diff --git "a/deduped/dedup_0840.jsonl" "b/deduped/dedup_0840.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0840.jsonl" @@ -0,0 +1,43 @@ +{"text": "The web-based collection of images is housed on the ASC website, which went live on November 5 The second edition of the Bethesda 'blue book' atlas maintains an easy to read format with bulleted morphologic criteria and half-page color illustrations. The content has been divided into chapters based on the major 2001 Bethesda System interpretive categories. Highlights of the new edition include: (1) incorporation of liquid based cytomorphologic criteria and images; (2) new sections addressing ancillary testing, educational notes and recommendations, computer assisted interpretation and anal-rectal cytology; and (3) inclusion of sample reports, references, and detailed legends for images. Overall, the second edition has tripled in size from the original version, with a total of 186 images of which 90% are new images and 40% are from liquid based specimens. Some are classic examples of an entity while others have been selected to illustrate interpretive dilemmas or \"borderline\" cytomorphologic features that may not be interpreted in the same way by all cytologists.In parallel with production of the Bethesda book atlas, the ASC-NCI task force has also developed a Bethesda web-based collection of images. Approximately 350 images, (40% of which are from liquid based specimens) along with linked explanatory notes, including all the images in the published atlas, can be viewed on this site. The website is user friendly and has several search modalities for viewing the images including searching by Bethesda terminology, atlas chapter headings, keyword(s), or preparation type. It also allows for individual self-assessment by participating in a \"self test\" in which viewers can compare their interpretation to other participants' responses.Step 1: individual Bethesda forum group members (32 participants) reviewed and selected images for their chapter from among those in the first edition of the atlas and new submissions; and Step 2: the images selected from Step 1 were reviewed individually by 13 task force members and scored on a scale of 1\u20135 for agreement with interpretation and quality of image. In all over 1000 images were reviewed of which 186 images were selected for the atlas and an additional 163 for the website.The image selection process for the book atlas and website involved a multistage review: A subset (n = 77) of the book atlas images were posted as \"unknowns\" on the Internet from mid July to mid September 2003 as part of a study \u2013 the Bethesda Interobserver Reproducibility Project (BIRP). The site was open to the cytopathology community to view the images and provide their interpretations. Immediately after submitting their response, participants were able to view a histogram of the distribution of results submitted by all prior participants for that image. Over 600 cytologists from around the world participated in BIRP. Summary histograms for each of the 77 images can be viewed on the Bethesda atlas website (select BIRP images from the left menu). Preliminary BIRP results presented at the ASC annual meeting in Orlando in November 2003 showed that Negative for Intraepithelial Lesion or Malignancy (NILM) and Low Grade Squamous Intraepithelial Lesion (LSIL) reference images attained the highest concordance scores, while glandular abnormalities demonstrated the most splay in distribution of interpretations. BIRP analyses are ongoing and further results should be available in 2004.ASC Bethesda 2001 Task Force:Ritu Nayar (Chair), Diane Solomon George Birdsong (Adequacy), Jamie Covell (Glandular Lesions), Ann Moriarty , Dennis O'Connor , Marianne Prey (Computer assisted interpretation), Steve Raab (Ancillary testing), Mark Sherman , Sana Tabbara , Tom Wright (Squamous lesions), Nancy Young (Non-neoplastic findings).ASC Consultants: ASC 2002/2003 Presidents: Diane Davey and Dave Wilbur.Information Technology representatives: Mike Montgomery (NCI) and Brandon Winbush (Northwestern University), Aquilent .The second edition of the 'blue book' atlas can be ordered through Springer-Verlag (1-800-SPRINGE) for $34.95."} +{"text": "The piperidine N atom deviates by 0.128\u2005(1)\u2005\u00c5 from the plane through its three neighbouring atoms. In the crystal structure, mol\u00adecules are connected by inter\u00admolecular Cethyn\u00adyl\u2014H\u22efO contacts to form chains extending in the [10The six-membered ring of the title compound, C \u00c5 b = 20.800 (3) \u00c5 c = 8.3662 (13) \u00c5 \u03b2 = 97.434 (10)\u00b0V = 1052.5 (3) \u00c53 Z = 4 K\u03b1 radiationMo \u22121 \u03bc = 0.07 mmT = 167 K 0.60 \u00d7 0.50 \u00d7 0.50 mm Siemens SMART 1K CCD diffractometerAbsorption correction: none18416 measured reflections3580 independent reflectionsI > 2\u03c3(I)3143 reflections with R int = 0.039 R[F 2 > 2\u03c3(F 2)] = 0.040 wR(F 2) = 0.112 S = 1.09 3580 reflections131 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.34 e \u00c5\u22123 \u0394\u03c1min = \u22120.17 e \u00c5\u22123 \u0394\u03c1 SMART (Siemens, 1995SMART; data reduction: SAINT (Siemens, 1995SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008SHELXTL (Sheldrick, 2008SHELXL97.Data collection: 10.1107/S1600536809004681/su2096sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809004681/su2096Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "The N\u2014O bond is in the plane of the five-membered ring. The mol\u00adecule is positioned about a pseudo-mirror plane at y = 0.375. In the crystal, mol\u00adecules are connected by inter\u00admolecular C\u2014H\u22efO contacts into layers parallel to (010).The five-membered ring of the title compound, C \u00c5 b = 19.058 (4) \u00c5 c = 6.5989 (11) \u00c5 \u03b2 = 104.333 (14)\u00b0V = 966.6 (3) \u00c53 Z = 4 K\u03b1 radiationMo \u22121 \u03bc = 0.07 mmT = 167 K 0.60 \u00d7 0.55 \u00d7 0.07 mm Siemens SMART 1K CCD diffractometerSADABS; Sheldrick, 2000T min = 0.870, T max = 0.995Absorption correction: multi-scan (16848 measured reflections3301 independent reflectionsI > 2\u03c3(I)2214 reflections with R int = 0.035 R[F 2 > 2\u03c3(F 2)] = 0.062 wR(F 2) = 0.157 S = 1.19 3301 reflections121 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.44 e \u00c5\u22123 \u0394\u03c1min = \u22120.23 e \u00c5\u22123 \u0394\u03c1 SMART (Siemens, 1995SMART; data reduction: SAINT (Siemens, 1995SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008SHELXTL (Sheldrick, 2008SHELXL97.Data collection: 10.1107/S1600536809026725/nc2152sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809026725/nc2152Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:"} +{"text": "Using dual-focus fluorescence correlation spectroscopy, we have analyzed the adsorption of three human blood serum proteins, namely serum albumin, apolipoprotein A-I and apolipoprotein E4, onto polymer-coated, fluorescently labeled FePt nanoparticles (~12 nm diameter) carrying negatively charged carboxyl groups on their surface. For all three proteins, a step-wise increase in hydrodynamic radius with protein concentration was observed, strongly suggesting the formation of protein monolayers that enclose the nanoparticles. Consistent with this interpretation, the absolute increase in hydrodynamic radius can be correlated with the molecular shapes of the proteins known from X-ray crystallography and solution experiments, indicating that the proteins bind on the nanoparticles in specific orientations. The equilibrium dissociation coefficients, measuring the affinity of the proteins to the nanoparticles, were observed to differ by almost four orders of magnitude. These variations can be understood in terms of the electrostatic properties of the proteins. From structure-based calculations of the surface potentials, positively charged patches of different extents can be revealed, through which the proteins interact electrostatically with the negatively charged nanoparticle surfaces. Recent years have seen enormous advances in the field of nanotechnology. A huge variety of nanoparticles (NPs), defined as objects with all three spatial dimensions in the range of 1\u2013100 nm, has been developed, with well-controlled physicochemical properties including size, shape, charge, chemical composition and solubility. Many of these NPs have already found their way into consumer products.Owing to their small size, NPs may potentially invade all parts of the human body including tissues, cells and even subcellular compartments. Consequently, they hold great promise as tools for biomedical applications such as targeted drug delivery or gene Upon incorporation into the body, NPs become exposed to biological fluids such as lung epithelial lining fluid or blood plasma, which contain a variety of dissolved molecules, especially proteins. Depending on the properties of its surface, a NP may adsorb proteins and other biomolecules from the fluid to a lesser or greater extent. A protein coating layer, the so-called \u2018protein corona\u2019, forms and can completely enshroud the NP \u201311. ConsTo be able to control the biological effects of NPs, such as prevention of uptake or targeted delivery to specific cells or tissues, it is of utmost importance to understand the structural and dynamic properties of the protein corona at the molecular level. Recently, we have used quantitative fluorescence microscopy, especially fluorescence correlation spectroscopy (FCS), to study protein adsorption of human serum albumin (HSA) on polymer-coated FePt NPs with an overall diameter of 11 nm . We foun\u03c4D. Based on the well-known spatial extension of the observation volume, the diffusion coefficient, D, and, by using the Stokes\u2013Einstein equation , the hydrodynamic radius of the fluorescent particle, RH, can be calculated. Consequently, a NP size increase due to protein adsorption onto the NP surfaces can be measured via an increase of \u03c4D. Knowledge of the molecule detection function (MDF), i.e., the probability to detect a fluorescence photon from a molecule at a given position in the sample volume, is key to the precise quantitative analysis of an FCS experiment , by the Hill equation [where the maximum number of proteins binding to the NP is denoted by equation ,K\u2019D, denotes the midpoint of the transition, i.e., the concentration of protein molecules free in the solution at half coverage. It quantifies the strength of the NP\u2013protein interaction. The Hill coefficient, n, controls the steepness of the curve; it contains information about the cooperativity of binding. The lines in RH(0) = (6.0 \u00b1 0.1) nm, was taken as a global parameter in the fit for all three proteins. The best-fit parameters in Here, the equilibrium dissociation coefficient, RH, is a characteristic of the particular protein species adsorbed. In our previous studies with HSA [Comparison of the data in with HSA and tranwith HSA , we notiLipid-free human apoA-I is the principal component of high-density lipoprotein (HDL) and plays an essential role in lipid transport and metabolism. This protein has a molecular mass of 28 kDa. X-ray crystallography revealed a two-domain structure, with a N-terminal domain forming a four-helix bundle and a structurally less well organized C-terminal domain , left 3\u201334. In sHuman apoE4 is another member of the family of soluble apolipoproteins . The 34 RH for the three proteins studied here, by ~6 nm = 0.021 \u00b1 0.003 \u00b5M and K\u2019D (apoA-I) = 140 \u00b1 60 \u00b5M . They carry carboxylic acid groups on their surfaces, making them water-soluble. The polymer shell was labeled with the amino-modified fluorescent dye DY-636 .FePt NP cores were synthesized according to published protocols and coat2+ and Mg2+, PAA Labs, C\u00f6lbe, Germany). All proteins were purchased from Sigma . NP solutions at (1 \u00b1 0.5) nM were mixed with equal volumes of solutions containing the proteins at varying concentrations. Because of the high affinity of apoE4 to the FePt NPs, the NP concentration was reduced to (0.1 \u00b1 0.05) nM to ensure that only a small fraction of apoE4 proteins was bound to the NPs even at the lowest protein concentrations studied. All protein solutions were prepared by repeated dilution of a single stock solution. The apoE4 dilution series was prepared 2 h before mixing with the NPs to allow the sample to equilibrate between monomers and oligomers [2fFCS measurements were performed in PBS buffer, pH 7.4 . Instead of using a single excitation laser, the light from two identical, orthogonally polarized pulsed 640 nm diode lasers was combined by a polarizing beam splitter . The lasFor data collection, a few microliters of the sample solution were placed between two standard microscope cover slips separated by a 200 \u00b5m thick mylar foil with a 1 mm wide channel for the sample solution in the middle.D, according to the Stokes\u2013Einstein relation,Samples were illuminated continuously for 8 min, with the power of each laser adjusted to 3 \u00b5W. For NP concentrations of 1 nM (0.1 nM), ~10,000 single molecule bursts were analyzed. The temperature was measured during the experiments and accounted for in the determination of the diffusion coefficient, RH, Boltzmann constant kB, temperature T, and viscosity \u03b7. Three independent series of measurements were taken and averaged.with hydrodynamic radius x,y-)plane along the optical axis, z, the MDF is, for both foci, modeled by a two-dimensional Gaussian function, U, of width w(z) and amplitude \u03ba(z)/w2(z),In conventional FCS, the MDF is typically approximated by a three-dimensional Gaussian profile. However, this assumption is rather crude. In 2fFCS, data fitting is facilitated by a new, semi-empirical two-parameter model describing the MDF . In eachwith\u03bbexc and \u03bbem are the excitation and the center emission wavelengths, respectively; n is the refractive index of the immersion medium and a is the radius of the confocal aperture divided by the magnification. R0 and w0 are a priori unknown model parameters that are determined by the fit.In these equations, As the emitted photons are registered as a function of time, they can be assigned to one of the two foci. Therefore, three correlation functions can be calculated from the data from each of the two foci, that is, the two auto-correlation functions and the cross-correlation function. Actually, a cross-correlation between two detectors is also performed to calculate the autocorrelation functions, so to avoid afterpulsing artifacts of the APDs. All three correlation functions are fitted globally, according to\u03b51 and \u03b52 take the proper weighting of the two polarization channels, due to the different excitation powers and detection efficiencies, into account. For the auto-correlation curves, the spatial separation of the two foci, \u03b4, is set to zero, and \u03b51\u03b52 is replaced by \u03b512 or \u03b522.The coefficients 3/g, as specified by the supplier , and of apoA-I (9.2 cm3/g) [The correlation analysis was performed with the SymphoTime software (PicoQuant). FCS experiments are notoriously sensitive to the presence of large aggregates, therefore, those parts of the time traces that showed excessively high intensities were excluded from the correlation analysis. Changes in viscosity due to the increasing protein concentration were taken into account by using a linear approximation for the contribution of the solute to the solution viscosity, based on the intrinsic viscosity of HSA of 4.2 cm2 cm3/g) . The vis"} +{"text": "Ligation-assisted endoscopic enucleation (EE-L) was developed for the pathological diagnosis and resection of small gastrointestinal tumors originating from the muscularis propria. The technique combines endoscopic band ligation and endoscopic enucleation. The aim of this study was to evaluate the efficacy and safety of EE-L in the diagnosis and resection of gastrointestinal tumors originating from the muscularis propria.A total of 43 patients were eligible for inclusion in this study from June 2009 to June 2011. Endoscopic ligation was first performed to force the tumor to assume a polypoid form with a pseudostalk. EE-L was then performed until the tumor was completely enucleated from the muscularis propria. Wound closure was performed using clips and adhesive tissue.All 43 tumors were completely enucleated. The mean enucleation time was 7.2 minutes . No perforation, massive hemorrhage, or peritonitis requiring further endoscopic or surgical intervention occurred. Histopathology, 19 lesions were identified as gastrointestinal stromal tumors and 24 lesions were identified as leiomyomas. The mean follow-up time was 20.4 months . No recurrence has occurred during the follow-up period.EE-L appears to be a safe, effective, and relatively simple method for the histologic diagnosis and removal of small gastrointestinal tumors originating from the muscularis propria. Some gastrointestinal tumors originating from the muscularis propria, such as gastrointestinal stromal tumors (GISTs), may be nonmalignant when diagnosed but have the potential to undergo malignant transformation. A majority of patients with gastrointestinal lesions originating from the muscularis propria prefer to undergo resection despite controversies over therapeutic decisions. Several endoscopic resection techniques have been proven feasible and safe for tumors originating from the muscularis propria, including endoscopic submucosal dissection -4, endosLigation-assisted endoscopic enucleation (EE-L) was developed by combining endoscopic band ligation and endoscopic enucleation to fully exploit the advantages of each technique. The present study investigated the efficacy and safety of EE-L in the diagnosis and resection of gastrointestinal tumors originating from the muscularis propria.Patients who underwent EE-L for gastrointestinal tumors originating from the muscularis propria at Shengjing Hospital of China Medical University from June 2009 to June 2011 were enrolled in this study. To be included in the study, the tumors had to originate in the muscularis propria layer of the gastrointestinal wall, and this had to be confirmed by endoscopic ultrasonography (EUS). All tumors eligible for participation based on EUS examination were no more than 10 mm in diameter because the diameter of the air-driven ligator cap was 10 mm. All patients in this series had a normal complete blood cell count and thrombin time without having taken warfarin, clopidogrel, aspirin, or any other nonsteroidal anti-inflammatory drug for at least 1 week before the procedure.This study was approved by the Institutional Review Board and Ethics Committee of China Medical University. All patients voluntarily chose their therapeutic course and provided written informed consent for their participation in this study. The operator performing the EE-L procedure in this study was familiar with both the endoscopic ligation and submucosal dissection techniques.Endoscopic ultrasound was performed with a linear-array scanning echoendoscope (Pentax EG3870UT equipped with a HITACHI 6500 EUB ultrasonography machine) or a radical scanning echoendoscope . Endoscopic ligation was performed with a standard endoscope with a 10-mm air-driven ligator cap . This ligator cap had a small tube to control the band, which was released after 2 ml of air had been injected into the tube. EE-L was performed using equipment including a hook knife , injection needle , forceps, snare , hemostatic forceps , and high-frequency generator . Wound closure was performed with endoclips and tissue adhesive composed mainly of alkyl alpha-cyanoacrylate . About 1.5 to 3.0 ml of adhesive was sprayed evenly over the wound by endoscopic catheters that were placed in the stomach and aimed at the wound surface.The lesion was first aspirated into the transparent cap attached to the tip of endoscope. The elastic band was then released around its base Figures\u00a0A, B. ThePathologic examination included identification of cell type, overall cellularity, nuclear atypia, immunohistochemical findings, and the mitotic index. Immunohistochemical analysis for CD117 (c-kit), CD34, smooth muscle actin, desmin, S-100, etc. were performed to differentiate tumors of mesenchymal origin.Endoscopic examinations were performed 5 days after the procedure to examine the wound surface and again 2 months after the resection to assess ulcer healing. To confirm the completeness of tumor resection, endoscopic examinations were performed for all patients once a year for the first 2 years. If no residual tumor or tumor recurrence was found, the patients diagnosed with leiomyomas required no further treatment; the patients diagnosed with GISTs were advised to undergo endoscopic examinations once every 2 years. If residual tumor or tumor recurrence was detected, EE-L could be performed.From June 2009 to June 2011, a total of 43 patients with 43 tumors underwent EE-L at Shengjing Hospital of China Medical University. Patient demographic characteristics, lesion features, pathological diagnosis, and clinical outcomes are summarized in Table\u00a0All 43 gastrointestinal tumors were resected by EE-L. The mean operation time was 27.9 minutes , and the mean enucleation time was 7.2 minutes . One patient experienced self-limiting, non-life-threatening hemorrhage 5 days after EE-L. No perforation, massive hemorrhage, or peritonitis requiring further endoscopic or surgical intervention occurred. The mean follow-up time was 20.4 months . No recurrence has occurred during the follow-up period.The histological diagnoses obtained for the 43 lesions were 24 leiomyomas and 19 GISTs. Mitotic counts in all 19 GISTs were <5 per 50 high-power fields; thus, all were classified as very low-risk.Gastrointestinal tumors of muscularis propria origin include leiomyomas, GISTs, neural tumors, and others. GISTs are the most common mesenchymal tumors of the gastrointestinal tract. Large GISTs with high mitotic rates are often associated with malignant behavior and display higher rates of recurrence and metastasis -18. TherLaparoscopic