings on Hcrt function within normal sleep/wake states as well as situations of aberrant sleep . In the second section, we discuss research establishing a role for Hcrt in mood and affect . Finally, in the third section, we briefly discuss future directions for the field and place an emphasis on analytical modeling of Hcrt neural activity. We hope that the data discussed here provide a broad overview of recent progress in the field and make clear the diversity of roles played by these neuromodulators. Their name was a combination ofhypo for their hypothalamic origin andcretin based on their sequence homology to the gut hormone secretin1. At the same time, Masashi Yanagisawa’s group at University of Texas Southwestern was characterizing ligands for orphan G-protein-coupled receptors as a means to determine their role in various physiological processes. The group found two extracts within the hypothalamus that bound and activated two orphan receptors with unknown functions. When supraphysiological doses of peptide were injected intracerebroventricularly, these peptides promoted food intake. Owing to this effect, the group named the peptides “orexins” based on the Greek word for appetite (orexis)2. Indeed, the two groups were describing the same peptides, and today hypocretin and orexin are synonymous. Here, we will review some of the most recent findings in the neurobiology of Hcrt in relation to arousal, emotional processing, and motivation and finally discuss future directions for analytical modeling of Hcrt networks.In 1998, two research studies published within a month of each other described a set of novel hypothalamic peptides. The first group to describe them was led by Gregor Sutcliffe at the Scripps Research Institute in La Jolla, California. The Sutcliffe group used subtractive RNA hybridization to characterize a cDNA clone with restricted expression in the dorsal and lateral hypothalamus (LH). This cDNA clone encoded a preproprotein termed preprohypocretin. This was the putative precursor to two peptides that they named hypocretin-1 (Hcrt1/Ox-A) and hypocretin-2 (Hcrt2/Ox-B) with respective receptors OX3. Chemogenetics uses modified G-protein-coupled receptors  that are largely activated by a metabolite of clozapine N-oxide (CNO) when injected systemically4. Excitatory or inhibitory DREADDs can be selectively expressed in neuronal populations of interest  which then can be manipulated by injections of CNO5. Additionally, the expression of calcium indicators allows the measurement of cell activity in relation to behavior via fiber photometry or microendoscopy6. Most recently, genome editing via CRISPR/Cas9 systems and developmental engineering can quickly produce knock-outs or knock-ins for multiple gene targets in a single generation10. As our review focuses primarily on advances made within the past 3 years, there is an overwhelming representation of these methodologies, which already have significantly advanced our understanding of the Hcrt circuit12.As new tools have become increasingly accessible to researchers at all levels, we have seen an explosion of studies using specific methodologies for the study of neural circuitry, namely the use of optogenetics and chemogenetics for the manipulation of neural circuits, fiber photometry and microendoscopy for the measurement of cellular activity via genetically encoded calcium indicators , and precise genetic tools  and high-throughput sequencing to characterize and manipulate genes. Optogenetics is a technique in which neurons are genetically modified to express light-sensitive ion channels . Subsequent photostimulation of these neurons can activate or inhibit cells on the basis of the wavelength and intensity of light used13. They receive major inputs from a diversity of afferents covering all of the major neurotransmitter systems14. The increasing database of research on Hcrt shows that these neuropeptides may not be necessary for the generation of sleep or wakefulness per se but rather for coordinating and stabilizing these states. Hcrt activity regulates sleep-to-wake transitions via its many interactions with other neuroanatomical and neurotransmitter systems16. Thus, many of the recent findings discussed here are a combination of studies done directly on Hcrt circuitry or studies done on other systems that either coordinate activity with or are modulated by Hcrt.Hcrt cell bodies reside exclusively within the hypothalamus and project broadly throughout the brain and spinal cord20. Narcolepsy is characterized by unexpected sleep episodes during times of wakefulness, excessive daytime sleepiness, rapid eye movement (REM)-like episodes that can co-occur with conscious wakefulness, and disrupted nocturnal sleep22. Further support for aberrant state boundaries in narcolepsy was recently published showing intrusions of REM sleep during wakefulness as well as intrusions of non-REM (NREM) sleep during wakefulness23. While it is established that Hcrt neuron degeneration contributes to the etiology of narcolepsy in many cases, recent evidence has characterized how sleep and wakefulness are impacted through the progression of Hcrt cell loss24. Studies in mice at different stages of Hcrt neuron degeneration found that loss of these neurons reduces the likelihood of long wake bouts but increases the likelihood of short wake bouts  as a result of waking primarily during the first 30 seconds of NREM sleep and a reduced likelihood of returning to sleep within the first 60 seconds of wakefulness24.Hcrt deficiency underlies the majority of cases of narcolepsy25. Optogenetic manipulations of Hcrt circuitry revealed that activation of