resection appears to be a safe and effective alternative method for the treatment of gastric tumors . HoweverLigation-assisted endoscopic mucosal resection is one of several endoscopic treatment modalities employed in the treatment of esophageal neoplasia . EndoscoEE-L was developed for the diagnosis and treatment of small gastrointestinal lesions originating from the muscularis propria. This technique combines endoscopic band ligation and endoscopic enucleation to fully exploit the advantages of each technique.Endoscopic band ligation of the tumor simplifies the endoscopic enucleation procedure and reduces the time required because the elastic bands firmly ligate the lesions and cause them to assume a polypoid form with pseudostalks during the entire enucleation process. After the tumor has been ligated, the overall endoscopic enucleation process progresses easily and smoothly. The enucleation time in the present study was short .Perforation is a recognized complication during endoscopic resection, even in the hands of an expert endoscopist -6. The ETissue adhesives are used for a variety of local applications including hemostasis, wound closure, and fistula repair. The most commonly utilized tissue adhesives in gastrointestinal endoscopy include cyanoacrylates, fibrin glues, and thrombin. Cyanoacrylates are widely used outside of the United States for gastric variceal bleeding and, to a lesser extent, ulcer bleeding and fistula closure . Tissue This study has some limitations. Like other endoscopic resection techniques for removal of tumors arising from the muscularis propria, EE-L is limited in that complete enucleation is defined solely by endoscopic observation. It was impossible to remove tumor tissue with a sufficiently safe margin using endoscopic resection. Therefore, long-term follow-up assessment should be performed to ensure the complete removal of GISTs. Although endoscopic resection cannot completely eliminate the need for continued surveillance in all patients, it can eliminate the need for surveillance in patients with leiomyomas. For patients with GISTs in this study, endoscopic resection achieved complete tumor resection based on gross observation, and no residual tumor or tumor recurrence was found during the long-term follow-up.This study shows that EE-L appears to be a safe, effective, and relatively simple technique for the pathological diagnosis and resection of small gastrointestinal tumors originating from the muscularis propria. Controlled clinical trials with larger sample sizes and longer follow-up periods are necessary to further examine the value and limitations of this technique.EE-L: Ligation-assisted endoscopic enucleation; GIST: Gastrointestinal stromal tumor; EUS: Endoscopic ultrasonography.JG, SS, ZL, SW, NG, XL, GW, and XY declare that they have no competing interests.SS carried out the study design, obtained funding, and performed the endoscopic and EE-L procedures. GJ participated in the study design and drafting of the manuscript. LZ participated in the study design and helped to draft and critically revise the manuscript. WS and GN helped with performance of the EE-L procedure and clinical management of the patients. LX carried out the data acquisition, performed the statistical analysis, and helped with clinical management of the patients. WG was involved in data acquisition and clinical management of the patients. YX participated in the study design, carried out the pathological diagnosis, and participated in data acquisition. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/13/88/prepub"} +{"text": "Indeed, it is established that the ubiquitously expressed JNK1 and JNK2 isoforms regulate energy expenditure and insulin resistance. However, the role of the neuron-specific isoform JNK3 is unclear. Here we demonstrate that JNK3 deficiency causes hyperphagia selectively in high fat diet (HFD)-fed mice. JNK3 deficiency in neurons that express the leptin receptor LEPRb was sufficient to cause HFD-dependent hyperphagia. Studies of sub-groups of leptin-responsive neurons demonstrated that JNK3 deficiency in AgRP neurons, but not POMC neurons, was sufficient to cause the hyperphagic response. These effects of JNK3 deficiency were associated with enhanced excitatory signaling by AgRP neurons in HFD-fed mice. JNK3 therefore provides a mechanism that contributes to homeostatic regulation of energy balance in response to metabolic stress.The cJun NHDOI:http://dx.doi.org/10.7554/eLife.10031.001 Consuming the right amount of food is important for health. Eating too little for a long time causes damage to organs, and overeating can cause harm as well, in the form of conditions such as obesity and type 2 diabetes. Several signaling molecules and brain regions are linked to controlling food consumption and ensuring the body receives the correct amount of nutrients to fuel its activities.Previous studies have found that two proteins called JNK1 and JNK2, which are found in most tissues of the body, can reduce how much energy cells use. This can trigger insulin resistance and fat accumulation, and so suggests that blocking the activity of these proteins may help to treat type 2 diabetes and obesity. However, the role of another JNK protein \u2013 JNK3, which is mostly found in the brain \u2013 was not known.Now, Vernia, Morel et al. have investigated the role of JNK3 in metabolism. It was found that JNK3 reduced the amount of food consumed by mice provided with a cafeteria (high fat) diet. Mice that lacked JNK3 ate far more food and gained more weight on a high fat diet than normal mice. However, JNK3 played no role in food consumption when mice were fed a standard chow diet. Treating normal mice with leptin \u2013 an appetite-suppressing hormone \u2013 caused them to lose weight, but did not affect mice that lacked JNK3.Examining the brains of the mice revealed that in normal mice, JNK3 in a specific sub-population of neurons decreases the production of proteins that promote eating. However, the proteins continued to be produced in mice that lacked JNK3, encouraging overeating.Overall, the results suggest that blocking the activity of all the JNK proteins will not help treat obesity and diabetes as shutting down JNK3 could encourage overeating. Therefore, future investigation into treatments for these conditions should focus on drugs that specifically target JNK1 and JNK2, and not JNK3.DOI:http://dx.doi.org/10.7554/eLife.10031.002 The regulation of energy balance (food consumption and energy expenditure) is important for health and survival. Sustained negative energy balance caused by cachexia and anorexia is associated with serious injury to multiple organ systems . SimilarIt is established that the arcuate nucleus (ARC) in the hypothalamus plays a key role in the regulation of energy balance . AgRP neAgRP and POMC neurons integrate signals from nutrients (e.g. glucose and fatty acids) and peripheral hormones to mediate opposite actions regulating downstream neuroendocrine circuits linking internal and environmental stimuli with the coordinated control of homeostatic satiety . Thus, lThe anorexigenic hormone leptin plays a key role in the regulation of food consumption. Leptin can act directly on AgRP and POMC neurons, but leptin can also act on other neurons in several brain sub-regions, including mid-brain and brainstem nuclei . Control2-terminal kinase (JNK) signaling pathway regulates feeding behavior. Previous studies have established that the ubiquitously expressed JNK1 and JNK2 isoforms play an important role in the metabolic stress response of peripheral tissues (Mapk10 gene) plays a key role in the maintenance of energy balance during consumption of a high fat diet (HFD) by promoting leptin signaling. Mapk10 gene ablation studies identify AgRP neurons as a site of JNK3 function. JNK3 is therefore a key mediator of homeostatic regulation of energy balance in response to metabolic stress.The purpose of the study reported here was to test whether the cJun NH tissues . HoweverLeptin is an anorexigenic hormone. Indeed, treatment of chow-fed mice with leptin suppressed feeding behavior and caused decreased body mass . In contWe considered the possibility that a stress-activated MAP kinase pathway may contribute to the regulation of leptin signaling in HFD-fed mice. It is established that feeding a HFD causes activation of the ubiquitously expressed isoforms JNK1 and JNK2 in peripheral tissues, including liver, muscle, and adipose tissue . However-/-Mapk10 (JNK3-deficient) mice. We found that -/-Mapk10 mice gained similar body mass when fed a CD, but these mice gained significantly greater mass when fed a HFD compared with WT mice or a HFD to wild-type (WT) mice or WT mice . 1H-MRS ean mass . Indeed, WT mice . Microsc WT mice .Mapk10 gene ablation selectively increased consumption of a HFD, but not a CD , reduced glucose turnover, reduced whole body glycolysis, increased hepatic glucose production, and decreased hepatic insulin action compared with HFD-fed WT mice & Cd68), increased expression of genes associated with M1-like macrophage polarization , and decreased expression of genes associated with M2-like macrophage polarization in the adipose tissue of HFD-fed -/-Mapk10 mice compared with HFD-fed control mice mice and Loxp/LoxPLeprb-cre Mapk10 (LepR\u2206J3) mice gained similar body mass when fed a CD. However, HFD-fed LepR\u2206J3 mice gained significant more body mass than LepRWT mice LepRolerance , increasolerance , increasolerance , increasolerance , and incolerance . White aRWT mice . Moreoveteatosis .+ neuronal sub-population relevant to JNK3-regulated HFD feeding behavior, we examined Mapk10 gene ablation in selected neurons within the hypothalamus. Gene expression analysis demonstrated that JNK3 was required for HFD-induced regulation of Agrp and Npy, but not Pomc (Loxp/LoxPAgrp-cre Mapk10 (Agrp\u2206J3) mice and Loxp/LoxPPomc-cre Mapk10 (Pomc\u2206J3) mice. We found that JNK3 deficiency in POMC neurons of HFD-fed mice caused no significant changes in feeding behavior, glucose intolerance, blood glucose concentration, hypertrophy of white and brown adipocytes, and hepatic steatosis compared with control Pomc-cre (PomcWT) mice (Agrp-cre (AgrpWT) mice mice . In contWT) mice . Metabolrol mice . Togethe-/-Mapk10 mice. This analysis demonstrated that JNK3 deficiency caused no change in mIPSC frequency or amplitude in CD-fed and HFD-fed mice in neurons have been examined. This analysis demonstrated that compound JNK-deficiency caused markedly increased survival responses associated with increased autophagy hyperphagia was found in mice with JNK3 deficiency in AgRP neurons, but not POMC neurons mice do not exhibit altered feeding behavior in WT mice would cause a small change in feeding behavior. However, the pro-apoptotic function of this activated Mapk8 allele and cause insulin resistance in peripheral tissues indicates that drugs that block JNK signaling may be therapeutically beneficial for the treatment of pre-diabetes . However-/-Mapk10 mice previously (tm1(FLP1)DymGt(ROSA)26Sor/RainJ (tm2(cre)RckLepr/J mice (tm1(cre)LowlAgrp/J mice . Homologous recombination of the 3\u2019 arm of the targeting vector was verified by PCR using the primers (3F: 5\u2019-CTGAGTGACGTGTGGAG-3\u2019 and 5R: 5\u2019-TCATTGGGTTGGGATATTC-3\u2019) followed by digestion with +XhoI . Cre-mediated recombination between the LoxP sites was detected by PCR using the primers 1F and 4R: 5\u2019-GATTCTCCCTGTCTGAG-3\u2019 . The Mapk10LoxP/LoxP mice were routinely genotyped by PCR using primers 1F and 2R .We established 1H-MRS . The mice were housed in a facility accredited by the American Association for Laboratory Animal Care (AALAC). The Institutional Animal Care and Use Committee (IACUC) of the University of Massachusetts and the University of Cincinnati approved all studies using animals.Male mice (8 wks old) were fed a chow diet or a HFD for 4 to 12 wks. Body weight was measured on a weekly basis and whole body fat and lean mass were non-invasively measured using The clamp studies were performed at the National Mouse Metabolic Phenotyping Center at the University of Massachusetts Medical School. A 2\u2009hr hyperinsulinemic-euglycemic clamp was conducted using overnight fasted conscious mice with a primed and continuous infusion of human insulin , and 20% glucose was infused at variable rates to maintain euglycemia .The analysis was performed by the National Mouse Metabolic Phenotyping Centers at the University of Massachusetts Medical School and the University of Cincinnati. The mice were housed under controlled temperature and lighting with free access to food and water. The food/water intake, energy expenditure, respiratory exchange ratio, and physical activity were measured using metabolic cages .rd ventricle . Mice were monitored daily and allowed to recover for 1 week after surgery. Mice received either solvent or Leptin (5 \u00b5g) in 2\u00a0\u00b5l delivered over 10 min. Leptin treatment by intraperitoneal (ip) injection was performed following 3 consecutive days of sham injection.Intracerebroventricular treatment with leptin was performed using mice with a cannula stereotaxically implanted into the 3Adipoq (Mm00456425_m1), Agrp (Mm00475829_g1), Arg1 (Mm00475988_m1), Ccl2 (Mm00441242_m1), Emr1 (F4/80) (Mm00802530_m1), Il1b (Mm00434228_m1), Il6 (Mm00446190_m1), Mapk8 (Jnk1) (Mm00489514_m1), Mapk9 (Jnk2) (Mm00444231_m1), Mapk10 (Jnk3) (Mm00436518_m1), Mgl2 (Mm00460844_m1), Mrc1 (Mm00485148_m1), Mrc2 (Mm00485184_m1), Npy (Mm03048253_m1), Pomc (Mm00435874_m1), and Tnf (Mm00443258_m1). The relative mRNA expression was normalized by measurement of the amount of 18S RNA in each sample using Taqman\u00a9 assays .Tissue isolated from mice starved overnight was used to isolate total RNA using the RNAeasy mini kit (Qiagen). Total RNA (500\u00a0ng) was converted into cDNA using the high capacity cDNA reverse transcription kit . The diluted cDNA was used for real-time quantitative PCR analysis using a Quantstudio PCR PCR machine (Life Technologies). TaqMan assays (Life Technologies) were used to quantify Blood glucose was measured with an Ascensia Breeze 2 glucometer . Adipokines and insulin in plasma were measured by multiplexed ELISA using a Luminex 200 machine .Glucose and insulin tolerance tests were performed by intraperitoneal injection of mice with glucose (1\u00a0g/kg) or insulin (1.5 U/kg) using methods described previously .Mice (8\u201312 week-old) were fasted overnight. Hypothalamic extracts were prepared using Triton lysis buffer . Extracts (30\u201350 \u00b5g of protein) were examined by immunoblot analysis by probing with antibodies to JNK3 and GAPDH . Activated JNK was isolated by immunoprecipitation with the mouse monoclonal p-JNK antibody G9 pre-bound to protein G Sepharose and detected by immunoblot analysis by probing with an antibody to JNK3 . Immunocomplexes were detected by fluorescence using anti-mouse and anti-rabbit secondary IRDye antibodies and quantitated using the Li-COR Imaging systemHistology was performed using tissue fixed in 10% formalin for 24\u2009h, dehydrated, and embedded in paraffin. Sections (7 \u00b5m) were cut and stained using hematoxylin & eosin . Paraffin sections were stained with an antibody to F4/80 that was detected by incubation with anti-rabbit Ig conjugated to Alexa Fluor 488 (Life Technologies). DNA was detected by staining with DAPI (Life Technologies). Fluorescence was visualized using a Leica TCS SP2 confocal microscope equipped with a 405\u00a0nm diode laser .2, 5% CO2) high sucrose solution . Then, 300 \u00b5m thick coronal sections were cut with a Leica VT1000S Vibratome and incubated in oxygenated aCSF at 34\u00b0C for 30\u2009min. The slices were maintained and recorded at room temperature (20\u201324\u00b0C). The intracellular solution for voltage clamp recording contained the following: 140\u00a0mM CsCl, 1\u00a0mM BAPTA, 10\u00a0mM HEPES, 5\u00a0mM MgCl2, 5\u00a0mM Mg-ATP, and 0.3\u00a0mM Na2GTP, pH 7.35 and 290 mOsm.Brain slice preparations were performed using 8\u201310-weeks-old mice anaesthetized with isoflurane before decapitation and removal of the entire brain. The brains were immediately submerged in ice-cold, carbogen-saturated were recorded in the presence of tetrodotoxin (1\u00a0\u00b5M) and picrotoxin (100\u2009\u03bcM) in whole cell voltage clamp mode. To record miniature inhibitory postsynaptic currents (mIPSCs), the neurons were recorded in the presence of TTX and kynurenic acid (1\u2009mM). The membrane potential was clamped at \u221260 mV. All recordings were made using a Multiclamp 700B amplifier, and data were filtered at 1.4 kHz and digitized at 20 kHz. Data was analyzed using Clampfit 10.2 and Origin Pro 8.6.Differences between groups were examined for statistical significance using the Student\u2019s test or analysis of variance (ANOVA) with the Fisher\u2019s test. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your submission has been favorably evaluated by Tony Hunter (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled \"JNK3 participates in an AgRP/NPY neuron circuit that suppresses feeding behavior in response to metabolic stress\" for peer review at The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:All the reviewers agree that this manuscript does a fine job of demonstrating that JNK3 function is important for body weight regulation and that its function in AgRP-expressing neurons is particularly important. However, the mechanism(s) by which JNK3 acts within AgRP neurons is not sufficiently developed. In particular the deficiency in Lepr signaling seems unlikely.Essential revisions:eLife, the authors need to delineate the mechanism by which loss of JNK3 signaling in AgRP neurons promotes obesity. The reviewers have suggested several approaches to address this issue. Alternatively, the authors may decide that publishing the more descriptive part of the study but not on a normal (control diet). The obesity is associated with elevated leptin, elevated insulin, glucose intolerance, insulin resistance, and many other parameters associated with obesity. The most important contribution is their systematic demonstration that the phenotype of global KO of the gene encoding JNK3 is replicated quite well by KO only in AgRP neurons. The authors go on to explore how loss of JNK3 function in AgRP neurons might lead to the hyperphagic obese phenotype, but those studies are less compelling.1) The authors show that leptin receptors can be activated (phosphorylated) in the JNK3 KO but the dose-response is apparently shifted \u2013 LepR is fully activated (phosphorylated) with 2.5 mg/kg leptin but only partially activated with 0.1 mg/kg . They attribute this hypo-sensitivity to a reduced level of Lepr mRNA . They show that phospho-cJun (transcription factor) normally binds to an AP1 site in the promoter of Lepr gene and hence the lack of phosphorylation of c-Jun could impair transcription of Lepr gene. There are two problems with this analysis: (a) The Lepr mRNA (rather than protein) measurements were made in hypothalamic samples, not in AgRP neurons where it is relevant. (b) Complete loss of Lepr in AgRP neurons has a relatively small effect on obesity and heterozygous Lepr db/+ mice do not become more obese on a HFD than WT mice. Thus, it seems unlikely that reduced levels of LepR account for the obesity observed in this JNK3 KO model.before they become obese. The experiment in 2) Their data in Reviewer #2:Vernia and colleagues present an interesting paper in which the conclusion are based on a broad set of experiments and well justified. They show that the loss of the neuronally expressed JNK3 is associated with increased obesity due to increased food consumption. This is brought about by the elimination of JNK3 in leptin receptor expressing neurons. They narrow this down further by doing an AgRP-specific elimination and compare that to a POMC neuronal-specific elimination. They demonstrate hyperphagia to be present in only Agrp-specific nulls for JNK3. They go on to demonstrate that this is in fact due at least in part due to reduced leptin receptor expression on AgRP-neurons. All of this happens exclusively in the context of high fat diet exposure.These are interesting findings that should be of broad interest to the field. It is surprising to find JNK3 necessary for leptin receptor expression and to see it distinct from the role of JNK1/2 in the periphery where their lack results in improvement in the metabolic phenotype due to reduced inflammatory tone.There is very little to criticize here. The authors address an important question, find a surprising answer of broad interest and base their conclusion on a broad set of genetic data.lepR expression in AgRP neurons, and it does not lead to reduced lepR expression in POMC neurons?The only area that falls short is a further analysis of the signaling mechanisms in place. What other component of leptin signaling are affected by the lack of JNK3? The downstream analysis could be broader. Where does the cell specificity come in? How does JNK3 deficiency lead to reduce Reviewer #3:In this manuscript Vernia et al. show that JNK3 plays a role in inhibiting high-fat diet-induced weight gain and does so through actions at AgRP/NPY neurons, which may include bolstering leptin actions. Overall, the metabolic studies convincingly show that JNK3 deficiency increases HFD-induced weight gain, hyperphagia, adipose hypertrophy, glucose intolerance and insulin resistance. The logical set of cell-type-selective JNK3 deletions provides compelling evidence that JNK3 actions specifically in AgRP neurons are necessary to reduce HFD-induced weight gain. Thus, the overall conclusion is well supported by the data presented in The primary weakness in the work is the cursory attempt to provide some mechanistic insight . The mEPSC recordings add very little to the paper as is. It is important to know that mIPSCs don't also increase and how the deletion affects basal regulation of POMC neurons. In Figure 7, the overall JNK3 knockout was used and thus, it is not possible to determine if leptin receptor expression is also reduced in POMC cells or rather, if maybe POMC cells do not express JNK3 or require JNK3 to bolster leptin sensitivity. In my opinion, these last 2 experiments do not need to be included to support the conclusions made, but if they are included, they need to be done more thoroughly so that they can be better interpreted.JNK3 is the neuron-specific isoform of JNK and its activity is dynamically regulated rather than constitutive like the other 2 isoforms. Together with the compelling evidence provided here that JNK3 helps reduce high-fat diet-induced weight gain through actions in AgRP neurons, this work adds significant new insight into JNK3 function and the hypothalamic regulation of energy balance in the face of metabolic challenge.2-terminal kinase 3 in response to metabolic stress\" for consideration by eLife. Your article has been evaluated by a Reviewing Editor and overseen by Tony Hunter as the Senior Editor.Thank you for submitting your work entitled \"Excitatory transmission onto AgRP neurons is regulated by cJun NHNpy and Agrp expression and enhance feeding behavior makes sense, especially because the previous work from the Lowell lab showing that removing NMDA receptors from AgRP neurons has the opposite effect. However, there are a few loose ends that need attention.In response to our reviews, the authors of this paper have now dropped data suggesting that reduced leptin receptor expression was responsible for the obesity of JNK3-deficient mice in favor enhanced glutamatergic (mEPSC) signaling . The autMapk10 KO mice rather than mice lacking Mapk10 selectively in AgRP neurons; hence, it is not possible to make a strong conclusion that the enhanced excitatory transmission is due to lack of JNK3 in AgRP neurons.1) Unfortunately, the electrophysiology experiments were performed on 2) The authors suggest in the Discussion that NMDA signaling is enhanced, but their experiments do not distinguish NMDA from AMPA signaling.3) The hypothesis that links JNK3 deficiency and the presumptive enhanced NMDA signaling is not clearly explained. In the fifth paragraph of the Discussion the authors say that JIP proteins might be involved and refer to Kennedy et al. (2007). If I understand that paper correctly, it suggests that lack of JIP proteins influences JNK localization (activity) and prevents phosphorylation of NR2 subunit of NMDA receptor. If so, it seems like lack of JNK3 would impair (not enhance) NMDA function in the present studies. The authors admit that \"further studies are required to identify the complete spectrum of JNK3 targets in AgRP neurons\". I would argue that they have not identified any direct JNK3 targets in AgRP neurons.Essential revisions:At the very least, the authors need to discern whether AMPA or NMDA receptor signaling is altered in JNK3-deficient AgRP neurons and provide some data or reasonable hypothesis indicating how lack of JNK3 could lead to enhanced AMPA/NMDA signaling. Essential revisions:For publication in eLife, the authors need to delineate the mechanism by which loss of JNK3 signaling in AgRP neurons promotes obesity. The reviewers have suggested several approaches to address this issue. Alternatively, the authors may decide that publishing the more descriptive part of the study \u20135 in anoThank you for providing this opportunity to improve our manuscript. We have carefully considered the reviewers\u2019 comments and we have thoroughly revised the manuscript by including new experiments to more fully establish our conclusions. Specifically, we demonstrate that JNK3 plays no role in the regulation of feeding behavior in chow-fed mice, but activated JNK3 in high fat diet-fed mice regulates excitatory transmission onto ARP/NPY neurons. Collectively, our data establish a new paradigm for the regulation of feeding behavior by metabolic stress.Reviewer #1: This paper provides evidence that lack of JNK3 expression in all cells, all neurons that express the leptin-receptor or just the sub-population of neurons that expresses AgRP leads to obesity when mice are reared on a high-fat diet (HFD) but not on a normal (control diet). The obesity is associated with elevated leptin, elevated insulin, glucose intolerance, insulin resistance, and many other parameters associated with obesity. The most important contribution is their systematic demonstration that the phenotype of global KO of the gene encoding JNK3 is replicated quite well by KO only in AgRP neurons. The authors go on to explore how loss of JNK3 function in AgRP neurons might lead to the hyperphagic obese phenotype, but those studies are less compelling. 1) The authors show that leptin receptors can be activated (phosphorylated) in the JNK3 KO but the dose-response is apparently shifted \u2013 LepR is fully activated (phosphorylated) with 2.5 mg/kg leptin but only partially activated with 0.1 mg/kg . They attribute this hypo-sensitivity to a reduced level of Lepr mRNA . They show that phospho-cJun (transcription factor) normally binds to an AP1 site in the promoter of Lepr gene and hence the lack of phosphorylation of c-Jun could impair transcription of Lepr gene. There are two problems with this analysis: (a) The Lepr mRNA (rather than protein) measurements were made in hypothalamic samples, not in AgRP neurons where it is relevant. (b) Complete loss of Lepr in AgRP neurons has a relatively small effect on obesity and heterozygous Lepr db/+ mice do not become more obese on a HFD than WT mice. Thus, it seems unlikely that reduced levels of LepR account for the obesity observed in this JNK3 KO model.We agree. These data have been removed from the revised manuscript. Conclusions about leptin receptor expression have also been removed from the text. The mechanistic analysis in the revised manuscript focuses on the role of JNK3 to regulate excitatory transmission onto ARP/NPY neurons.2) Their data in We have revised the manuscript to address these important points:1H-MRS analysis on day 3 demonstrated no obesity phenotype (A) We performed a time course analysis using metabolic cages to examine when hyperphagia is detected in mice fed a high fat diet. This analysis demonstrates that hyperphagia is detected within 2 days of high fat diet consumption . 1H-MRS henotype . These dB) We performed a pair-feeding study using high fat diet-fed WT and JNK3-deficient mice. This analysis demonstrates that no obesity phenotype was detected when the WT and JNK3-deficient mice consumed the same amount of food . These dC) We have not performed in vivo recording and calcium imaging of AgRP neurons, as requested; we believe that such studies are beyond the scope of the present manuscript. We did examine cFos staining, but no differences in the number of cFos-positive AgRP neurons were detected between WT and JNK3 KO mice. We have expanded the electrophysiological analysis of AgRP neurons from chow diet-fed mice and high fat diet-fed mice in the revised manuscript . This anReviewer #2: [\u2026] There is very little to criticize here. The authors address an important question, find a surprising answer of broad interest and base their conclusion on a broad set of genetic data. The only area that falls short is a further analysis of the signaling mechanisms in place. What other component of leptin signaling are affected by the lack of JNK3? The downstream analysis could be broader. Where does the cell specificity come in? How does JNK3 deficiency lead to reduce lepR expression in AgRP neurons, and it does not lead to reduced lepR expression in POMC neurons?Based on the comments of the other reviewers, we have deleted the section of the original manuscript on LepRb expression. In the revised manuscript, we have focused the data presentation and the central conclusions of our study on the finding that excitatory transmission onto AgRP neurons is regulated by the JNK3 signaling pathway in HFD-fed mice. This observation provides a mechanism for the HFD-specific hyperphagia of JNK3 KO mice. Further studies will be required to identify direct molecular targets of JNK3 signaling; this analysis is beyond the scope of the present study.Reviewer #3: [\u2026] The primary weakness in the work is the cursory attempt to provide some mechanistic insight . The mEPSC recordings add very little to the paper as is. It is important to know that mIPSCs don't also increase and how the deletion affects basal regulation of POMC neurons. In Figure 7, the overall JNK3 knockout was used and thus, it is not possible to determine if leptin receptor expression is also reduced in POMC cells or rather, if maybe POMC cells do not express JNK3 or require JNK3 to bolster leptin sensitivity. In my opinion, these last 2 experiments do not need to be included to support the conclusions made, but if they are included, they need to be done more thoroughly so that they can be better interpreted.We agree that the leptin receptor expression data do not strengthen our study. Based on this criticism and the comments of reviewer #1, we have deleted these data from the revised manuscript. We have revised the manuscript to include new data to document mIPSCs and we hJNK3 is the neuron-specific isoform of JNK and its activity is dynamically regulated rather than constitutive like the other 2 isoforms. Together with the compelling evidence provided here that JNK3 helps reduce high-fat diet-induced weight gain through actions in AgRP neurons, this work adds significant new insight into JNK3 function and the hypothalamic regulation of energy balance in the face of metabolic challenge.Thank you for these comments.In response to our reviews, the authors of this paper have now dropped data suggesting that reduced leptin receptor expression was responsible for the obesity of JNK3-deficient mice in favor enhanced glutamatergic (mEPSC) signaling . The aut1) Unfortunately, the electrophysiology experiments were performed on Mapk10 KO mice rather than mice lacking Mapk10 selectively in AgRP neurons; hence, it is not possible to make a strong conclusion that the enhanced excitatory transmission is due to lack of JNK3 in AgRP neurons.2) The authors suggest in the Discussion that NMDA signaling is enhanced, but their experiments do not distinguish NMDA from AMPA signaling.3) The hypothesis that links JNK3 deficiency and the presumptive enhanced NMDA signaling is not clearly explained. In the fifth paragraph of the Discussion the authors say that JIP proteins might be involved and refer to Kennedy et al. (2007). If I understand that paper correctly, it suggests that lack of JIP proteins influences JNK localization (activity) and prevents phosphorylation of NR2 subunit of NMDA receptor. If so, it seems like lack of JNK3 would impair (not enhance) NMDA function in the present studies. The authors admit that \"further studies are required to identify the complete spectrum of JNK3 targets in AgRP neurons\". I would argue that they have not identified any direct JNK3 targets in AgRP neurons.Our focus on JNK3 function in AgRP neurons is based on the findings that:Agrp and Npy expression, but not Pomc expression, in the response to leptin administration (1) JNK3-deficiency causes changes in stration or feedistration ;2) mEPSC amplitudes are increased in AgRP neurons of JNK3-deficient mice ; and3) JNK3-deficiency in AgRP neurons is sufficient to cause hyperphagy on a HFD .+ Mapk10LoxP/LoxP NPY-GFPAgRP-cre mice would be interesting, but these mice are not available in our colony; we consider these studies to be beyond the scope of the present study.Together, these data provide strong evidence for a function of JNK3 in AgRP neurons. We agree that electrophysiological studies of These data represent a key finding that provides a mechanism for JNK3 function mediated by the regulation of AMPA responses. It is established that AMPA receptor phosphorylation by JNK controls AMPA receptor trafficking and function .We apologize for the poor presentation of the conclusions of our study in the Discussion section of our manuscript where we suggested that NMDA receptors may be regulated (based on the two papers we cited) without adequately addressing the role of AMPA receptors. We have revised the manuscript to make our presentation clearer. Moreover, we have included new data in the manuscript that demAlthough an altered AMPA response in AgRP neurons contributes to the JNK3 phenotype, we cannot exclude the possibility that there is also an altered NMDA response. We feel that dissecting the specific contributions of AMPA and NMDA receptors, while interesting, is outside the scope of this study. In addition, while our recordings show alterations in mEPSC amplitudes, further studies are needed to identify the full spectrum of JNK3 targets that could potentially result in this observation. Such targets may include the JNK scaffold protein JIP1 and JIP2 , but we consider studies of these scaffold proteins to be beyond the scope of the present manuscript.Essential revisions:At the very least, the authors need to discern whether AMPA or NMDA receptor signaling is altered in JNK3-deficient AgRP neurons and provide some data or reasonable hypothesis indicating how lack of JNK3 could lead to enhanced AMPA/NMDA signaling.We agree \u2013 this is an important point. We did not make conclusions about AMPA vs. NMDA currents in the previous version of this manuscript. We have therefore revised the manuscript to include new data documenting that the mEPSC currents that we detect are mediated by AMPA receptor responses, not NMDA receptor responses . These d"} +{"text": "We report the use of optical imaging to assess ChoK\u03b1 status in cells and in vivo using JAS239, a carbocyanine-based ChoK\u03b1 inhibitor with inherent near infrared fluorescence. JAS239 attenuated choline phosphorylation and viability in a panel of human breast cancer cell lines. Antibody blockade prevented cellular retention of JAS239 indicating direct interaction with ChoK\u03b1 independent of the choline transporters and catabolic choline pathways. In mice bearing orthotopic MCF7 breast xenografts, optical imaging with JAS239 distinguished tumors overexpressing ChoK\u03b1 from their empty vector counterparts and delineated tumor margins. Pharmacological inhibition of ChoK by the established inhibitor MN58b led to a growth inhibition in 4175-Luc+ tumors that was accompanied by concomitant reduction in JAS239 uptake and decreased total choline metabolite levels as measured using magnetic resonance spectroscopy. At higher therapeutic doses, JAS239 was as effective as MN58b at arresting tumor growth and inducing apoptosis in MDA-MB-231 tumors, significantly reducing tumor choline below baseline levels without observable systemic toxicity. These data introduce a new method to monitor therapeutically effective inhibitors of choline metabolism in breast cancer using a small molecule companion diagnostic.Choline kinase alpha (ChoK\u03b1) overexpression is associated with an aggressive tumor phenotype. ChoK\u03b1 inhibitors induce apoptosis in tumors, however validation of their specificity is difficult Aqueous cellular extracts were separated by thin layer chromatography (TLC) and the 14C-PC production measured by autoradiography on a Fujifilm FLA-7000 using previously described methods *100%/pre-treatment ratio.Mice bearing subcutaneous 4175-Luc+ or MB-MDA-231 tumors (\u223c300 \u03bcL) underwent pre-treatment MRS scans in a 9.4 T horizontal bore magnet equipped with 40 G/cm gradients interfaced to an Agilent Direct-Drive console (Agilent) operating vnmrj 2.3.C software. Multi-slice gradient and spin echo images were used for tumor localization. sequence . An unsuIn vivo MRS was used to establish baseline tCho levels in a cohort of 10 mice bearing 4175-Luc+ tumors. Mice were injected i.p. for 5 consecutive days with either 100 \u03bcL saline or 2 mg/kg MN58b. A follow-up MR scan was acquired 1 week after treatment. After the final MRS, each animal was injected i.v. with a trace dose (20 nmol) of JAS239 in ethanol/saline. After 24 h, the mice were euthanized and each tumor was analyzed for NIRF as described above.A cohort of 15 mice was inoculated with 4175-Luc+ tumors. Beginning 3 days post-inoculation, animals received a 100-\u03bcL i.p. injection of either control DMSO/saline, 2 mg/kg MN58b or 4 mg/kg JAS239 in DMSO/saline daily for 5 days. Tumor volumes were assessed by caliper measurement.T2-weighted images. The animals were separated into treatment groups, and for 5 consecutive days each group was injected i.p. with 100 \u03bcL of DMSO/saline vehicle (n = 5), 2 mg/kg MN58b (n = 4), or 4 mg/kg JAS239 (n = 3). Initial cohort sizes were 5 per condition, but some animals had to be removed from the study because the treatment was so effective that we were unable to place voxels within post-treatment tumors that were too small. Follow-up MRI/MRS scanning was performed on day 7 to assess tumor growth inhibition and changes in metabolite levels. After imaging, blood was collected into heparin-coated tubes by cardiac puncture. Resected tumors were embedded in Optimum Cryo-Temperature (Sakura Finetek), flash-frozen in liquid nitrogen, and stored at \u201380\u00b0C. Blood samples were submitted to the Ryan Veterinary Clinic Diagnostic labs for toxicology.A cohort of 15 mice was inoculated with MDA-MB-231 cells. When a volume of 300 \u03bcL was reached, tumors were scanned using MRS. Tumor volumes were measured using Hematoxylin and eosin staining and immunohistochemistry was performed as described previously . Caspaset-test unless noted otherwise. A P-value of \u2264 0.05 was considered to be statistically significant. All error bars represent mean \u00b1 SEM.Statistical analyses of treated versus control values were performed in Microsoft Excel 2010 (Version 14.0.7128.5000 32-bit) using an unpaired Student's"} +{"text": "Understanding individuals\u2019 experience of accessing care and tending to various other needs during chronic illness in a rural context is important for health systems aiming to increase access to healthcare and protect poor populations from unreasonable financial hardship. This study explored the impact on households of access to free healthcare and how they managed to meet needs during chronic illness.Rich data from the life stories of individuals from 22 households in rural south-western Uganda collected in 2009 were analysed.The data revealed that individuals and households depend heavily on their social relations in order to meet their needs during illness, including accessing the free healthcare and maintaining vital livelihood activities. The life stories illustrated ways in which households draw upon social relations to achieve the broader social protection necessary to prevent expenses becoming catastrophic, but also demonstrated the uncertainty in relying solely on informal relations.Improving access to healthcare in a rural context greatly depends on broader social protection. Thus, the informal social protection that already exists in the form of strong reciprocal social relations must be acknowledged, supported and included in health policy planning. Low-income countries such as Uganda, along with other sub-Saharan African countries, are experiencing an increase in chronic illnesses . ChronicHealthcare systems around the world are seeking to ensure that everyone has access to the health services they need without experiencing unreasonable financial hardship, and providing free treatment and medication at health facilities is one of the strategies used in countries like Uganda , 8. HoweThere is limited knowledge on the extent to, and the mechanisms through which social relations minimise barriers to healthcare access and provide financial protection for rural populations in a low-income setting . In thisExtant literature indicates that communities struggling to access services such as medication and treatment tend to activate and nurture alternative strategies, such as drawing on their social connections in order to meet their needs , 14, 15.Social relations represent one way in which populations with limited access to healthcare resolve their social, economic and financial problems. Through these social relations, individuals and households are able to access a pool of resources embedded in the community when the need arises, and in ways that match their needs, protecting them from a downward socio-economic spiral. The support provided may include borrowing or lending a bicycle to transport the ill person, nursing care for the sick, food, accommodation, etc. Due to the unpredictability of future need, these exchanges are mostly indirect, meaning that the support is based on need and not necessarily on what the recipient has previously contributed. For example, an individual who receives care when they are ill may repay this social debt with food to someone in need, rather than the person who provided the care. This type of exchange is known as a generalised reciprocity , which iStudies in the early 1990s noted that family networks were not effective safety nets for people affected by HIV at that time, because of the poverty of household members and kin . HoweverThe rural districts in Uganda are served by health centres that range from level I (lowest \u2014village level) to level IV (highest\u2014district level) and generally focus on outpatient services. The point of access to the health system is the level I health centre, which refers complicated cases to a health centre at the next level. However, the public health facilities are few in the rural areas, and are characterised by: lack of transportation for emergency cases from home or with referral between facilities, constant shortage of medication and chronic understaffing by health workers, with a ratio of one health worker to more than 20,000 people . TherefoThe Ugandan government is seeking to provide universal access to healthcare services by applying strategies such as elimination of user fees in public health facilities, providing free treatment and introducing health insurance. However, in rural areas, where 84% of Ugandans live, non-medical needs