this neuronal population induces wakefulness in mice while optogenetic inhibition promotes NREM sleep26. Likewise, chemogenetic studies targeting Hcrt neural activity have shown that injections of CNO in mice expressing excitatory (Gq) DREADDs promote wakefulness but that engagement of inhibitory (Gi) DREADDs decreases wakefulness and increases time in NREM sleep27. Thus, Hcrt clearly plays a critical role in the regulation of sleep-to-wake transitions, but its various effects on these processes are regulated by the many brain regions and neurotransmitter systems with which it interacts. Indeed, research has demonstrated important interactions between Hcrt and histaminergic neurons within the tuberomammillary nucleus (TMN), cholinergic and GABAergic neurons of the basal forebrain (BF), dopamine (DA) neurons within the ventral tegmental area (VTA), and norepinephrine (NE) neurons of the locus coeruleus (LC), among others28  show normal sleep-to-wake transitions upon optogenetic stimulation of Hcrt neurons36. These data suggest that histaminergic signaling in the TMN may serve a redundant function in Hcrt-mediated arousal. Recent findings also show that histaminergic regulation of wakefulness within the TMN may be via co-transmission of GABA. Small interfering RNA (siRNA)-mediated knockdown of the vesicular GABA transporter (VGAT) or genetic knockout of the VGAT gene in histaminergic neurons results in hyperactivity and sustained wakefulness37. Future studies should characterize how manipulations of GABA transmission in the TMN impacts Hcrt-induced wakefulness specifically.As we discuss below, histaminergic neurons of the TMN play a role in arousal, but the ways in which Hcrt influences TMN-mediated arousal are not clear. TMN histaminergic neurons become active during wake onset and are silent during sleep38. Similarly, the region expresses both Hcrt receptors, and there is a higher density of OX2R than OX1R39. This difference may be meaningful, as studies in organotypic slice cultures show that Hcrt depolarizes cholinergic cells of the BF via actions at OX2R but not OX1R38. However, injections of Ox-A into the BF of rats resulted in wakefulness in regions of the BF that show stronger expression of OX1R40. Chemogenetic studies demonstrate that activation of cholinergic neurons of the BF decreases electroencephalogram (EEG) delta power  and promotes cortical desynchronization without behavioral wakefulness41. In contrast, activation of GABAergic neurons in this region produces sustained wakefulness whereas inhibition increases NREM sleep42. Further genetic targeting studies show that subsets of GABAergic neurons in the region exhibit a diversity of responses across arousal states45. For example, parvalbumin-positive (PV+) GABAergic neurons are more active during wakefulness and REM sleep than during NREM sleep whereas somatostatin-positive (SOM+) GABAergic neurons are reciprocally silent during wakefulness. Predictably, optogenetic activation of PV+ GABA neurons powerfully induces wakefulness whereas activation of SOM+ GABAergic neurons promotes NREM sleep49. Modern genetic tools will continue to allow more detailed examinations of the impact of neuronal heterogeneity within regions in the context of Hcrt-mediated arousal.The BF is an attention- and arousal-sustaining structure containing cholinergic, GABAergic, and glutamatergic cells that are depolarized by Hcrt51.In vitro electrophysiological recordings show that Hcrt1 and Hcrt2 treatment increases VTA DA neural firing52. Hcrt1 injections into the VTA increase time awake and levels of DA at axonal terminals in the prefrontal cortex54. Although Hcrt neurons project to systems for all the monoamines and drugs that increase DA transmission increase wakefulness, DA was thought not to be involved in normal sleep/wake regulation until recently60. Work from our laboratory has shown a role for VTA DA neurons in promoting arousal and the initiation of sleep-preparatory behaviors61. Optogenetic activation of VTA DA neurons induces emergence from anesthesia, and chemogenetic activation of the VTA induces and consolidates wakefulness63. Further manipulations have demonstrated that VTA effects on wakefulness are through a D2 receptor-mediated mechanism63. Future work using projection-specific manipulations of Hcrt fibers within the VTA should better characterize their role in VTA-mediated arousal.The BF receives projections from midbrain DA neurons which may underlie the coupling of motivation to arousal states. Indeed, Hcrt axons project to midbrain DA neurons, and DA cell bodies express Hcrt receptors65. Direct administration of Hcrt1 into the LC increases firing rates while optogenetic silencing of these neurons with concurrent excitation of Hcrt cells prevents Hcrt-evoked sleep-to-wake transitions68. Additional studies have shown that noradrenergic activity is required to promote wakefulness and Hcrt-induced arousal in zebrafish. Using DA b-hydroxylase (dbh) (the rate-limiting enzyme in NE synthesis) mutant zebrafish, researchers found that these animals had dramatically increased sleep yet lower arousal thresholds69. Additionally, wakefulness induced by genetic overexpression of Hcrt and optogenetic activation of Hcrt neurons is blocked by the inhibition or knocking out of NE in zebrafish larvae69. However, further investigations have shown that overexpression of Hcrt or activation of Hcrt neurons has no significant effect in dbh mutant zebrafish35. Thus, future work should continue to parse out the roles in which NE functions in sleep/wake regulation and how it may serve specifically within the Hcrt circuit to help regulate wakefulness