such as transportation to health facilities, food and sustaining livelihoods while experiencing illness are still a burden for households , 24. ProThis study was nested within the Rural Livelihoods Study (RLS) , which wPeople live in semi-permanent structures built from locally available materials. The community consists mostly of people from the Baganda tribe, with 15% being of Rwandese origin, who are well assimilated. Religious affiliation is mostly Christian, with a significant Muslim minority (28%). Levels of literacy are low and the main income-earning activities are growing bananas, coffee and beans, and trading fish. In 2013, the prevalence of diabetes in this community was 0.4%, hyperglycaemia 2.9%, high blood pressure 22.5% and HIV 7.3% because they pay all my bills when I am hospitalised \u2026Patricia started to access free basic treatment and medicines from the MRC clinic in 2000 and in 2004, when the MRC started providing ARV treatment to eligible clients, she was one of the beneficiaries.In addition to free ARVs and hospital care, Patricia was provided with transport to the health facility for follow-up visits and sometimes when she needed a medical consultation. With this available free treatment and transport, Patricia accessed adequate healthcare and did not fall into financial hardship. While free treatment and medication were available to everyone in the study area, free transport was only available to those on ART, and households with chronically ill members also had other needs such as food and help with personal care. One way of meeting these needs was through drawing support from their friends, neighbours and relatives.Free treatment and medication was available at the MRC health facility for all households in the study area. For services not offered at the facility, individuals received referrals to other health centres where they could receive appropriate healthcare, with the costs covered by the MRC. However, while treatment and medication were free to all, individuals who were part of the RCC and had enrolled for antiretroviral therapy (ART) received additional support, such as transportation. This eliminated most of the potential barriers to accessing care, such as unavailability of treatment and medication and high cost of treatment and transportation. Patricia\u2019s story illustrates how chronically ill individuals surveyed in this study accessed healthcare and the kinds of resources they needed.When I am sick my neighbour goes to the MRC clinic to report about my sickness and a clinic van is sent to pick me up\u2026It was also common for individuals such as Patricia to get such support from neighbours, friends and relatives with food and care, as we can see if we continue to look at her story .However, due to the general food shortage in the community and her physical absence from her land, people in that village stole Patricia\u2019s coffee and banana crop. She stopped hiring workers, cultivation on her land stopped and therefore she had insufficient income to buy food. When we inquired about her major source of food, she said:My neighbour who sells meat gave me one kg of meat for free \u2026 My life is deteriorating. I don\u2019t see why I should be bothered (to grow food) when all I need is a little food for me.In addition, Patricia had an \u2018auntie\u2019 who frequently visited her and helped to sell alcohol and do other house chores such as cooking food and helping with personal hygiene, as well as bedside care when Patricia was hospitalised. In return for the help, this \u2018auntie\u2019 grew her crops on Patricia\u2019s land in the other village.Thus through her social relations, Patricia benefited from the wider social protection she needed in order to meet other basic needs in her situation, including communication with the health facility, care and food. This support from neighbours and \u2018family\u2019 minimised the financial risks in accessing care. Patricia was in a position to repay the support her \u2018auntie\u2019 provided by allowing her to grow crops during the planting seasons. Through this exchange of services, Patricia was able to maintain a relationship that supported her during periods of illness.In addition to the need for treatment, individuals also needed to communicate with the health facility and needed transportation (for those not on ART), food and care while they were ill, which were usually not affordable to individuals. To meet such non-medical needs, household members mostly drew support from their social relations in various ways. In Patricia\u2019s household, in addition to treatment and transportation, she needed to communicate with the health facility and did not have access to a phone/reception. She reported:Kalooli, in his 80s at the time of interview, lived with his teenage grandson in a three-room house. He owned about 1.5 acres of land, most of which had been cultivated by a neighbour for the previous two years on a crop harvest-sharing basis, whereby Kalooli received part of the crop. During the 1990s, Kalooli sold part of his land to support his household when his wife, son and daughter became ill and died. During the interviews, Kalooli was ill on a number of occasions and was therefore unable to work and provide for the household. His grandson did casual work for neighbours for food and money. Kalooli was not entitled to transportation from the clinic and he claimed that he could not receive hospital care as he had no one to provide bedside care in hospital and supply food (which is not included). He described his access to care during one of the visits when he was ill:If I had someone to take care of me there I would have gotten hospitalised. They [the hospital management] cannot admit you when you have no one to care for you, cook and provide food.Kalooli depended on his neighbours to transport him to the health centre and to provide care, including food, help with finding other treatment and personal hygiene.When I was ill and unconscious my neighbours called a health worker from the neighbourhood to treat me.Providing and receiving generalised reciprocal support maintained connections with the social network. Households such as Kalooli\u2019s regarded being able to give and receive support as an \u2018investment\u2019 with benefits that could be reaped when need arose in the future.Helping my neighbour is like giving a loan, which will be repaid when I am in need. If you do not give [support] what will you do when it [crisis] happens to you? In the past when I trapped mudfish I gave my neighbour some fish for free, even when they wanted to pay, and now those neighbours meet all my needs when I am ill\u2026. I have debts that are not yet paid because I have been ill; I have to give bark-cloth to the home where a man died in this village; and I have not yet given a bunch of matooke to my neighbour whose son was recently married.Households in the study area thus endeavoured to maintain connections with their social network in order to access resources embedded in the social relations when they are in need. During times of illness, Kalooli\u2019s household depended heavily on support from neighbours. While hospitalisation costs would have been covered by the MRC, Kalooli could not access this service as he had no-one to accompany him to hospital and provide care and food. His case revealed the dynamics involved in maintaining social connections, including giving support to others when they need it. This enabled households in the study area to meet their needs, especially during crisis such as chronic illness, confirming the critical role of social relations in providing the wider social protection required by households with limited financial resources. The stories told by Patricia and Kalooli were common among the study respondents and illustrate the importance of having free treatment and medicines but also of having support with non-medical needs during chronic illness.It was common for households experiencing chronic illness to cite friends, neighbours and relatives as major sources of support. Some households, such as that of Kalooli described below, believed that they needed to build and maintain good relationships with relatives and friends in order to meet their future needs.Bettina, who was in her 60s at the time of interview, lived with her older sister, her niece Peace and Peace\u2019s four children. The household had 7.5 acres of land where they grew crops such as coffee and bananas and reared cows and goats for sale and subsistence. They had all moved to live in the household, now headed by Bettina, at different times so as to meet their needs, including a need for care.Bettina had relocated to the study area in 1987 to help her parents, who were chronically ill and lived on their own. They needed treatment, food, care, personal help with household chores, transportation to medical facilities and work in the fields to produce food, among other things. At that time, she was 45 years old, childless and experiencing partner violence in her marriage, all of which prompted her to move back to her village. At her parents\u2019 home, Bettina had accommodation and a field to cultivate crops for subsistence and income, and helped her parents in other various ways.Bettina\u2019s mother died in 1991 following a snake bite. Her father fell off his bicycle in 1990 and suffered a spinal injury and five years later he died. Therefore, in 1995 the status of the household changed from being male-headed to female-headed, with Bettina as the head. During his illness, Bettina took her father to hospital on a bicycle, washed him and provided food. She also paid a local herbalist to provide treatment using herbal concoctions, and personal care like washing the patient. She sold her coffee stocks so as to meet the bills for care.By moving to stay with her parents, Bettina provided the support they needed during chronic illness, including accessing treatment and medicines, food, transportation and personal care. This protected them from exposure to financial risks from e.g. having to sell assets to address their needs. At the same time, she met her own need for accommodation and food.Some households that did not have a member with chronic illness, but had other needs, offered care services to households that were experiencing chronic illness and had resources, in exchange for other support. Unlike Patricia, Bettina had not experienced illness directly in her household initially.With food, coffee bushes and income from crop sales, Bettina had the resources to support not only her parents, but also other relatives with various needs. In 1993, her brother, who lived in the fishing islands in Lake Victoria, moved to the household because he had developed HIV-related complications and needed to access the free treatment from the MRC and care from Bettina. Bettina transported him to the MRC clinic to receive treatment and medicine. Her brother also continued commuting to the lake and spent most of the day fishing, not only to generate income for the household, but also to keep away from being seen by the people in his community since he had developed rashes on his skin, a symptom the local people associated with HIV infection. Bettina commented:The MRC counsellors advised him to stop going to the lake but he would not. He had developed rashes all over his body and did not want to be seen by people, he also needed money for hiring people to plant potatoes.For one year this brother was bedridden and Bettina took care of him until he died in 1994. When asked about his contribution to the household Bettina responded:He was the one planting potatoes when he was not sick, he brought fish which we sold and part of it was for home consumption . . . however his body did not respond to the treatment from MRC because he consumed alcohol. He was therefore too weak; he could not wash our father who was bedridden. Imagine, I am a woman but I had to care for him and also wash my father\u2026An older sister to Bettina, who lived alone in another village, also joined the household. She had chronic illnesses including back pain and visual impairment, and needed to access the free treatment and other support including transportation to the health facility, care and food.In 2003 Bettina herself developed chronic illness and therefore needed healthcare services. Both Bettina and her sister accessed the free medicines available at the MRC. However, the household needed other help to provide e.g. food, care and transportation. In order to meet these needs, their teenage niece called Peace (aged 17 years) joined the household to provide care for Bettina and her sister. Peace had left school due to lack of money to pay the fees and needed support for accommodation and food. She carried out most of the work in the fields and household chores. She also organised transportation to the MRC clinic and provided personal care and food. At the same time, she met her needs for accommodation and food. During the following three years, Peace had two sets of twins with a man who lived in another village and did not provide any support for the children. Bettina and her sister provided childcare and lodging for Peace\u2019s children in exchange for the care that they received.Bettina and her sister had ailments that were not addressed within the available basic health services at the MRC. Their brother who lived in the city took them in turns to the city to get treatment. During one of the visits, Bettina\u2019s sister commented:People can die before their time due to lack of treatment. People in this village said that I was suffering from old age. At the city hospital I received blood, urine and stool tests and they gave me medicines. Now I feel better.In summary, since Bettina\u2019s household had accumulated resources, including food, income, accommodation and manpower, it was able to support several individuals who had health related and non-health related needs such as care, food, accommodation, child care and transportation. All these were gathered under one roof and thus the household was at one point in time supporting others and at another point in time receiving support. Accounts such as Bettina\u2019s were common among the study households.In some households experiencing chronic illness, the individual who was chronically ill moved to live with relatives (or non-relatives) that had resources and were therefore in position to provide support. Bettina\u2019s household (as the story unfolds further) also illustrated a situation where individuals who were chronically ill moved into the household to access support.Over time, as Bettina and her sister needed more care, Peace could not provide the care and do all the field work by herself, as well as take care of her children. Therefore Bettina identified a single mother from the neighbourhood who had no land to cultivate and who moved around looking for work in exchange for food. This woman was recruited to help Bettina\u2019s household with work in the fields and household chores. The woman did not always get paid immediately for her work, but Bettina provided support to her whenever she needed it. Bettina explained:There is a neighbour who works in the fields for free. She is my friend. She stays in this village but she does not own land so she goes around looking for food and money. I provide her with cassava and potatoes and other necessities like soap and salt even on occasions when she has not worked for me. I sometimes hire her to hoe (weed) through the cassava, potatoes and banana plantation. When the harvest is ready she gets to harvest the crop in return for her labour.Bettina\u2019s household also regularly provided food for another neighbour who was ill and lived on her own. This neighbour gave nothing to Bettina in return because she was not able. When asked about giving out support Bettina responded:There is an elderly woman that I always give food. She is sickly and weak and lives on her own. All her children live in the city and rarely visit her\u2026 I do not get anything from her in exchange for the food.Bettina thought that it was good will to support others in the community:In this community you will always get something for free when need arises, such as seeds when the planting season is here, food when the harvest is ready \u2026Continuously expanding the number of people with whom to exchange support was an important form of \u2018insurance\u2019 for households experiencing chronic illness and for households that had other needs. The continuation of Bettina\u2019s story illustrates that.The food, housing and land which Bettina\u2019s household possessed placed it in a position to create new relations and exchange care for support, and also to provide support to those who needed it without receiving anything in return.Eseri was in her early 30s at the time of interview and, like Bettina, she had moved into the study area to live with and care for her elderly grandmother, along with her four children. Her grandmother was chronically ill and lived alone on 11.0 acres of land with coffee and matooke, while Eseri needed food and housing for her household. Like Bettina, Eseri provided care for her grandmother, including personal care and food, took her to hospital and also tended the plantation. In exchange, she had housing and food for her family. Following the death of her grandmother two years later, she inherited the house and two acres of the land. Most of the land was rocky and therefore not suitable for growing crops. Unlike Bettina\u2019s household, which had land to cultivate, Eseri needed to find other ways to support her household with food and money. She identified a couple in the neighbourhood who were chronically ill and helped them cultivate and pick coffee. The couple provided Eseri food and money in return, and even on some occasions when Eseri had not helped them. A year later the man died, leaving a widow in her 60s. Eseri had this to say during one of the interview visits:The bad news here is that my neighbour died. He was very kind, he always gave me salt, cooking oil, matooke for free \u2026 His wife is not as kind, but she now lives on her own. She is old, sickly and weak. I always prepare food, fetch water and wash clothes for her. When I cook we (Eseri and her household) all eat from there. When I work on her farm I receive matooke for my family or a bucket of coffee, which I sell and buy food. Her sons live in the city and visit once in a while\u2026Eseri spent at least four days a week in the other household, providing personal care, food, taking the widow to hospital and tending the farm. In return, she received the food that her household needed and also money for her other needs. Eseri\u2019s household was also able to receive support in advance that she paid back when she was able:This month my family and I had malaria and diarrhoea. I was too weak to do any work and the children were too weak to go to school. The widow that I always help provided the money, 20,000 UGX [equivalent to six USD] which I used to buy medicines and food from the shop. The youngest did not respond to the medicines, I had to use the rest of the money to take him to the MRC clinic. We are now digging in the widow\u2019s coffee plantation to repay the debt.Thus Eseri had helped two households with individuals suffering chronic illnesses. In the first case she relocated her family to her grandmother\u2019s house. She therefore accessed accommodation and food and later inherited and owned property. Later when her grandmother died, Eseri helped a neighbouring household experiencing chronic illness, which enabled her to continue accessing food and money. In essence, through continuously expanding her social relations, Eseri was able to support her household. At the same time, she provided the wider social protection that her grandmother and neighbour needed during chronic illness, including accessing free treatment and medicines.In the same way, households that did not have a chronically ill member but had other needs, such as food and accommodation, were able to turn to relatives and create new friends with whom to exchange support.Bettina and Eseri\u2019s households demonstrate the importance of continuously creating reciprocal social relations to meet increasing and changing household needs and also show that these social relations are not static, e.g. not having close relatives around did not mean that a household was vulnerable. The new relations sometimes transcended the close kin network and made it possible for households to meet their needs. It is also clear from their stories that everyone is expected to support people in need if they can, but that a basic level of trust in each other is a precondition for this support.This study demonstrated the importance of social relations for a health system that is endeavouring to provide universal access to healthcare for low-income rural populations. The data from the households surveyed indicate that the free treatment and medicines provided at the health facility were very important for individuals experiencing prolonged illness. However, such individuals also needed broader social protection in order to meet other needs during illness and to prevent costs becoming catastrophic for the household. The households had developed an informal type of social protection composed of a network of kin, friends and neighbours.It is obviously important for a health system to target specific components of healthcare such as access to treatment and medicines. However, any health system that is aiming at achieving broader outcomes, such as universal access to healthcare, needs to go beyond conventional healthcare practices and include a wider set of social and economic interventions. This will insure the population against risks and future insecurities. Recognising and supporting community health actors as part of a national health system is gaining ground due to their connections with others in the community and their non-bureaucratic elements composed mainly of trust, reciprocity and sustainability .A previous study on five sub-Saharan African countries found that \u2018hidden\u2019 actors played a key role in access to care and hence contributed to improved child survival . The exaThe unforeseen situations that increasingly typify the day-to-day lives of individuals in poor rural households, such as food shortages, prolonged illness, prolonged droughts and sudden death, force households in the area to develop an informal insurance system built on trust and general reciprocal exchange of support. This support based primarily on need extends beyond kinship and beyond direct exchange of services. Through trust and participation in reciprocal activities, individuals remain connected and cooperate for long-term mutual benefit. Such reciprocal activities in the study context of rural Uganda include care for the sick and frail, labour and food exchange, midwifery, child care, money-lending, funerals and weddings.Furthermore, it is notable that the strong reciprocal social relationships that emerged in this study took place in the context of free healthcare from MRC, which could have been expected to solve most healthcare needs and lower the need for having to rely on kin, neighbours and the general community. It shows that availing the free healthcare addressed only one aspect of what households with sick individuals needed in this setting. It is also likely that the free healthcare allowed household members\u2019 to concentrate time and resources in other areas of need. Thus, the provision of free health care could have had the additive effect that people were able to care for each other\u2019s non-medical needs more.A comprehensive social protection model that provides health services so that access by rural households is improved and that provides financial protection might be too resource-intensive for a setting with limited resources. Moreover, given that experiences with chronic illness and the types of needs can be context-specific, strategies and responses that are locally developed and embedded in that specific context are important to consider . In the In this analysis we found that involvement of neighbours and fostering of children in need had become increasingly important. The involvement of neighbours may have been driven by the increasing fragmentation of families due to out-migration, hence the neighbours becoming more important due to their proximity. Their participation in reciprocal exchanges of support increased households\u2019 social assets. Drawing on neighbours was common when the needs within the close kin exceeded the available resources. Studies of orphan welfare in rural Uganda , 40 founIn this community you will always get something for free when need arises\u201d. Individuals gave support without expecting immediate reciprocation, or even any reciprocation, from the recipient. In the same way, they were not always able to redeem support directly from the households and individuals they had previously helped when they needed support themselves. Their good will to give had been registered and was regarded as a good reputation by others in the community, and would most likely lead to being helped when in need. This way of investing for future eventualities is essential in relation to health care, especially due to variations in timing of individual health needs, such as during prolonged illness, and when the person in need can no longer contribute something in exchange for the care. Most importantly, this also illustrates a cohesive attribute of social relations that makes them a potential resource for a health system that is aiming to improve access to healthcare for its population.The life history accounts in the present study indicate that there are several benefits from reciprocal social relations. One of them is being able to trust and expect that needs would be met by others, which gave individuals the motivation to invest in generalised reciprocal support, for example when Bettina commented on helping the elderly lady who could not help her back: \u201cThus generalised reciprocal relations and trust provide a form of social protection to household members during prolonged illness, and hence financial protection. However, the individual and household material deprivations due to overall social and economic factors and the national political choices should not be downplayed. Such deprivations can influence the ability to participate in reciprocal activities and hence undermine access to health services and protection from catastrophic expenditure on health services. Moreover, social networks can be socio-economically costly, and these costs are borne more heavily by those with fewer economic resources and marginalised groups, such as women, children and the elderly. In this study, lack of resources such as land, food and accommodation was common among the women that relocated or cared for neighbours and friends. Social relations have also been associated with negative health outcomes such as stress, especially in situations where individuals with limited resources find it a burden to contribute to the network. Therefore, care should be taken to assess the quality of social relations and strengthen the aspects that are beneficial for healthcare strategies, while finding approaches to minimise those aspects that are harmful to health.This analysis of the experience of individuals in accessing free medication and treatment, their needs and the way in which they meet these needs during chronic illness provides unique insights for health systems seeking to improve access in rural communities in similar contexts. It was found that improving access to healthcare while providing financial protection for the rural poor depends on broader social protection. In the rural population studied, this broader social protection was achieved through reciprocal exchange of support to address healthcare needs, hence mediating access to free medical care and also meeting other needs."} +{"text": "Ureaplasma diversum ATCC 49782 was undertaken as a step towards understanding U. diversum biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)\u2014Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome . In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of U. diversum was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable U. diversum. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, U. diversum has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis.Whole genome sequencing and analyses of Ureaplasma diversum is a bovine ureaplasma that was first isolated in 1969. Initially, it was defined as a non-pathogenic species, but recently it has been shown to cause damage to bovine tissue cells and organs [U. diversum is frequently found in the genital tract of cattle and is associated with major genital disorders in these animals [U. diversum have shown infertility, placentitis, fetal alveolitis, and abortion or birth of weak calves [U. diversum may cause low sperm motility, seminal vesiculitis, and epididymitis [U. diversum and reproductive disorders in bovine remains controversial, mainly because high rates of positive vaginal cultures were also detected in animals with normal reproductive rates [ animals , 10, 11.k calves , 12, 13.ve rates .U. diversum is a facultative intracellular microbe, i.e. can be detected inside cells or adhered to their surfaces [U. diversum exerts a temporal modulation of the host programmed cell death. Invasion of bovine spermatozoids by U. diversum has also been linked to low sperm viability, suggesting that U. diversum may contribute to the death of these cells [U. diversum also was capable of inducing significant TNF-alpha production in the uterus of experimentally infected mice [U. diversum was undertaken as the first step towards understanding the mechanisms by which this microorganism causes disease and establishes infection, as well as to gain new insights into the biochemical pathways.surfaces . Recentlsurfaces , but as se cells . U. diveted mice , which ited mice , 17, 18.ted mice , 20. TheU. diversum ATCC 49782 are summarized in The general genome features of U. diversum ATCC 49782 and the human ureaplasma serovars [U. diversum and a slight higher G+C content when compared to other ureaplasmas . U. diversum showed 782 CDSs; but U. urealyticum and U. parvum have an average of 608 CDSs. The hypothetical CDSs in U. diversum are 279, in U. urealyticum the average is 230 and in U. parvum the average is 201. These genetic differences may reflect the host specificity of U. diversum.Comparisons among parvum) indicateU. diversum and human ureaplasmas. CDSs that are conserved among the three species do not have the same organization, suggesting significant genomic reorganization , an ammonia/ammonium transporter, and a FOF1-ATPase. Ureaplasma generates 95% of its ATP through the hydrolysis of urea by urease [U. diversum to acquire this metabolite from the environment is unknown.Analyzing the predicted set of aplasmas . The maiy urease , 24. Hydy urease , nickel y urease . It is bU. diversum is incomplete; the U. diversum genome does not contain orthologs for the pyruvate dehydrogenase complex. Therefore, the production of ATP from the oxidation of pyruvate to acetate is unlikely. Other mycoplasmas and ureaplasmas also do not contain this enzyme, but this in vitro activity has been described [U. diversum is incomplete. Only two putative PTS components genes were annotated . The PTS is found only in bacteria which catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives [The sources of the 5% of ureaplasma ATP production not from the urea hydrolysis are most probably obtained from substrate phosphorylation . CDSs coescribed . Moreoveivatives . Thus, iU. parvum [As observed in other mycoplasma species as well as with . parvum , the pen. parvum . TherefoU. diversum. This enzyme has been related to the interconversion of NAD+ and NADP+ in nicotinate/nicotinamide metabolism, which plays a critical role in maintaining the NADH/NADPH pool balance inside the bacterial cell [The Nicotinate/Nicotinamide metabolism appears to be incomplete but functional for NADPH generation. The presence of the enzyme NAD kinase was observed in ial cell .de novo biosynthesis of purines or pyrimidines were not identified in U. diversum genome. We also did not identify the enzyme ribonucleoside diphosphate reductase responsible for the conversion of ribonucleosides to deoxyribonucleosides. Like U. urealyticum, U. diversum could import all its deoxyribonucleosides and/or deoxyribonucleoside precursors, or have a different mechanism for converting ribonucleosides to deoxyribonucleosides. Moreover, the absence of the enzyme thymidine phosphorylase suggests that thymidine is the precursor imported for dTTP production [As expected, CDSs for the oduction or anothde novo synthesis of saturated and unsaturated fatty acids [U. diversum. Among the few identified pathways, metabolic reactions from glyceraldehyde-3-phosphate to cardiolipin are mostly preserved, but one enzyme is missing, the phosphatidyl glycerophosphatase. The role of this enzyme may be replaced by the enzyme cardiolipin synthase, which can convert cytidine 5' diphosphate diacylglycerol directly to cardiolipin in the presence of phosphatidylglycerol. Moreover, phospholipids are of exclusive bacterial origin, therefore excluding the possibility that it is provided by the serum lipids normally present in broth media, the only possibility is that it is synthesized [Mycoplasmas are thought to be completely incapable of fatty acid biosynthesis from acetyl-CoA, probably due to the loss of genetic material. Phospholipids, glycolipids and sterols are the three major lipid constituents of cell membranes. This pathway has been poorly described in mycoplasmas and not all enzymes are identified . Ureaplaty acids ; howeverthesized .U. diversum , phospholipase (gudiv_472), hemolysin (gudiv_91), and a Mycoplasma Ig binding protein (MIB)\u2014Mycoplasma Ig protease (MIP) system (gudiv_161/gudiv_162 and gudiv_612/gudiv_613) were found and are described below in more detail. In addition, CDSs that are likely related to the production of a capsular structure (gudiv_216) and surface molecules were observed. A scheme of these virulence factors is found in To date, only few features have been associated with virulence of ic cells . After aU. diversum. Nevertheless, the analysis of the organization of the U. diversum urease gene cluster revealed the same organization of the human ureaplasma urease gene clusters [ureA (gudiv_255), ureB (gudiv_254), and ureC (gudiv_253). Furthermore, downstream of ureC, four CDSs coding for accessory proteins were found activities have been reported in human ureaplasmas, however, no genes showed significant similarity to known sequences of PLC, PLA1, or PLA2 [U. diversum genome. This is somewhat surprising, considering a recent report showing that U. diversum reference strains, including the ATCC 49782 sequenced herein, and clinical isolates have high phospholipase activity [in vitro invasion of Hep-2 cells by U. diversum [U. diversum are able to interfere with prostaglandin E2 and prostaglandin F2a production by bovine endometrial cells and induce premature labor [U. diversum, genes encoding phospholipase D family protein (PLD) (gudiv_472) and triacylglycerol lipase (gudiv_748) were identified. Therefore, in contrast to human ureaplasmas, the PLD is likely the true virulence factor of U. diversum linked to cell invasion and premature labor in intrauterine infection, suggesting the phospholipase activity detected in our previous study [An array of potent hydrolytic enzymes has been identified in mycoplasmas, including phospholipases, proteases and nucleases . Phospho or PLA2 . Ortholore labor . In U. dU. diversum. The predicted protein sequence has 63.1% identity with the hemolysin found in human ureaplasmas. In U. parvum serovar 3, this hemolysin has been characterized as an \u03b1-hemolysin (hlyA) with both hemolysin and cytotoxic activities [U. diversum genome. It seems that the mycoplasma or ureaplasma hemolysins belong to a unique class of bacterial toxins; more studies are needed to better understand the role of this enzyme in the pathogenesis of bovine ureaplasmas.Hemolysins cause lysis of erythrocytes by forming pores of varying diameters in the host cell membrane . There itivities . Other mtivities , 41, butMollicutes: the MIB-MIP system [U. diversum genome. However, experimental studies should be conducted to better understand the role of this enzyme in the pathogenesis of bovine ureaplasmas.Recently, a new virulence factor has been described in P system . MIB actP system . AccordiU. diversum acts as a virulence determinant by binding to and internalizing within human airway cells [Among the detected lipoproteins, one CDS was identified as a membrane nuclease lipoprotein (gudiv_93). Membrane nuclease activity in mycoplasmas was first reported in pulmonis . Nucleasoduction , but it ch other , and havsepticum . Additioay cells .M. pulmonis. Antigenic variation of surface proteins is thought to be a survival strategy for many mycoplasmas [M. bovis possess a large family of variable surface lipoproteins designated as Vsps, which are encoded by 13 different genes. The Vsps undergo phase variation in the respiratory tract of infected calves, which limits the microorganism elimination [Another important lipoprotein that has been identified is a putative variable surface antigen lipoprotein (gudiv_179) with 36% identity to the VsaA protein of oplasmas . M. bovimination . The expmination .U. diversum ATCC 49782 (gudiv_653) (in vitro and in vivo [U. diversum features a similar organization with the MBA locus of U. parvum serovar 14 (UPA14) and the variable surface antigen lipoproteins (gudiv_179) are in the same paralogous gene family in U. diversum. These two proteins, added to fourteen other paralogous proteins , may play a critical role in modulating the immune response against U. diversum (Although with borderline similarity (33%) a multiple banded antigen like protein (MBA-like) was annotated in the genome of div_653) . This pr in vivo , 55. The in vivo . Analyzi in vivo , we can (UPA14) . In partdiversum .U. diversum, a glycosyltransferase enzyme (gudiv_216) was found [M. mycoides subsp. mycoides, the presence of glycosyltransferase is believed to be related to the attachment of galactan to the capsule [U. diversum. By using electron microscopy and the red ruthenium dye, a polysaccharide capsule extending from 11 to 17 nm outside of U. diversum cells was observed for the first time and a glucose-1-phosphate uridylyltransferase that uses glucose-1-phosphate to generate UDP-glucose [in silico analysis, we did not observe genes related to the biosynthesis of polysaccharides. However, the presence of two genes of a sugar transporter was observed . We believe it is likely that these sugars are captured by U. diversum and are incorporated using glycosyltransferase to form the capsule.The chemical composition of the capsular material of acterium . The bioectively . Probabl-glucose . In our U. diversum surface molecules, we infect murine macrophages J774 with viable and nonviable microorganisms. In the analysis of cytokine production, there was an increased production of pro-inflammatory cytokines in both conditions compared to the negative control (p<0.05), but there was no statistical difference between the profile triggered by the viable and nonviable ureaplasma that, when recognized by the innate immune system, trigger the production of pro-inflammatory cytokines from manifold cells, causing inflammation [Mollicutes to induce the secretion of inflammatory cytokines such as IL-1\u03b2, TNF-\u03b1 and IL-6 have been reported in studies with different species, e.g. U. diversum [U. urealyticum [M. fermentans [In order to better understand the immune response against eaplasma . Therefoammation \u201367. The rol p<0.0, but thealyticum , 69, andrmentans .U. diversum cells. It can be observed that U. diversum induced a higher gene expression of TLR2 when compared to the uninfected cells and inflammasome pathway genes were also evaluated using murine macrophages J774 infected with viable ed cells (p<0.05)llicutes . Howeverand TLR4 . Moreovermentans , 72.U. diversum. A study of the development of gastric tumors using monocytes exposed to M. hyorhinis confirmed a high production of IL-1\u03b2 and IL-18-induced NLRP3-dependent mechanisms [Acholeplasma laidlawii have the capacity to promote the release of IL-1\u03b2 by the presence of cytosolic ASC , promoting inflammasome activation [U. diversum. This study is the first report of the activation mechanisms involved in the immune response against Ureaplasma diversum. These findings can contribute to the studies on macrophage activation for the regulated secretion of cytokines to prevent tissue damage caused by an exacerbated immune response. The results described herein will also support further research to understand the possible effector mechanisms that lead to effective protection against U. diversum infection.The results of the inflammasome expression assay demonstrate the up-regulation of genes related to chemokine binding domains, interleukin-6, inhibitors of kinases and B cells, NF-kB, and interferon regulatory prostaglandin endoperoxide . In contchanisms . It has tivation . In the U. diversum on a genetic basis. Furthermore, extensive analyses of these data helped us to detect important biological features of this mollicute and its mechanisms of pathogenicity. The results of this study will help to better understand this microbe, and can direct future research on the pathogenicity of this bacterium in cattle. Moreover, it will also aid the development of new strategies for treatment, prevention and control of ureaplasma infections.The whole genome sequence described herein represents a valuable new resource for the study of Ureaplasma diversum ATCC 49782 was originally isolated from a cow with clinically acute granular vulvitis in 1978 [U. diversum ATCC 49782 was first cultured in 2 mL of ureaplasma medium (UB) at 37\u00b0C, followed by propagation in 3,000 mL of the same broth. At the logarithmic growth phase (based on colorimetric changes), the culture was centrifuged at 20,600 x g for 30 minutes at 25\u00b0C. The DNA was extracted using a PureLink\u2122 Genomic DNA Mini Kit following the manufacture\u2019s instructions. in 1978 . The vir in 1978 . U. diveU. diversum strain ATCC 49782 has been deposited in the GenBank\u00ae database under the accession no. CP009770.The whole genome was sequenced from a paired-end library using Illumina HiSeq 2000 at the Purdue University Genomics Core Facility. Average reads of about 100 bases were assembled using ABySS 1.2.7. After assembly resulting from a 4,552X genome coverage, eight remaining gaps were closed using conventional PCR, followed by sequencing using Sanger method in both directions. The genome sequence of U. parvum serovars 3, may be outdated , we used tblastn to search for possible misannotated sequences when an ortholog was not found. A diagram of the overall structure of genome was generated using a DNAPlotter version 1.4 from Artemis 12.0, Sanger Institute [First-pass annotation was achieved using the NCBI prokaryotic genome annotation pipeline (PGAP). An initial set of CDSs was identified using PRODIGAL and analnstitute .U. diversum, U. urealyticum and U. parvum were performed using Sybil [Whole genome synteny comparisons among ng Sybil . In SybiPhylogenetic analyses of the 41 mollicutes were also performed using a multiple sequence alignment of 32 concatenated protein sequences from each organism . These pThe ureaplasma was cultured in 50 mL of UB media. Cells in the logarithmic growth phase were collected by centrifugation at 20,600 x g for 30 minutes, and the pellets were suspended in 3% of glutaraldehyde and 0.2% of ruthenium red in 0.1M-cacodylate buffer (pH 7.4). Ruthenium red is a polycationic dye that binds polysaccharides and is frequently used to detect bacterial capsules . After f2HPO4; 1.47 mM KH2PO4) (to avoid contamination of the polysaccharides derived from the culture medium), and resuspended in 10 mL of the same buffer. The extraction of capsular polysaccharide continued by the phenol-chloroform method [-1 of trifluoroacetic acid, at 100\u00b0C for 8 h. The samples were dried under nitrogen stream and then dissolved in water 300 \u03bcL and the pH was elevated to 8 by addition of NH4OH. The samples were reduced with NaBH4 at room temperature for 4 hours, then the cationic resin was added to remove salts, the samples were dried under nitrogen stream and followed by dissolution/dryness process in methanol to remove the residual borate. The alditol products were acetylated by addition of 200 \u03bcL of acetic anhydride-pyridine , held overnight at room temperature. Methanol (200 \u03bcL) was added to stop reaction and the samples were dried under nitrogen stream. The alditol acetates were analyzed by gas chromatography coupled to mass spectrometry with Ion Trap analyzer. The chromatography was developed in a DB-225-MS fused silica capillary column . The temperatures were: injector 250\u00b0C and the oven programmed from 50\u00b0C to 220\u00b0C at 40\u00b0C min-1. The monosaccharides were identified on the basis of their mass spectra at electron ionization and authentic standards (Sigma-Aldrich).To extract and purify the capsular component, the bacterial strain was cultured in 2 liters of UB media and then centrifuged to retain the pellet. The pellet was washed twice in PBS were performed with viable bacterial strain and bacterial strain inactivated by heat (100\u00b0C for 30 minutes) for 24 hours (MOI 1:100). The inactivation was confirmed by absence of a positive culture on ureaplasma medium (UB). Negative controls without bacteria inoculation were also used. After 24 hours, the supernatant of the bottles was collected and frozen at -80\u00b0C to perform the ELISA. The cells were washed with trypsin and the cell suspension was placed in microtubes with RNA later for RNA extraction procedures.The dosage of the cytokines TNF-\u03b1, IL-1\u03b2, IL-6, IL-10 was set using Ready-SET-GO enzyme-linked immunosorbent assay kit . The mRNA from cells was extracted using TRIzol Plus RNA purification kit , following the protocol supplied by the manufacturer. The cDNA was obtained using a retro-transcription (RT) from the mRNA using the SuperScript \u00ae III Reverse Transcriptase kit. cDNA was used in a qPCR reaction to determine gene expression of TLR's 1\u20139 and 11 according to a previously described protocol . The RT-2 Profiler\u2122 qPCR Array kit for the expression of 84 key genes involved in the function of inflammasomes, protein complexes involved in innate immunity, as well as general NOD-like receptor (NLR) signaling (Gene expression of the inflammasome pathway was verified by quantitative PCR (qPCR) methodology. The cDNA obtained was subjected to analysis using Mouse Inflammasomes RTignaling . This arS1 FigStatistical significance (p<0.05) is represented by the asterisk (*) .