in particular.Noradrenergic neurons of the LC are strong promoters of arousal70. Indeed, measures of muscle tone along with cortical activity are the most common endpoints for characterizing various arousal states. A hallmark of waking is low-amplitude, high-frequency EEG activity with high muscle activity. REM sleep, also known as paradoxical sleep, is characterized by a near complete loss of skeletal muscle activity and an EEG resembling wakefulness. Hcrt-deficient narcoleptics show cataplexy 74. Similarly, individuals with REM sleep behavior disorder (RBD) show muscle tone problems. Under normal conditions, REM sleep is devoid of skeletal muscle tone; however, in RBD, an individual acts out their dreams by moving their limbs or talking, which can be dangerous for the individual enacting their dreams as well as anyone in their surroundings75. Noradrenergic activity is necessary for motor behavior76. Indeed, NE depletion has been shown to have a stronger motor-impairing effect than dopaminergic lesions with MPTP  infusions of NE-induced hyperactivity, and loss of NE neurons is associated with motor learning deficits in aged rats80. Likewise, increasing noradrenergic tone has been shown to reduce cataplectic episodes81. As discussed above, noradrenergic neurons of the LC are powerfully regulated by Hcrt; Hcrt dysfunction predictably alters both arousal and motor control. Moreover, Hcrt neurons project to dorsal raphe nucleus (DRN) serotonergic neurons where they may further influence motor behavior. Indeed, restoration of OX2R into serotonergic DRN neurons of dual Hcrt receptor knockout mice suppresses cataplexy-like episodes yet has no effect on sleep/wake fragmentation. Likewise, optogenetic stimulation of serotonergic DRN terminals in the amygdala (AMY) suppresses cataplexy-like arrests in Hcrt-deficient mice, and optogenetic inhibition blocks the cataplexy-reducing effect of Hcrt receptor restoration in serotonergic DRN neurons82. Additional chemogenetic manipulations of this amygdalar circuit show that GABAergic populations of the central AMY are responsible for the production of cataplexy in mice but may not be the only circuit that can drive emotionally driven cataplexy83. Together, these findings demonstrate a key role for amygdalar circuits in the production of cataplexy; however, they do not rule out other nuclei or circuits that may influence emotionally driven cataplexy. Indeed, the neural infrastructure exists for Hcrt activity to modulate AMY activity via its connections from the LC and DRN, and future studies should characterize the influence of Hcrt in emotion-driven cataplexy.Despite evidence demonstrating innervation of motor control systems by the Hcrt neurons, the coupling of arousal states with motor control is poorly understood84), and drug administration schedules  model of panic provocation89. Additional studies within the CO2 model that screened selective Hcrt receptor antagonists (SORAs) and dual Hcrt receptor antagonists (DORAs) found that both a SORA1 (compound 56) and a DORA-12 attenuate anxiety-like behaviors but that a SORA2 did not90. Importantly, these data provide a promising treatment route, as animals treated with SORA1 and DORA-12 showed no significant changes in sleep90. Currently, the levels of benzodiazepines needed to achieve anxiolytic effects are also sedating; as discussed here, OX1R antagonists can have anxiolytic effects without impacting sleep90.Hcrt plays a role in the coordination of stress responses. Plasticity in the Hcrt system is thought to contribute to long-term dysregulation of arousal seen in certain psychiatric disorders91. However, the benefits of modafinil treatment may go beyond this, as it has been shown to stimulate adaptive stress responses in an animal model of PTSD93. In a model of orofacial pain-induced anxiety, rats given injections of capsaicin into the upper lip showed increased anxiety-like responses on the elevated plus maze. Administration of Hcrt exacerbates this response while treatment with OX1R antagonists inhibits orofacial pain-associated anxiety94. In another study, differential effects of OX1R antagonism were observed. The OX1R antagonist SB-334867 influenced arousal (mobility/immobility in an open field) but not anxiety-like behavior (center exploration) in conditions of mild stress in male rats95. Yet Hcrt knockout mice show increased anxiety in the open-field test, light-dark box test, and predator scent avoidance test despite intact fear learning96. Likewise, OX1R receptor knockout mice show increased anxiety and reduced social interaction, increased startle responses, and altered depressive-like behavior97. Although genetic knockout results do not completely contradict findings from pharmacological studies, they do showcase the necessity to use the newest genetic techniques to parse out the role of Hcrt in anxiety. Two points must be made with regard to these findings: first, knockout models may result in compensatory mechanisms that may explain how Hcrt-null or OX1R-deficient mice display lower anxiety. Second, models of stress discussed here vary greatly, and the conclusions drawn from these works may reflect the differences in the circuits underlying different types of anxiety. Thus, findings must be interpreted within the context of pharmacological, genetic, and behavioral manipulations used in these studies.Although the mechanism of action of the wake-promoting drug modafinil is mainly through activation of DA circuitry, it also activates Hcrt neurons and is used for the treatment of narcolepsy. Treatment with modafinil after a traumatic experience reduces the incidence of post-traumatic stress disorder (PTSD), a disorder characterized by anxiety and hyperarousal. The anxiolytic effect of this treatment may be due to its interference with normal sleep-dependent memory processes98. Together, these data suggest that the activity of Hcrt on stress may be context or stressor specific but additionally that individual differences at baseline may influence stress resilience.Recent work is also characterizing how individual differences in baseline Hcrt activity may pose resilience or susceptibility to stress. Rats that show low expression of preprohypocretin mRNA are resilient to social stress, and further manipulations show that chemogenetic inhibition of Hcrt reduces depressive-like behavior in otherwise stress-susceptible rats100. These processes necessitate and evoke arousal states to monitor reinforcers and facilitate learning101. Reciprocally, motivational states impact arousal so as to facilitate the seeking of rewards and the avoidance of punishments103. As discussed above, LH-Hcrt neurons send excitatory projections to the VTA51. Thus, the VTA may be an optimal region by which Hcrt can influence motivated arousal states. The majority of recent advances made in this field have investigated the effects of Hcrt manipulations on motivation for cocaine and ethanol (EtOH). To date, these studies suggest that Hcrt1 plays a role in motivation for drug reward, especially when drug presentation is dependent on effortful responses on the part of the animal. Here, we discuss the role of Hcrt in addiction and motivation, focusing on cocaine, alcohol, and opioids.The mesolimbic DA system, which originates in the VTA and projects to the striatum, is a key region for the processing of reward and reinforcement104. Similarly, Hcrt-deficient mice show reduced cue-induced cocaine-seeking behavior following a period of abstinence, suggesting a role for Hcrt in relapse behavior105. Additionally, these animals show blunted cocaine intake at the highest dose and reduced behavioral responses to cocaine after abstinence105. Additional work from Navarro and colleagues further supports the role of Hcrt in relapse behavior106. In particular, their work shows that cocaine acts at and alters activity of corticotropin-releasing factor receptor (CRF1R)/OX1R heterodimers within the VTA. Action of cocaine at these sites disrupts Hcrt/CRF crosstalk even 24 hours after a single systemic injection and may be a mechanism underlying stress-induced cocaine relapse106.Hcrt knockdown attenuates cocaine self-administration under progressive ratio schedule  but not under a fixed ratio schedule1R antagonism via SB-334867 only decreases cocaine demand in the presence of cues. SB-334867 treatment also blocks cue-induced reinstatement of drug seeking—an effect most pronounced in high-demand animals . This suggests that OX1R increases the reinforcing efficacy of cocaine-associated cues but not of cocaine alone. This supports the notion that Hcrt plays a role in the ability of conditioned cues to elicit motivational responses107. Recentin vivo measurements of DA activity are beginning to inform the mechanisms that may underlie these observed effects on cocaine reinforcement. For example, Hcrt knockdown within the VTA delays acquisition of cocaine self-administration and reduces motivation for cocaine under a progressive ratio schedule while reducing DA release in the ventral striatum, DA uptake, and cocaine-induced DA reuptake inhibition at striatal terminals108. Similarly, OX1R blockade with RTIOX-276 attenuates motivation for cocaine and reduces the number of DA transients, DA release evoked by cocaine cues, and cocaine-induced DA reuptake inhibition as measured by fast scan cyclic voltammetry (FSCV)109. Suvorexant, a DORA, attenuates the motivational properties of cocaine as measured by progressive ratio and place conditioning. Additionally, treatment with Suvorexant also reduces the hedonic properties of cocaine as measured by ultrasonic vocalizations. Additionally, DORA treatment reduced cocaine-induced elevations in ventral striatal DA110. Work by Prince and colleagues suggests that effects of the DORA may be mediated by OX1R, as blockade of OX2R receptors alone has no effect on DA signaling or self-administration of cocaine111. However, blocking of OX1R or both OX1R and OX2R decreases motivation for cocaine as measured by self-administration under a progressive ratio schedule and reduces the effects of cocaine on DA signaling as measured by FSCV111.Indeed, Hcrt may play a unique role in cue-reward associations, as OX112. OX1R antagonism with SB-334867 reduces EtOH self-administration in alcohol-preferring rats113. Similarly, the OX1R antagonist GSK1059865 reduces EtOH drinking in EtOH-dependent mice114. In a model of EtOH seeking and preference, activation of the LH is correlated with degree of seeking in context-induced reinstatement and degree of preference in home cage EtOH preference testing. Interestingly, cue-evoked reinstatement shows no correlation with Hcrt activation in any region. This suggests that there is a relationship between Hcrt activity in the LH and EtOH seeking and preference behavior but that cue-induced reinstatement for alcohol may be mediated by a different mechanism115. Interestingly, EtOH consumption increases OX2R mRNA within the anterior paraventricular nucleus of the thalamus and local antagonism of OX2R reduces total EtOH intake116.In the case of EtOH, Hcrt antagonism generally reduces EtOH consumption. In a voluntary EtOH intake model in zebrafish, it was seen that intake of EtOH increases Hcrt expression in the hypothalamus117. The interactions of these neurotransmitters