(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file."} +{"text": "In an Editorial, Edward Maibach and colleagues discuss the important role of health professionals in future responses to threats of climate change. In the 2015 Paris Climate Agreement, 195 nations agreed to work together to hold the rise in global temperature to \u201cwell below 2 degrees Celsius.\u201d Recent research by the Intergovernmental Panel on Climate Change (IPCC) suggested the need for an even more ambitious goal: limiting the warming to 1.5\u00b0C to avert the considerable risks to human health, livelihoods, food and water supplies, security, and economic growth that are likely with 2\u00b0C of warming [Therefore, this moment is one of extraordinary consequence. Actions taken by all nations over the next decade will determine whether global health will continue to improve or whether it will instead decline\u2014possibly catastrophically so\u2014as a result of climate change.Lancet Countdown [Numerous recent scientific assessments\u2014including the IPCC 1.5\u00b0C report [ountdown \u2014have madEmissions of heat-trapping carbon pollution reached an all-time high in 2018 . Global This prospect should be of grave concern to\u2014and should serve as a clarion call for\u2014all health professionals. As health professionals, we pledge to do no harm. We often aspire further, to serve humanity by preventing needless harm and suffering and to foster conditions that give every person a fair chance at the blessings of good health\u2014especially those who are most vulnerable. Climate change directly imperils these aims.Our collective contributions to human health and well-being over the past century have been remarkable. We have ushered in major improvements in health and longevity. Now, an entirely new challenge demands our full attention\u2014the challenge of stopping and protecting people against the harms of climate change. Over the next decade, the nations of the world must embrace and accomplish three difficult but achievable objectives so that human civilization and the ecosystems on which we depend can continue to flourish: creating a clean energy economy, drawing down excess atmospheric carbon, and preparing for and adapting to health impacts.The world\u2019s transition to a clean energy economy must be greatly accelerated . This can be done with wind, solar, geothermal, hydro, improved energy storage capabilities, smart electrical grids, and nuclear energy\u2014although there are legitimate concerns about the inclusion of nuclear energy. This global transition has already started but must be greatly accelerated and must be completed within the next the next few decades. The Solutions Project has developed potentially viable plans for every state in the United States and for most nations in the world to demonstrate the feasibility of this objective ,8.The excess heat-trapping pollution in our skies must be harvested and put back into the ground and/or used to make solid products\u2014reducing this pollution to the levels seen in preindustrial times, a reduction of about one-third from current levels. This can be done through natural and technological carbon-capture technologies, including and perhaps especially improved land use, forestry, and agricultural practices. This is a global project that will take decades to accomplish, but the sooner it begins at scale, the sooner levels of heat-trapping pollution will begin to drop. Project Drawdown has identified a range of the most cost-effective approaches that are ready for widespread adoption .Every community, state, and nation must prepare itself for the unavoidable health impacts of climate change that are already happening and will continue to occur with greater frequency and severity over the next several decades (or longer)\u2014as a result of inertia in the climate system. In the US, for example, the Centers for Disease Control and Prevention\u2019s (CDC) Building Resilience Against Climate Effects (BRACE) framework provides recommended practices that can guide communities in this effort . The heaHealth professionals and the organizations that represent us must become tireless champions for these objectives and for the global agreements that will enable them\u2014especially the Paris Climate Agreement and the Sustainable Development Goals. In a world rife with rapidly eroding trust, we remain trusted honest brokers who place public interests above all others . In thisThe voices of health professionals as champions and advocates for human health and sustainable ecosystems\u2014in communities, states, nations, and global negotiations\u2014is imperative. When our voices are absent, those most loudly heard by the public and policy makers are those of major corporations\u2014especially fossil fuel companies whose business models are currently tied to the extraction and pollution that is directly harming human health and is jeopardizing the very possibility of continued human prosperity. The claim that, for the foreseeable future, the fossil fuel economy is the only viable economy is made in full knowledge that fossil fuel use causes irreparable harm to human health and to the earth\u2019s climate system, on which all human life and prosperity depends.Those with strong vested interests in the fossil fuel economy are likely to assert that we health professionals should \u201cstick to our own lane\u201d and stay out of complicated policy dialogues that we are ill-prepared to understand, much less advance. For us to acquiesce to that assertion would be irresponsible: protecting the health and well-being of people individually and collectively is our lane. We need not become experts in areas of policy outside our lane. Rather, we must only participate in the relevant policy discussions to ensure that the existentially important objectives we are advocating for are properly and fully considered. Failure to protect the health of people in this generation\u2014and potentially the next hundred generations\u2014is not an option.In addition, we should lead by example\u2014in our practices, our hospitals, our health departments, and our own lives. Organizations have already arisen from within our ranks\u2014including Health Care Without Harm, Practice Green Health, and My Green Doctor\u2014that are helping countless numbers of our peers to lead by example. We should all embrace this opportunity\u2014both because the practice of healthcare and public health should not continue contributing to the problem and because leading by example is the most effective form of leadership.An ancient proverb holds that wrapped in every crisis is an opportunity. The crisis of climate change is a perfect example. Those who argue that responding to the climate challenge is risking our prosperity have it exactly wrong. That is because the actions that must be taken will not only ensure the long-term viability of human civilization but will also soon produce greater health and wealth for all nations that rise to the challenge. Our air and water will be cleaner, our health better, our productivity greater, and our economies stronger. Climate solutions are health solutions, and health solutions are economic solutions. Health professionals are among the people best positioned to make sure that the public and policy makers understand this. Carpe diem."} +{"text": "Poor diet including inadequate fruit and vegetable consumption is a major contributor to the global burden of disease. Aboriginal and Torres Strait Islander Australians experience a disproportionate level of preventable chronic disease and successful strategies to support Aboriginal and Torres Strait Islander people living in remote areas to consume more fruit and vegetables can help address health disadvantage. Healthy Choice Rewards was a mixed methods study to investigate the feasibility of a monetary incentive: store vouchers, to promote fruit and vegetable purchasing in a remote Australian Aboriginal community. Multiple challenges were identified in implementation, including limited nutrition workforce. Challenges related to the community store included frequent store closures and amended trading times, staffing issues and poor infrastructure to support fruit and vegetable promotion. No statistically significant increases in fruit or vegetable purchases were observed in the short time frame of this study. Despite this, community members reported high acceptability of the program, especially for women with children. Optimal implementation including, sufficient time and funding resources, with consideration of the most vulnerable could go some way to addressing inequities in food affordability for remote community residents. Food security is a major global issue . StrategThe life expectancy gap of 10\u201311 years between Aboriginal and Torres Strait Islander people and non-Indigenous Australians is well known . Recent Many factors influence the nutritional status of Aboriginal and Torres Strait islander people, including socioeconomic disadvantage and other historical, social, environmental and geographical factors ,7,8. HeaThere is a well-established link between increased fruit and vegetable consumption and improved health outcomes ; consequIn the context of increasing health care costs and government budget cuts threatening progress in the prevention of chronic disease , it is iStrategies to make fruit and vegetables more accessible to Aboriginal and Torres Strait Islander families living in remote communities have the potential to reduce health inequality and subsequent health care costs. Here we report on a feasibility trial of a monetary incentive to promote fruit and vegetable purchasing in one remote community. To our knowledge, the effectiveness of immediately rewarding healthy purchasing behaviours has not yet been explored in a remote Aboriginal and Torres Strait Islander community context. Apunipima Cape York Health Council (Apunipima) community nutrition project staff conducted a study in 2015 to assess the feasibility of implementing a fruit and vegetable incentive in a very remote Australian community store in far north Queensland, located around 2500 km from the nearest major city. This remote community has approximately 1400 residents with most (90.4%) identifying as Aboriginal and/or Torres Strait Islander . The comA mixed methods approach was used and included collection of qualitative data using semi-structured interviews, participant observation, a weekly electronic survey on store and wider community contextual information and a quantitative assessment of store sales data. Feasibility of the intervention was assessed in terms of acceptability, voucher uptake, implementation issues and impact on fruit and vegetable sales. All customers of the store were eligible to participate. Study implementation was led by Apunipima community nutrition staff. The Healthy Choice Rewards (HCR) program offered community store customers an incentive of a fruit and vegetable voucher to the value of AUD $10 each time a set minimum amount was spent on fruit and vegetables. Store staff participated in semi-structured interviews prior to the study to inform the reward system design and determine what supports would need to be in place for implementation. Two phases of the minimal amount spent were trialed: phase one required a AUD $20 spend on fresh fruit and vegetables to receive a AUD $10 HCR voucher to be redeemed on the date of purchase; phase two required a AUD $15 spend on fresh fruit and vegetables to receive a AUD $10 HCR voucher to be redeemed within three days. Frozen, tinned and dried fruit and vegetables were not included as part of the minimum spend as they could not be easily distinguished in the store\u2019s electronic grocery management system. The vouchers were redeemable for fresh, frozen, tinned or dried fruit and vegetables and excluded tinned fruit in syrup and frozen potato chips and wedges. Vegetable packs valued at AUD $10 were available for sale. The store was reimbursed for the value of any vouchers used. The incentive was available for 32 weeks; phase one ran for 15 weeks, followed immediately by phase two for 17 weeks. The HCR vouchers appeared as black and white plain text print outs at the end of customer store dockets. The reward offer was promoted in English and local language using posters, flyers, radio advertisements and electronic register screen displays at the store.Project staff visited the community monthly during the intervention period to promote HCR. During the visits they delivered healthy cooking demonstrations, distributed healthy recipe flyers, spoke with community members on how to utilise the offer and assisted store staff in merchandising the fruit and vegetable display. Between visits the project team provided weekly phone and email support to the store manager to maintain program promotion and assist with processing the vouchers. To determine the feasibility of the HCR program the primary outcome measures included: acceptability of the voucher incentive to customers and store staff, voucher uptake and redemption and identification of the opportunities and challenges of implementation. A secondary measure included per capita total fruit and vegetable intake derived from store sales purchasing data. n = 34) received a fruit and vegetable voucher to the value of AUD $10 to acknowledge their time contributed. In addition to the customer satisfaction interviews conducted at the completion of the program, four customer interviews were conducted during phase one to inform intervention changes for phase two. Following completion of both phases of the intervention, we invited store staff and store customers (community members) to provide feedback through semi-structured face-to-face interviews (customer or staff satisfaction interviews). Demographics on age, gender, Aboriginal and Torres Strait Islander status, and employment were collected. Project staff (one of whom is a Torres Strait Islander woman) with training and experience interviewing Aboriginal and Torres Strait Islander community members conducted the interviews. To promote the feedback opportunities to the community, the team engaged in local activities including performing a healthy cooking demonstration and organising a group fishing trip with a healthy lunch. All customer and store staff interviewees [James Cook University Human Research Ethics Committee (HREC H5938) and the combined Northern Territory Department of Health and Menzies School of Health research Human Research Ethics Committee (HREC 2014-2313) granted ethical approval for the study.n = 6) reported they wanted the offer to occur again. Community members identified that healthy eating was important for health but there were many challenges and competing priorities to eat a healthy and nutritious diet. They also provided suggestions for improving the program.A total of 28 post program customer satisfaction interviews were completed. The majority of customers interviewed identified as Aboriginal and/or Torres Strait Islander people (82.1%) and were women (71.4%). Additionally, 68% of responders reported being employed at the time of the interview. Of those interviewed, more women that were employed than not employed reported using the HCR voucher. All respondents reported they would like the offer to continue and 61% of respondents indicated that HCR encouraged their family to consume more fruit and vegetables. All store staff interviewed following the completion of the project , those relying on meals provided by family (such as the frail elderly) and even people who struggled with addictions such as alcohol, drugs or gambling. Feedback from the customer interviews suggested that future incentives may be more effective if the reward system was tailored specifically for women with children and used electronic store loyalty cards instead of paper-based vouchers. Other recommendations from the interviews included: increased flexibility of redemption parameters, more support from store staff (such as explaining the voucher and helping determine AUD $10 worth of fruit and vegetables so it is more convenient for the customer), offering higher incentives and strengthening promotion through increased community involvement. Store staff observed an increase in customer interest in HCR following promotion by the visiting nutrition team and noted that customers reported that uptake of the incentive could have been be improved with greater promotion. Voucher redemption rates averaged 28.6% for the duration of the study. A total of 2150 vouchers were issued and 632 redeemed. Redemption rates were higher during phase two of the study compared to phase one, averaging 30% and 27% respectively. The highest redemption rate (44%) was recorded on a week when project staff were at the store performing cooking demonstrations raising awareness about the project and assisting with merchandising of fresh produce.Four of the six staff interviewed reported having issues with the reward offer and required more support to run the offer. Issues identified by store staff included: being unsure of how to process the voucher in the store electronic grocery management system; having too many customers at once to help other customers claim their voucher; limited time to prepare the AUD $10 fruit and vegetable packs for customers to redeem; customers complaining of losing their receipts and customers refusing the voucher as it meant they needed to queue up a second time to redeem their reward.Several challenges that impacted on project implementation at the store level were observed by project staff including store infrastructure issues; support for store staff to run the offer; and support for store managers to promote fresh produce. Fresh produce displays were impacted by transport issues; infrastructure issues, such as limited equipment to display produce; the hot climate affecting the temperature control of open display refrigeration units; and store air-conditioning and refrigeration units often breaking down. Supporting store staff proved challenging due to a shortage of trained and experienced staff; high turnover and low attendance among store staff; variable expertise among store management in merchandising of fresh produce and limited capacity of Community and Store Nutritionists to provide sufficient support to store staff. In addition, due to issues impacting the community during the study period such as community unrest, forced store closures and amended trading hours were reported on 23 out of 32 weeks.Implementation of the project was strengthened by the existing rapport of project staff with community and regular presence in the community; strong partnerships with industry and the research sector; and support from community, local Council and the Store Group to implement the project. p = 0.01. Non-significant reductions in sales of vegetables and overall food and drink sales were also observed. The voucher incentive was not successful in increasing fruit and vegetable store sales during the study