are beyond the scope of this review; however, of major relevance is the point that these neurotransmitters have opposing yet complementary actions on VTA cellular excitability121. OX1R antagonism with SB-332867 modulates demand for the opioid drug remifentanil in low takers but not in high takers122. Additionally, intra-VTA injections of the OX1R antagonist SB-334867 attenuate morphine conditioned place preference (CPP) acquisition and expression. Interestingly, in the case of opioid reward, OX2R antagonism via TCS-OX2-29 also significantly attenuates morphine CPP acquisition and expression, suggesting that both receptors within the VTA are important for expression of morphine reward123. Similarly, systemic treatment with the OX2R antagonist NBI-80713 dose-dependently reduces heroin self-administration in a long-access paradigm. Long-access heroin self-administration paradigms are thought to mimic compulsive drug taking; thus, OX2R antagonism may be particularly effective at influencing drug-associated compulsivity. Similar effects have been observed in the hippocampal dentate gyrus (DG), which receives Hcrt projections from the LH and interacts with the VTA to play an important role in the linking of drug reward with contextual cues124. In a stress- and drug-induced model of morphine reinstatement, intra-DG administration of OX1R and OX2R antagonists attenuates drug priming-induced reinstatement dose-dependently with no effect on stress-induced reinstatement125. Similarly, morphine CPP increases Hcrt1 release in the DG and OX1R antagonism via SB-334867 ameliorates morphine CPP. These findings suggest that Hcrt actions at the DG may influence the learning of drug-context associations126.The interactions of Hcrt with opioid rewards are particularly interesting, as the endogenous opioid dynorphin (Dyn) is expressed in 94% of Hcrt neurons and Hcrt and Dyn are thought to be co-released at Hcrt terminals within the VTA127. Blomeley and colleagues used optogenetics and electrophysiology to characterize a direct Hcrt→DA D2 excitatory circuit that is necessary for the expression of risk avoidance behavior in mice127. Indeed, increased DA D2 neuron activation caused animals to avoid risks such as crossing a predator-scented chamber to attain a food reward and chemogenetic silencing of accumbal DA D2 cells inhibited Hcrt-mediated avoidance. Importantly, these data showcase how Hcrt can influence adaptive behavioral inhibition even in the presence of rewards. These data open up new opportunities of research, such as characterizing the effects of Hcrt on different subregions of the NAc, which is a heterogeneous structure with distinct electrophysiological properties129. Additional lines of research should investigate how Hcrt-mediated motivation in the NAc is impacted by diurnal rhythms as well as sleep disturbance and how the Dyn system interacts in this region to modulate motivation130.Finally, additional work has begun to delineate the effect of Hcrt on motivation at VTA terminal sites such as the nucleus accumbens (NAc)131. Hcrt/ataxin-3 transgenic mice (a progressive model of narcolepsy), which become Hcrt deficient at 12 weeks old, show impaired avoidance memory in a two-way active avoidance paradigm in which an animal has to perform a specific motor response to avoid an aversive stimulus. Hcrt1 administration reverses memory deficits, suggesting that Hcrt plays a role in hippocampal-dependent consolidation of two-way active avoidance memory132. Chemogenetic activation of Hcrt neurons improves short-term memory for novel locations, a function that putatively supports foraging and exploration133.Studies suggest that Hcrt deficiency is associated with memory deficits. Hcrt deficiencies negatively impact working memory as tested in a non-matching-to-place T-maze task1R antagonist SB-334867 whereas administration of Hcrt1 prevented these spatial memory deficits134. Importantly, injections were directed at the trigeminal nucleus caudalis, which is a central relay for orofacial pain. Thus, the observed effect on memory may be via alterations in the experience of pain itself rather than the formation of a pain-associated memory134. In a similar study by Raoof and colleagues, orofacial pain memory was mediated by Hcrt at the level of the hippocampus (HPC). Intra-hippocampal injections of Hcrt1 inhibit pain-induced memory impairments as measured by the MWM. However, treatment with the OX1R antagonist SB-334867 had no effect on learning and memory135. Indeed, the HPC is a critical region for memory function and Hcrt action at this site may influence memory processes via its influence on the induction of long-term potentiation (LTP).In vitro studies show that OX1R antagonists significantly decrease the firing rates of hippocampal CA1 neurons, showing that the effect of Hcrt on these neurons is excitatory136. Additionalin vitro electrophysiology studies demonstrate that Hcrt1 may bidirectionally modulate HPC CA1 function. Specifically, moderate doses of Hcrt1 inhibit LTP while subnanomolar concentrations result in re-potentiation via OX1R and OX2R137. It is important to note that the Hcrt manipulations discussed here may have influenced sleep and therefore resulting memory problems may be sleep dependent and thus only indirectly dependent on Hcrt.Pain negatively influences memory processing in ways that may be influenced by Hcrt. In the Morris water maze (MWM) , orofacial pain-induced memory impairments are exacerbated by the OXComputational modeling of the Hcrt network remains a relatively unexplored frontier. Development of analytical models of Hcrt function will inform our interpretation