period compared to sales for the same time period in the previous year. In fact, despite including voucher purchases, a 7% reduction in total fruit sales was observed between the two periods, decreasing from 41 to 38 g/person/day (0.27 to 0.25 serves/person/per day), This feasibility study describes a monetary incentive strategy to promote fruit and vegetable consumption in a remote Aboriginal community in Australia. While we were unsuccessful in increasing fruit and vegetable purchases during the intervention period, qualitative data indicates that there was a high level of acceptability of the program by community members. This study also highlights the many challenges to be considered in implementing food subsidy strategies to improve nutritional health in the remote community context. The HCR project was completed in 2015 as part of implementing a key objective of the Cape York Food and Nutrition Strategy\u2014to ensure equitable food affordability, availability and access comparable to urban Australia . This prAlthough the project staff made frequent trips to the community, store staff and management identified the need for more support. Furthermore, interview data suggested that voucher uptake could have been improved with strengthened promotion. These findings are consistent with other studies demonstrating that consumers need to be made aware of promotional offers . LimitedWomen who were employed were most likely to report using the HCR in customer satisfaction interviews. Qualitative data indicated that healthy eating was considered by community members to be more of a priority for women with children or young families. Given that improving access to nutritious food for at-risk mothers, infants and children is a key priority of the 2013\u20132023 National Aboriginal and Torres Strait Islander Health Plan , these fThis study provided information of fruit and vegetable consumption data for this community which differ from other information sources. Average fruit and vegetables sales were estimated to be equivalent to 0.25 serves/person/day for fruit and 0.92 serves/person/day for vegetables during the study period. These results are lower than self-reported data from the 2012\u20132013 National Aboriginal and Torres Strait Islander Nutrition and Physical Activity Survey (NATSINPAS) which reported Aboriginal and Torres Strait Islander people living in remote areas across Australia consume on average 0.9 serves of fruit and 1.7 serves of vegetables per day . The NATThe limitations of this study are that it was conducted in one remote community only and for a short time period, with limited staffing. These factors reflect the currently limited resources available for nutrition promotion in this setting, compared to previous investments . With adAnother strength of this project was the strong partnerships and relationships formed by the project team with the community, particularly with the local community store as HCR was supported by store managers, staff and at management levels of the store group. Store Managers play an important role in food supply in remote Aboriginal and Torres Strait Islander communities and are therefore essential partners in helping to improve dietary intake . This prRemote community stores have an important influence on community health through their ability to control the availability and accessibility of both healthy and unhealthy foods . SignifiThis mixed methods feasibility study showed high levels of acceptability of the program by community. It also resulted in the identification of several challenges to be considered when implementing a food subsidy strategy or incentive in remote Australia. Investing in remote retailers to overcome the challenges in providing healthy foods is critical to the success of any efforts to improve fruit and vegetable consumption in remote Aboriginal and Torres Strait Islander communities. Additionally, increased investment in a nutrition prevention workforce to implement healthy remote store practices and support retailers to promote nutrition is required. Feedback from customer interviews suggested that future incentives may be more effective if the reward program was tailored specifically for women with children. A larger scale controlled study targeting women and children may provide greater insight into the use and appropriateness of a fruit and vegetable subsidy in the remote community context. Consumer food subsidy schemes can help overcome financial barriers and increase affordability of healthy food and drink in remote areas. The high rates of food insecurity in remote Aboriginal and Torres Strait Islander communities are largely a consequence of high rates of unemployment and low incomes compounded by high food costs. Government commitment is needed to reduce the underlying social inequality and to address the affordability of healthy food choices to help close the gap in Aboriginal and Torres Strait Islander health."} +{"text": "The correct name is: Anlan Yang. The correct citation is: Sah RK, Yang A, Bah FB, Adlat S, Bohio AA, Oo ZM, et al. (2019) Transcriptome profiling of mouse brain and lung under Dip2a regulation using RNA-sequencing. PLoS ONE 14(7): e0213702."} +{"text": "Mycobacterium tuberculosis. The function of T cells usually decreased and even exhausted in severe TB such as multiple drug resistant TB (MDR-TB), which might lead to the failure of treatment in return. The mechanism of T cell dysfunction in TB is still not clear. In this study we set up a mouse model of T cell dysfunction by persistent M. tuberculosis antigen stimulation and investigated the therapeutic role of interleukin 2 (IL-2) in it. C57BL/6 mice were primed with Mycobacterium bovis Bacillus Calmette-Gu\u00e9rin (BCG) and boosted repeatedly with a combination of M. tuberculosis fusion proteins Mtb10.4-HspX (MH) plus ESAT6-Ag85B-MPT64 <190\u2212198>-Mtb8.4-Rv2626c (LT70) or MH plus ESAT6 and CFP10 with adjuvant of N, N\u2032-dimethyl-N, N\u2032-dioctadecylammonium bromide (DDA) plus polyinosinic-polycytidylic acid (Poly I:C). Following persistent antigen stimulation, the mice were treated with IL-2 and the therapeutic effects were analyzed. The results showed that compared with the mice that received transient antigen stimulation (boost twice), persistent antigen stimulation (boost more than 10 times) resulted in decrease of antigen specific IFN-\u03b3 and IL-2 production, reduction of memory CD8+ T cells, over-expression of immune checkpoint programmed cell death protein 1 (PD-1), and impaired the protective immunity against bacterial challenge. Treating the T cell functionally exhausted mice with IL-2 restored antigen-specific T cell responses and protective efficacy. In conclusion, persistent stimulation with M. tuberculosis antigens induced T cell dysfunction, which could be restored by complement of IL-2.Tuberculosis (TB) is a chronic disease mainly caused by Mycobacterium tuberculosis, which activates protective cell-mediated immune responses is a typical chronic infectious disease caused by esponses . Howeverpatients . CD4+ anrculosis . We and rculosis . We suppT-cell exhaustion was primarily identified in lymphocytic choriomeningitis virus (LCMV) infection , and als+ T cell function and enhance viral control , T cell immunoglobulin mucin 3 (TIM-3), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), are highly expressed on exhausted T cells during chronic viral infection and tumor progression \u201316. Up-r control . PD-1 ( <190\u2212198>-Mtb8.4-Rv2626c) plus LT7Rv2626c) or MH plAll animal experiments were carried out under the guidelines of Council on Animal Care and Use, and the protocols were reviewed and approved by Institutional Animal Care and Use Committee of Lanzhou University. Animals were monitored daily and received free access to water and food throughout the study.M. tuberculosis fusion protein MH without affinity tag on Q-sepharose high performance column, hydrophobic chromatography (HIC) on butyl-sepharose high performance column and gel filtration chromatography (GFC) on Superdex 75 prep grade column were performed with AKTA Purifier 100 .Antigens were prepared as previously described , 31. Thenity tag was highThe method for purification of LT70 without affinity tag included6 bacterial colony forming units (CFU) once via subcutaneous administration and boosted with antigens. Two combinations of antigens were applied to boost BCG in different schedules.Specific pathogen free C57BL/6 female mice (6\u20138 weeks old) were primed with BCG at a dose of 5 \u00d7 10M. tuberculosis antigens including 10 \u03bcg of MH plus 10 \u03bcg of LT70 in 100 \u03bcl via intraperitoneal injection daily for 2 weeks (IL-2 treatment group). Then, all groups of mice were challenged with BCG 1 week after IL-2 treatment. Three weeks later, the immune responses were evaluated. Boosting BCG with the antigens only at 6th and 7th weeks was used as a control of transient antigen stimulation (Ag transience group). The PBS was used as the sham control (Naive group).In the first schedule , 6 weeks of LT70 with the of LT70 via subcM. tuberculosis antigens 5 \u03bcg of ESAT6, 5 \u03bcg of CFP10, and 10 \u03bcg of MH in 100 \u03bcl via intraperitoneal injection daily, starting from 8th week daily for 20 weeks (IL-2 treatment group). One week after IL-2 treatment, the immune responses were evaluated. Boosting BCG with the antigens only at 6th and 7th weeks was used as a control of transient antigen stimulation (Ag transience group). The adjuvant of DDA plus Poly I:C was used as the sham control (DP group).In the second experiment , 6 weeks\u03bcg of MH with the\u03bcg of MH via subc5 cells per well in RPMI-1640 supplemented with penicillin, streptomycin, and 10% newborn calf serum and stimulated with Ag85B and results were expressed as the mean of the spot forming cells (SCFs) per 5 \u00d7 105 cells \u00b1 standard error of mean (SEM).Lymphocytes were freshly isolated by gradient centrifugation from spleens. Mouse IFN-\u03b3 pre-coated ELISPOT kit was performed according to the instruction of the manual , 34. Theth Ag85B and Ag85B (5 \u03bcg/ml) for 4 h at 37\u00b0C for 3 days and the concentrations of IL-2 in culture supernatant were detected by ELISA according to the manufacturer's protocol .Lymphocytes isolated from spleen cells were plated in 24-well plates at 5 \u00d7 10Multiparameter flow cytometry was performed according to a standard protocol , 37. For+ T cells recognizing epitope 4\u201312 of MTB10.4 were analyzed by flow cytometry. As antigen specific memory T cells are too scanty to be detected directly, the immunized mice were stimulated with BCG (for MH plus LT70 immunization) or MH plus ESAT6 and CFP10 (for MH plus ESAT6 and CFP10 immunization) 3 days before the immunodetection to induce memory T cells proliferating and converting into effector T cells (Teff) or effector memory T cells (TEM). Cells were stained with MTB10.44\u221212 PE-conjugated Pro5\u00ae Pentamer and incubated at 22\u00b0C for 10 min. Subsequently, surface staining of these cells was performed using the following anti-mouse monoclonal antibodies including APC-conjugated anti-CD3 , PerCP-Cy5.5-conjugated anti-CD8 at 4\u00b0C for 30 min. Then, cells were resuspended in PBS and analyzed using NovoCyte flow cytometer.CD8Cells were resuspended in staining buffer (PBS containing 5% FBS) and blocked with mouse IgG. Subsequently, the anti-mouse monoclonal antibodies including APC-conjugated anti-CD4 (GK1.5), PE-conjugated anti-CD8 (SK1), FITC-conjugated anti-PD-1 (J43), mouse IgG1 isotype control, were added and incubated with cell suspensions at 4\u00b0C for 30 min. Cells were then resuspended in PBS and analyzed using LSR Fortessa flow cytometer . Results are calculated as percent positive cells as indicated.7 CFU of BCG per mice via caudal vein 6 weeks after the last antigen stimulation or 1 week after IL-2 treatment. Enumeration of CFU in the lungs was determined by serial 10-fold dilutions of whole-organ homogenates on 7H11 medium at 3 weeks after infection. Bacterial load is presented as log10 CFU \u00b1 SEM.To detect the immune protective capability against mycobacterial infection in persistent antigen stimulated mice, the mice constantly stimulated with MH plus LT70 were challenged with 1 \u00d7 109 CFU of BCG per mice via caudal vein 6 weeks after the last antigens stimulation or one week after IL-2 treatment. The mice without antigen stimulation were used as a na\u00efve control. The survival rates throughout the 90 days were analyzed.Mice constantly stimulated with MH and LT70 were challenged with 1 \u00d7 10p < 0.05 was considered statistically significant.All values are expressed as mean \u00b1 SEM. Differences between the variance were analyzed by one-way ANOVA using SPSS13.0 software. A value of M. tuberculosis antigens MH plus LT70 or MH plus ESAT6 and CFP10 repeatedly for more than 10 weeks. One or three weeks after the last stimulation, mice were euthanized to detect the antigen specific immune responses compared with transient antigen stimulation , although there were no obvious changes observed comparing transient antigen stimulation (0.55 \u00b1 0.25) with persistent stimulation for 12 weeks . In these two models, it was confirmed that persistent stimulation by either of the two combinations of antigens caused a decrease in the production of IFN-\u03b3 and IL-2.The C57BL/6 mice were primed with BCG once and boosted with esponses . As for on group . Meanwhiion mice . As for + T cells, the number of antigen specific CD8+ memory T cells was analyzed using the method as previously reported decreased clearly in comparison to transient antigen stimulation group ; After stimulation for 10 more weeks, the numbers of TB10.44\u221212 specific CD8+ T cells (0.1 \u00b1 0.04) were significantly lower than antigen transient group increased significantly at late stage of persistent antigen stimulation (p < 0.05) than both antigen transient group (4.32 \u00b1 0.65) and naive group (2.93 \u00b1 0.17), while the percentage of increasing PD-1 on CD4+ T cells was more obvious than that on CD8+ T cells. The expression of PD-1 on CD8+ T cells in persistent antigen stimulation group (1.7 \u00b1 0.50) was significantly higher than that in naive group was significantly higher compared with the group of transient antigen stimulation .+ T cells was detected again by flow cytometer. The results showed that in persistent MH plus LT70\u2013stimulated mice, the percentage of CD4+ T cells expressing PD-1 was 25.93 \u00b1 1.66, comparing that of 9.05 \u00b1 1.5 in IL-2 treatment group, which indicated that the expression of PD-1 on CD4+ T cells decreased following IL-2 treatment than persistent antigen group without IL-2 treatment with T cell dysfunction (6.75 \u00b1 0.18 Log10 CFU; p < 0.01; p < 0.5; M. tuberculosis antigens.The protective efficacy against BCG challenge after IL-2 treatment was detected. There was a dramatic decrease in the bacterial load in the IL-2 treatment group (5.95 \u00b1 0.11 LogM. tuberculosis antigens MH plus LT70 or MH plus ESAT6 and CFP10 and the immune responses were analyzed. The results showed that following persistent antigen stimulation the number of memory T cells was reduced, and the production of antigen specific cytokines IFN-\u03b3 and IL-2 decreased. Meanwhile, the expression of T cell-surface inhibitory receptor PD-1 (+ T cells increased. All these led to a decrease of protective efficacy against BCG challenge. Considering the possible effect of adjuvant DDA and Poly I:C, the adjuvant was used as control. Repeated stimulation with adjuvant DDA and Poly I:C did not induce immune dysfunction. Our study confirmed that M. tuberculosis persistent antigen stimulation induced T cell dysfunction in mice.We hypothesize that continuous antigen stimulation might be the direct reason causing immune dysfunction in TB. To examine the hypothesis, C57BL/6 mice were persistently stimulated with tor PD-1 on CD4+ M. avium (M. tuberculosis infection. They found that due to ESAT-6 was constantly produced throughout M. tuberculosis infection, ESAT-6-specific T cells were driven toward terminal differentiation and got functionally exhausted at late stage of infection (Accumulating data show that T-cell exhaustion developed in late state of chronic infections of bacteria and viruses such as LCMV, HBV, HCV, HIV \u201342, and M. avium . These cnfection . Our stunfection .+ T cells was expressed increasingly. Consequently, dysfunctional T cells are unable to clear the pathogens and provide effective protection. In our lab, T-cell dysfunction was also proved to occur in C57BL/6 mice primed with BCG once and boosted with Mycobacterium bovis BCG PPD weekly for 16 weeks (M. tuberculosis infection induced CD4+ T cell functional exhaustion in animal experiment (M. tuberculosis infection antigen-specific T cells expressed high level of PD-1 and had limited capacity to produce IL-2 (M. tuberculosis antigens induced T cell dysfunction, which provides a simple animal model of T cell dysfunction of TB. Besides protein antigens, other antigens in mycobacteria, such as lipid antigens (In this study, persistent stimulation with MH plus LT70, or MH plus ESAT6 and CFP10 induced T-cell dysfunction. The functionally exhausted T cells lose their function of producing cytokines. At the same time, the inhibitory receptor PD-1 on the surface of CD416 weeks . It was periment . During uce IL-2 . Our resantigens , may als+ T cells and IL-2 secretion; Then the lose of IFN -\u03b3 production.Among the antigens applied in this study, ESAT6 has strong immunogenicity and was found to be produced along chronic infection, which drove antigen-specific T cells toward terminal differentiation and functional exhaustion at last . CFP10 a+ T cell exhaustion in malignant pleural effusion of lung cancer (+ T cell numbers and function during persistent viral infection (+ T cell exhaustion than PD-L1 blockade alone during chronic infections (+ T cells increased, the expression of PD-1 on CD4+ T cells decreased, the immune protection against BCG challenge improved. It suggests that IL-2 could reverse T cell dysfunction induced by persistent M. tuberculosis infection such as in MDR-TB. This explains the effectiveness of IL-2 against MDR-TB and promotes developing novel strategy for intervention against severe TB. Certainly, IL-2 plays a complex role during this process. IL-2 could play an adjuvant role during M. tuberculosis antigen stimulation. Moreover, IL-2 might contribute to the hematopoiesis and T cell development in bone marrow. Our recent work showed that IL-2 treatment helps to balance the cytokines homeostasis in bone marrow and recover the dysfunctional hematopoiesis impaired by persistent antigen stimulation (It is important to find that IL-2 could restore the T cell dysfunction induced by persistent antigen stimulation in mouse model. IL-2 was proven to be effective to reverse CD8g cancer . Recombig cancer , reversenfection , such asnfection , 51, simnfection , 53, andnfection . Combinifections . Moreovefections . Combinifections \u201356. IL-2fections . In thismulation .M. tuberculosis antigen stimulation could lead to T cell dysfunction and supplementing IL-2 contributed to restoring the T cell dysfunction and the exhausted immunity.Persistent All datasets generated for this study are included in the manuscript/supplementary files.All animal experiments were carried out under the guidelines of Council on Animal Care and Use, and the protocols were reviewed and approved by Institutional Animal Care and Use Committee of Lanzhou University. Animals were monitored daily and received free access to water and food throughout the study.XL, FL, LM, HN, LP, CG, and XM performed experiments. BZ designed experiments. FL, XL, JC, YZ, and BZ wrote and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "The genotypes of the H9N2 avian influenza viruses have changed since 2013 when almost all H9N2 viruses circulating in chickens in China were genotype 57 (G57) with the fittest lineage of each gene. To characterize the H9N2 variant viruses from 2011 to 2014, 28 H9N2 influenza viruses were isolated from live poultry markets in China from 2011\u20132014 and were analyzed by genetic and biological characterization. Our findings showed that 16 residues that changed antigenicity, two potential N-linked glycosylation sites, and one amino acid in the receptor binding site of the HA protein changed significantly from 2011\u20132014. Moreover, the HA and NA genes in the phylogenetic tree were mainly clustered into two independent branches, A and B, based on the year of isolation. H9N2 virus internal genes were related to those from the human-infected avian influenza viruses H5N1, H7N9, and H10N8. In particular, the NS gene in the phylogenetic tree revealed genetic divergence of the virus gene into three branches labeled A, B, and C, which were related to the H9N2, H10N8, and H7N9 viruses, respectively. Additionally, the isolates also showed varying levels of infection and airborne transmission. These results indicated that the H9N2 virus had undergone an adaptive evolution and variation from 2011\u20132014. Escherichia coli, Chlamydia psittaci, Ornithobacterium rhinotracheale, Staphylococcus aureus, Haemophilus paragallinarum and others [The subtype H9N2 low-pathogenic avian influenza viruses have been prevalent in China since 1994. H9N2 viruses can cause great economic losses to the domestic poultry industry when co-infected with H5 or H7 influenza viruses or other pathogens including d others \u20139. Additd others \u201314. TherHemagglutinin (HA) is an important surface protein that plays a vital role when influenza viruses are introduced into host cells. According to epidemiological and genetic studies, the HA genes in H9N2 viruses can be divided into three distinct lineages in China including A/chicken/Beijing/1/94-like , A/quail/Hong Kong/G1/97-like , and A/duck/Hong Kong/Y439/97-like . Since 2To understand the evolutionary characteristics of the H9N2 avian influenza viruses from 2010\u20132014, molecular changes, the antigenic