of data gathered through empirical study and drive the development of testable hypotheses. In particular, computational modeling of Hcrt networks will prove essential for our understanding of the following three questions: (1) how do internal or external physiological states influence arousal? (2) How does the heterogeneity of the system  contribute to network dynamics? (3) How does Hcrt function as a volume transmitter to produce both generalized and specific effects? Ultimately, integration of these models with experimental approaches will allow for understanding of the network as a whole as well as monosynaptic interactions.138. This model, however, cannot account for overlapping states of arousal such as those observed in narcolepsy or RBD in which REM sleep can co-occur with conscious awareness140. Additionally, this model does not factor in the many systems that interact to influence arousal. These observations make it necessary to revise the binary nature of the flip/flop model. Studies have expanded the model by characterizing a circuit with hierarchical gating of additional neural circuits, feedback, and redundancy141. This hierarchical model provides a framework on which to add motivational influences on arousal states. Indeed, animals can adapt their sleep on the basis of internal and external variables such as migration or predator avoidance or to increase the likelihood of mating144. Recently, an alternative has been proposed in which sleep-to-wake transitions are predicted on the basis of inputs with different “weights” onto an integrator neuron145. An integrator neuron would continuously compute probabilities of wakefulness on the basis of functional connectivity of the system as well as physiological factors such as stress or circadian phase. Diversity of neuronal responses to stimuli can be integrated within this model to account for the heterogeneity of the system. In this vein, Schöne and Burdakov acknowledge the necessity of an adaptive behavioral control system that can respond to unpredictable changes in the environment146. Thus, they propose a model of brain arousal control modules organized in a feedback loop by which Hcrt can gate relevant information on the basis of environmental and homeostatic needs146. We look forward to the future advancement of this area of Hcrt research that will undoubtedly expand our understanding as an adaptable regulator of arousal.Current models have described Hcrt as functioning within a “flip/flop” model where it stabilizes wakefulness, preventing aberrant switches between mutually exclusive states148. This type of release is thought to allow for modulation of neural activity via long time courses and greater distances149. VT may happen via cellular pores, diffusion through the plasma membrane, exocytosis, or reversal of transporter proteins149. To date, actions of Hcrt at the dorsal lateral geniculate nucleus (DLG) and the DRN  have been theorized to be exerted via VT151. Observations of Hcrt1 immunoreactivity in many non-synaptic varicosities located far from synapses with axons forming asymmetric synapses suggest that DRN excitation via Hcrt1 may be via this mechanism150. Indeed, the DRN plays an important role in the regulation of arousal and both synaptic and VT mechanisms may support long-term cortical arousal36. In a separate set of findings, Hcrt was found to powerfully modulate neurons of the DLG despite only sparse expression of Hcrt nerve terminals in the region, suggesting that these actions are via VT151. Additionally, a recent study of melanin-concentrating hormone (MCH), a hypothalamic peptide important for the regulation of feeding, shows that MCH neurons project to ventricular regions where they increase MCH levels in the cerebrospinal fluid (CSF) and stimulate feeding152. MCH neurons are intermingled with Hcrt neurons in the LH, and the authors measure that 40% of Hcrt neurons also project to the CSF where they are poised to signal via VT to influence distal targets152. Further investigations should determine whether Hcrt acts via VT and, if so, how its activity is influenced by (1) temporal and spatial release dynamics, (2) diffusion and dilution parameters, and (3) transporter kinetics in order to characterize its effective radius.Volume transmission (VT) is a mechanism of neural signaling by which neurotransmitters can exert actions on cells in close proximity as well as distant targets. In VT, neurotransmitters signal via diffusion within extracellular fluidAs reviewed here, the ever-growing database on Hcrt continues to broaden our conceptualization of these peptides as more than just regulators of sleep-to-wake transitions. Technical advances have allowed ever more precise measurement and manipulation of these circuits which will continue to inform our understanding of this circuit. To date, therapeutic advances have allowed the effective targeting of Hcrt circuitry for the treatment of narcolepsy and insomnia, and research discussed here provides evidence for the potential of this system for the treatment of anxiety, addiction, and memory deficits. Integration of these findings with analytical models will provide a novel means for explaining and interpreting biological observations so as to gain a holistic understanding of their role in physiology and behavior.


Lung ultrasound may be a reasonable alternative to chest radiography for the identification of acute respiratory distress syndrome (ARDS), but the diagnostic performance of lung ultrasound for ARDS is uncertain. We therefore analyzed the clinical outcomes of ARDS diagnosed according to the Berlin Definition, using either chest radiography (Berlin-CXR) or lung ultrasound (Berlin-LUS) as an alternative imaging method.This was a retrospective observational study in a 20-bed medical intensive care unit (ICU). Patients who required noninvasive ventilation or invasive ventilation for hypoxemic respiratory failure on ICU admission from August 2014 to March 2017 were included. Both chest radiography and lung ultrasound were performed routinely upon ICU admission. Comparisons were made using either the Berlin-CXR or Berlin-LUS definitions to diagnose ARDS with respect to the patient characteristics and clinical outcomes for each definition. ICU and hospital mortality were the main outcome measures for both definitions.P = 0.041; hospital mortality 34.2% versus 24.2%, P = 0.027). Compared with Berlin-CXR, Berlin-LUS had a positive predictive value of 0.66  and a negative predictive value of 0.71 (0.65–0.77). Among the 216 Berlin-CXR ARDS patients, 150 patients (69.4%) also fulfilled Berlin-LUS definition.The first admissions of 456 distinct patients were analyzed. Compared with the 216 patients who met the Berlin-CXR definition , 229 patients who met the Berlin-LUS definition  and 79 patients who met the Berlin-LUS but not the Berlin-CXR definition  had similar outcomes. In contrast, the 295 patients who met either definition had higher mortality than the 161 patients who did not meet either definition  contains supplementary material, which is available to authorized users. Acute respiratory distress syndrome (ARDS) has high mortality and requires specialized therapy. The current diagnostic criteria for ARDS follows the Berlin definition, which requires chest radiography or computed tomography for determination of bilateral pulmonary infiltrates . AlternaChest radiography has certain drawbacks. Firstly, chest radiography has poor sensitivity for the detection of pulmonary infiltrates compared with other imaging modalities such as computed tomography . SecondlLung ultrasound may be a useful alternative to chest radiography for the identification of ARDS. It has been established as an important tool for the evaluation of critically ill patients, with higher sensitivity and specificity than chest radiography for the diagnosis of pneumonia, using computed tomography as the reference standard , 7. It hApart from a small pilot study  and a reThis study involved a retrospective analysis of patients who required noninvasive ventilation or invasive ventilation for hypoxemic respiratory failure , on admission to our 20-bed medical ICU within our 1200-bed tertiary hospital from August 2014 to March 2017 (32 months). Only the first admissions to ICU during the study period for patients whose diagnosis of respiratory failure was within 1 week of a known clinical insult or new/worsening respiratory symptoms, not fully explained by heart failure or fluid overload, were included for analysis. Chest radiography, lung ultrasound, and arterial blood gas measurements were performed routinely upon ICU admission, all within 60 min, and on the same ventilator settings. Ethical approval for the study was obtained from the National Healthcare Group Domain-Specific Review Board (DSRB reference 2013/00132). The need for informed consent was waived.Data were obtained from the hospital case notes and ICU electronic patient records . Patient demographics, main diagnoses from the ICU admission notes, comorbid conditions, the source of ICU admission , inspired oxygen fraction, arterial blood gases, chest radiography results, and lung ultrasound results were collected at the time of ICU admission. Patients on noninvasive ventilation had positive end-expiratory pressure values recorded at the time of ICU admission. Patients on invasive mechanical ventilation had plateau pressures and positive end-expiratory pressure values recorded at the time of ICU admission. Clinical outcome data collected included ventilator-free days through 28 days, ICU length-of-stay, hospital length-of-stay, ICU mortality, and hospital mortality.ARDS was defined in two ways: using a combination of the Berlin definition with chest radiography (Berlin-CXR) or modified using lung ultrasound as an alternative imaging method (Berlin-LUS). For both definitions, diagnosis of respiratory failure was within 1 week of a known clinical insult or new/worsening respiratory symptoms, not fully explained by heart failure or fluid overload. For both ARDS definitions, patients on noninvasive ventilation could be classified as having mild ARDS but not moderate or severe ARDS . With rePortable chest radiography was routinely performed at the bedside by the on-call radiographer, with images reported within a few hours by a radiologist who had no direct contact with patients. All radiological interpretations and confirmation of reports were performed by specialist radiologists in our hospital.Lung ultrasound was also routinely performed at the bedside using the Sparq Ultrasound System  equipped with a 2–4 MHz broadband sector phased array transducer, and concurrently reported using a reporting template within the ICU electronic patient record by trained respiratory therapists  with respect to patient characteristics, treatment parameters, and clinical outcomes. Imaging findings of patients who met the Berlin-CXR but not the Berlin-LUS definition, and vice versa, were described.Univariate comparisons of proportions, means, and medians were respectively performed using the Pearson chi-squared, Student To analyze the appropriateness of using at least one region affected by multiple B lines (> 2 B lines per region) or consolidation to define ARDS, the lung ultrasound criterion for ARDS was increased progressively from at least one region to all six regions of each hemithorax affected by multiple B lines (> 2 B lines per region) or consolidation, and the clinical outcomes of patients identified to have ARDS were described. The diagnostic accuracy of various lung ultrasound-based definitions was then tested against Berlin-CXR as the reference standard for ARDS.During the 32-month study period, out of 1677 ICU admissions of 1550 patients, the first admissions of 456 distinct patients were analyzed  patients met the Berlin-CXR but not the Berlin-LUS definition Table . Among tWith regard to initial treatment parameters, those who had ARDS required more noninvasive ventilation support, higher initial positive end-expiratory pressure, and higher initial plateau pressure compared with patients who did not have ARDS  or consolidation on lung ultrasound  were slightly lower than those reported in the LUNG SAFE study , which could be due to a greater proportion of mild ARDS in our study (39.4%) compared with the LUNG SAFE study (30.0%).Among 1550 patients admitted to our ICU within the study period, 216 patients met the Berlin-CXR definition for ARDS. This proportion of 13.9% was slightly higher than the 10% found in the Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG SAFE) , which mIn our study, as for the Kigali Modification, we considered sonographic consolidation in addition to B lines since the former could also be a pathologic entity for ARDS , 8. AddiAnother reason for the limited concordance between chest radiography and lung ultrasound is that lung ultrasound could only detect abnormalities in contact with the visceral pleura, while chest radiography could detect parenchymal abnormalities away from the pleura  . AdditioChest radiography interpretation could also be falsely positive in ARDS due to the limited reliability of chest radiography, even when read by experts. In a prior study involving 21 international experts, the chance-corrected interobserver agreement was moderate (kappa 0.55) . In anotWhile crafting the definition for ARDS, the Berlin definition workgroup primarily aimed to develop a robust and replicable definition. The workgroup secondarily supported their consensus definition using predictive validity for patient mortality. This means that, among patients with respiratory failure, the definition for ARDS should identify patients who are at higher risk of death. In their landmark paper for the Berlin definition, the workgroup found that the Berlin definition was not only able to identify patients with higher mortality but was further able to stratify patients with increasing mortality according to worsening oxygenation. We found similar results when we used the Berlin definition for our patients: the definition predicted hospital mortality, and patients with severe ARDS had worse mortality compared with patients with mild ARDS.P = 0.141) and positive end-expiratory pressure was only slightly lower .As for the Berlin definition, we found that modifying the chest imaging criterion—by replacing chest radiography with lung ultrasound—could also predict worse patient outcomes, with increased ICU mortality and increased ICU length-of-stay achieving statistical significance. Hospital mortality for patients also trended higher but did not achieve statistical significance, probably due to a limited sample size. As shown in Table Interestingly, in our study  resulted in a 37% increase in the number of ARDS cases over the Berlin-CXR definition alone while maintaining a specificity of 67%. Identification of patients with ARDS using lung ultrasound, who would otherwise have been missed using chest radiography, could allow for appropriate disposition and treatment, and could increase the number of patients who may be recruited for research.The main strength of our study was the availability of an established practice of performing both chest radiography and systematic lung ultrasound for patients on admission. This allowed us to perform a comparative analysis using various definitions for ARDS. Another strength is the larger number of patients in our study compared with prior published work on lung ultrasound in ARDS , 12.Our study had some limitations. Firstly, we did not adjudicate the chest radiography and lung ultrasound reports that have been reported by specialist radiologists and our trained respiratory therapists, respectively. It is likely that interobserver variability within each imaging modality would exist  and thatFurther work to be done includes testing the different definitions for ARDS in other settings and for patients with nonmedical conditions. Future studies could also test the predictive validity, feasibility, clinical benefit, and epidemiological impact of a combined chest imaging approach using both chest radiography and lung ultrasound.In conclusion, for the identification of ARDS with the Berlin definition, both chest radiography and lung ultrasound were equally related to mortality. The moderate overlap of patients when chest imaging modalities differed suggest that chest radiography and lung ultrasound should be complementary rather than used interchangeably.Additional file 1:Table S1. Lung ultrasound reporting template. (DOCX 18 kb)Additional file 2:Table S2. Treatment parameters and clinical outcomes using various definitions of acute respiratory distress syndrome. (DOCX 21 kb)Additional file 3:Table S3. Comparative analysis using lung ultrasound findings for ARDS identification. (DOCX 23 kb)Additional file 4:Table S4. Diagnostic accuracy using various definitions of acute respiratory distress syndrome compared with Berlin-CXR. (DOCX 22 kb)