residues, potential glycosylation sites, and receptor-binding sites in HA from 28 H9N2 strains isolated from 2011 to 2014 were analyzed. Moreover, the infection and airborne transmission from eight H9N2 isolates in this study were identified.The specific-pathogen-free (SPF) chickens and chicken embryos used in this study were purchased from Beijing Center for Laboratory Animals. Procedures involving the care and use of animals were approved by the Jiangsu Administrative Committee for Laboratory Animals (permission number SYXK-SU-2007-0005) and complied with the Jiangsu Laboratory Animal Welfare and Ethics guidelines of the Jiangsu Administrative Committee of Laboratory Animals.50) for the isolates were determined by propagating serial dilutions of viruses in eggs (0.2 mL per embryo) and calculated as described by Reed & Muench [From 2011 to 2014, tracheal or cloacal swabs collected from live poultry markets in China, including Jiangsu, Anhui, Heilongjiang, and Fujian, were placed into phosphate buffered saline (PBS) containing 2000 units/mL penicillin, 2 mg/mL streptomycin, 50 \u03bcg/mL gentamycin, and 1000 units/mL mycostatin. After freezing, the supernatants were collected through a 0.22 \u03bcm pore size filter after centrifugation at 1000 g for 10 min at 4\u00b0C. Next, 200 \u03bcL of the supernatant was inoculated in the allantoic cavities of 10-day-old SPF embryonated chicken eggs. After 72 h of incubation at 37\u00b0C, the viruses were tested for HA activity. The isolated viruses were identified as H9 subtype by hemagglutination inhibition (HI) assay using antisera against H9 subtype, H5 subtype avian influenza viruses (AIV), and Newcastle disease viruses (NDV). The 50% egg infective doses into cDNA using 12-base universal primers for influenza A viruses (U12 A/G: AGCG/AAAAGCAGG). The gene fragments were amplified by polymerase chain reaction after reverse transcription using specific primers, sequenced, and assembled by Takara Biotechnology [Total viral RNA was extracted from allantoic fluids infected with purified isolates using the TRIZol Reagent . Viral RNA was reverse-transcribed , 26. TheTo investigate the antigenic drift, the mutation levels for each of 18 reported antigenic residues from 28 isolates were quantified by comparison with 1081 full-length H9N2 HA sequences deposited in GenBank from 2010 to 2015 . The three commercial vaccine strains SH/F/98, SD/6/96, and GD/SS/94 were used as controls.http://www.cbs.dtu.dk/services/NetNGlyc/). Some key residues from all gene fragments were also investigated.The potential N-linked glycosylation sites (PNLGSs) in HA were predicted using the NetNGlyc 1.0 Server online software (v/v) for 24h at 37\u00b0C. The antisera were collected from vaccinated SPF chickens in two weeks after the booster vaccination and used to characterize the antigenicity of the 28 H9N2 isolates and the two commercial vaccine strains. The HI assay was expressed as the reciprocal of the highest serum dilution in which HA was inhibited. Then, antigenic cartography was performed with the Antigen Map program (http://sysbio.cvm.msstate.edu/AntigenMap), which uses matrix completion multidimensional scaling to map HI titers in two dimensions [To investigate the antigenic relationship between the H9N2 isolates and two commonly used vaccine strains (SH/F/98 and GD/SS/94), the nine H9N2 isolates and two commercial vaccine strains (SH/F/98 and GD/SS/94) were selected according to the genotype of the isolates and the year of isolation. The antiserum of each strain was made, then the HI titer of each straun were detected as following description. Briefly, 3-week-old SPF chickens were immunized twice by subcutaneous injection of 0.3 mL of oil-emulsion of inactive whole virus vaccine of the indicated virus, which was inactivated by adding 0.2% formalin (mensions .6 EID50 of each virus or a PBS control. Tissue samples (trachea and lung) from the inoculated chickens were collected at 3 days and 5 days post-inoculation (dpi). Briefly, three SPF chickens were euthanatized using CO2 asphyxiation at designated times, and half of the tissues were harvested, washed, and ground into 20% (w/v) suspension in 1 mL PBS. Virus titers in the trachea and lung were determined in 10-day-old SPF embryonated chicken eggs. The other half of the tissues were fixed with 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 mm, and processed for staining with hematoxylin and eosin for histopathological examination. These experiments were repeated three times.To study the viral replication and pathogenicity of the H9N2 influenza virus in chicken, groups of six 3-week-old SPF chickens were orally, intranasally, or intratracheally inoculated with 0.2 mL of 106 EID50 of the indicated virus. The airborne contact group was placed in a poultry isolator adjacent to the infected group with a distance of 50 cm between poultry isolators. At day 3 and 5 post-inoculation, tracheal and cloacal swabs from chickens were collected in 1 mL of PBS containing antibiotics and, following one freeze-thaw cycle, were centrifuged at 3000 rpm for 10 min. Of the resulting supernatant, 0.2 mL were taken, and the EID50 titers of the tracheal and cloacal swabs collected from the indicated passaged virus were determined by serial titration of viruses in SPF embryonated chicken eggs using the Reed & Muench method [For studying the viral transmission, fifteen 3-week-old SPF chickens were divided into five groups: (i) inoculated group (three chickens), (ii) direct contact group (three chickens), (iii) airborne contact group (three chickens), (iv) PBS control group (three chickens), (v) inoculated group for sera (three chickens). To prevent cross-contamination, the chickens from the experimental groups (inoculated group and direct contact group were in the same poultry isolator) were housed in a separated poultry isolator after fumigation with the formaldehyde-potassium permanganate. The infected group was inoculated orally, intranasally, and intratracheally with 10h method . Sera coh method . The expAllantoic fluids infected with tracheal or cloacal swabs taken from chickens were harvested from embryonated SPF chicken eggs. The viruses were identified as H9 subtype by HI assay using chicken H9 antiserum and were identified as N2 via PCR using specific primers. The isolated viruses did not react with H5 or Newcastle disease virus antiserum. Twenty-eight H9N2 strains from chickens in live poultry markets were isolated in this study. HA genes from the 28 H9N2 isolates and seven other genes (genes neuraminidase (NA), polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acidic (PA), nucleoprotein (NP), matrix (M), nonstructural (NS)) from the 9 representative isolates for the 28 H9N2 isolates were sequenced and submitted to GenBank under the accession numbers MG280611-MG280630 and MG280639-MG280709 .The phylogenetic analysis included the HA nucleotide sequences from the 28 field isolates and the other 42 reference strains, which include 18 reference strains from different lineages, three vaccine strains, three G57 reference strains, and 18 field strains isolated from 2010\u20132015. The results showed that HA genes from all 28 H9N2 isolates were clustered into the BJ/94-like lineage. According to the year of isolation, the viruses in the phylogenetic tree could mainly be clustered into two independent branches labeled A and B . The viruses in branch A which were isolated from 2011\u20132012 shared a 92%-93.5% sequence identity with the three vaccine strains: SH/F/98, SD/6/96, and GD/SS/94. However, the strains in branch B which were collected from 2013\u20132015 only had a 90%-92% nucleotide identity with the three vaccine strains .Based on the HA phylogenetic analysis, nine isolates were selected, and a phylogenetic analysis of these was performed on the seven genes of interest . The phylogenetic analysis of the NA nucleotide sequences showed that the viruses in the phylogenetic tree could be mainly grouped into two independent branches labeled A and B , which were closely related to the year of isolation . The strResults from a reciprocal HI assay demonstrated that antisera against the SD/C9QH/11, JS/TM58/13, JS/JT95/13, or AH/WB/14 strains had low reactivity with most of the isolates and the two vaccine strains, SH/F/98 and GD/SS/94. The two vaccine strains and most of the isolates reacted well with the antisera against JS/TM71/14 or JS/TM59/13 . The antThe mutations for each of the 18 reported antigenic residues were analyzed by comparison with 1081 full-length H9N2 HA sequences deposited in GenBank . In comparison to three commercial vaccine strains , the 18 antigenic residues in the HA protein of the isolates were changed significantly from 2011\u20132014, including H66Q, G90E, S127R, S145N, D153G, Q164K/R, N167G/S/K, A168D/N, E181G, A198T/V, T200R, N201D, D216E, T220I, Q235M, R254K, N256D, and S283R . In addiCompared with the three vaccine strains, the receptor-binding sites (RBS) with amino acids Y109, W161, T163, A191, L202, and Y203 were conserved among the 28 isolates, though a mutation occurred at position A198. Among the 28 isolates, 11 strains possessed an A198V mutation, and 8 strains possessed an A198T mutation. The 198T position began to be dominant in 2013, and the proportion of 198V or 198A decreased yearly since 2012 or 2011, respectively . The amiThe hemadsorbing sites , active center (140\u2013157), and antigenic determinants in the NA protein from 9 representative isolates in the two branches were also analyzed . All theSome critical residues in internal genes contributing pathogenicity or infectivity were analyzed, and the results showed that 627E and 701D in PB2 were observed among nine isolates (701N in PB2 was observed only in the virus JS/TM59/13), which are characteristics of low-pathogenic AIVs. In contrast, D253N and Q591K in PB2 were not observed, which could increase AIV pathogenicity . 588V in50 counts from oropharyngeal and cloacal swabs, tracheal and lung homogenates, and tracheal and lung histological specimens were conducted on 3 or 5 dpi. The EID50 results from the tracheal and lung homogenates on 3 and 5 dpi showed that all isolates replicated better in tracheae than in lungs. In particular, the JS/JT12/11, ZJ/618/12, JS/YZ640/12, JS/TM58/13, and JS/JT95/13 strains reached higher EID50 on 3 and 5 dpi than other strains in the tracheae. The AH/WB/14 and JS/TM71/14 strains reached lower EID50 on 5 dpi in the tracheas than on 3 dpi, while no SD/C9QH/11 virus was detected on 5 dpi in the trachea. Only the ZJ/618/12 strain was detected in lung homogenates on 3 and 5 dpi, and the JS/TM58/13 or JS/JT95/13 viruses were detected in lung homogenates on 3 dpi or 5 dpi, respectively, while little or no virus was detected in the lung homogenates on 3 and 5 dpi in the groups infected with the rest of the isolates and some field strains isolated in recent years were selected for phylogenetic analysis. The HA segment of the 28 isolates belonged to G57-like viruses. Additionally, the locations of all the strains in the phylogenetic tree are closely related to the year of isolation, and the later the isolation, the farther they are located from the original G57 ZJ/HJ/07 strain in the phylogenetic tree. Specifically, the NA gene has gradually evolved into an undefined lineage. In comparison to other lineages of each gene, the fittest and most advantaged one of each gene may help H9N2 viruses to survive easily in hosts. Additionally, our findings also showed that 16 antigenic residues, two PNLGSs, and one RBS residue changed significantly from 2011\u20132013, which might be related to the emergence of the G57 lineage. It is generally believed that the RSSR sequence at the HA cleavage site is a characteristic of low-pathogenic AIVs . In thisThe virulence, infectivity, and transmission of the H9N2 virus are affected by multiple factors, particularly genetic factors. In this study, most isolates possessed residues in critical sites that increase the infectivity and transmission. Subsequently, animal experiments showed that the isolates caused different degrees of histopathological changes concerning host tissue tropism and better replication in the upper respiratory tract. Better airborne transmission and replication in the host can create an opportunity for H9N2 viruses to survive easily.Currently, H9N2 viruses are circulating in China without an effective vaccination strategy. H9N2 viruses escape selection pressure from virus-host interactions by evolution, which may have directly resulted in the emergence of an epidemic G57 virus. To understand the evolution of the H9N2 virus from 2011\u20132013 when the G57 virus became predominant, we performed a molecular analysis of H9N2 isolates in this study. From 2011\u20132013 changes in antigenic residues, PNLGSs, and some critical residues might have contributed to the increased H9N2 virus survival and evolution. These changes might help the G57 virus to become the predominant strain and could potentially cause enhanced infectivity and transmission by the circulating H9N2 virus.In summary, our findings suggest that the H9N2 virus surface glycoproteins have undergone evolution and variation from 2011\u20132014, which might be related to the G57 virus outbreaks and emergence since 2013. The H9N2 virus internal genes were closely related to those from the human-infected avian influenza viruses H5N1, H7N9, and the emerging influenza virus H10N8. In particular, some NS genes from H9N2 viruses were closely related to the H10N8 and H7N9 viruses, respectively."} +{"text": "To evaluate why no families could be recruited for a nurse\u2010led and family\u2010centred support programme in Austria which aimed to prevent an age\u2010inappropriate caring role for young carers.A qualitative study incorporating qualitative e\u2010interviews and telephone interviews.N\u00a0=\u00a017) and with adult family members of children with caring responsibilities (N\u00a0=\u00a04). Data collection and analysis were guided by the \u201cSocial Marketing Framework.\u201d Relevant statements were assigned to the main categories: product; price; promotion; place; and working with partners.Twenty\u2010one interviews were conducted with statistically significant project stakeholders (The lack of awareness towards young carers, the unfamiliar, open outcome approach of the intervention, the inappropriate language used in promotional materials and the families' fear of stigma while seeking support were identified as central obstacles for successful recruitment of families and implementation of the support programme. However2The needs of young carers have been the focus of a considerable number of dedicated support programmes, with services categorized according to their goals and intervention types a literature review, (b) a problem and need analysis and (c) an analysis of the current practice (\u2026) in order to empower and relieve families and finally inform them about supportive measures available for their adapted situation. All interviewees regarded the FGC to be a valuable addition to existing support measures for young carers and their families. The coordinators reported great interest from colleagues, and they were convinced of its potential positive impact. It was considered to be a helpful tool for the provision of context\u2010specific support, as highlighted by one member of the partner organization's managerial board:(Family member 4)And then we developed that plan. It was also for my husband, that he knew \u2018OK, I know where I have to go today.\u2019 \u2013 daycare for adults people for example. It gave us a daily routine. There was someone in the house the whole time. The neighbors, my mother or the children. But the children no more than three days a week. The development of a strategy and the inclusion of alternative private networks are key elements of the FGC, and the potential usefulness of such an intervention was confirmed by the mother of a young carer, who had developed her own type of conference with one of her ill husband's therapists:The woman included friends and neighbours in the plan to create breaks for herself and her children. This enabled her to get more time for herself and to go to work without a guilty conscience. This also released the children, who usually cared for their father with dementia on a regular basis, from their caregiving duties.4.1.2(Coordinator 1)For the families it appears just as a clever\u2010talking method. They cannot imagine how talking works or what positive effects it can have. The conference follows an open outcome approach, and communication between all participants is one of its central elements. Coordinators received training to facilitate an open outcome. However, while they said they would have felt comfortable in using the method, a conversation\u2010based, open outcome intervention is an unfamiliar approach for caring families. The coordinators assumed this to induce bias against the FGC approach from the family's point of view, which might have hindered their participation:This \u201cjust talking\u201d assessment was likely associated with other conversation\u2010based interventions, similar to the services provided by psychotherapists and psychiatrists where they may have repeated conversation on their situations without a concrete outcome. While this is not negative evaluation per se, it is considered very unusual in the context of caring. The coordinators also stated that the expectations of families receiving this type of assistance were primarily to access care or receive advice; these expectations were shared by some coordinators who were unfamiliar to the provision of care that did not include their usual interventions, such as support and counselling. In this context, the coordinators assume that families are prejudiced against such procedures and that there is a general lack of experience with \u201csolution\u2010open\u201d support in nursing interventions.4.24.2.1(Coordinator 1)Especially when they (families) feel a burden owing to caring, the extent and effort of the intervention were certainly deterrent. The FGC was framed as a pilot project, and for the families, it would have been free of charge. However, other factors were identified as \u201chidden costs,\u201d such as the high time commitment required, which might have deterred potential participants. Based on their past experiences, the coordinators indicated that many adult carers often refused to participate in support measures, as caring itself is very time\u2010consuming:One family who was interested in the intervention withdrew their participation when the health status of the ill family member declined. All additional support, even when assumed to be helpful, was regarded as a minor priority for these families.4.2.2(Coordinator 11)That's why families often have a defensive attitude against all sorts of interventions in this regard. The interviewees shared the perception that families with caregiving children lived their lives in secrecy and were therefore cautious in making use of interventions like ours. Participation was associated with admitting to and revealing the extent of their situation. They were further understood to be afraid of being convicted of integrating their children into caring of another family member:(Coordinator 3)In the worst case (\u2026) they (families) fear the potential interference from (the) youth welfare office. Even worse, participation was assumed to be displaying their potential inability to protect their children, which led to a fear of being criticized from their immediate environment as well as the health or social care professionals involved, who might not have the appropriate level of awareness regarding the young carers:4.34.3.1\u201ctoo scientific\u201d and therefore not accessible enough for a group of people who do not usually have much time to read\u2014much less so if the material is difficult to understand.Information leaflets were created to have written, easily available promotion material. The leaflet covered information about the intervention aims, target group, procedure, involved organizations and how to contact them. The text was used in combination with basic graphic design elements to attract attention. However, the text was regarded as (Coordinator 5)I gave them the leaflet and reminded them to think about it. But then they (the family) have forgotten about it (the leaflet) just because they thought it did not apply to them. The leaflets were also intended to facilitate families' self\u2010referrals to the programme. It became apparent, however, that the leaflets did not enable families to recognize themselves as the target group. Families with caring children often do not see themselves as such, as the following quote demonstrates:(\u201cFamilienkonferenz Pflege\u201d) implies an order of hierarchies and regulations which is, in fact, the opposite of what was intended:(Coordinator 10)Not conferencing or things like that. It is much simpler like sitting together and talk about families' situations. Another reason of non\u2010participation was assumed to be the programme's name. The term \u201cconference\u201d was regarded to be a deterrent as the German translation of the term 4.44.4.1All the coordinators were very experienced homecare nurses, who had received special training in case management or other clinical specialties. However, we did not consider that the coordinators, as group leaders, spent comparatively little time to work on\u2010site with the families. This was later perceived as an aspect hindering extensive engagement in the recruitment process. The coordinators often had to deal with other projects, in addition to their daily routines as group leaders. This led to low priority being given to the intervention, and the coordinators tended to lose sight of it after a while.4.4.2(Coordinator 2)This is the time when children usually attend school and are not at home. The places where potential participants could have been recruited were broadly diversified. However, the most common place of immediate recruitment was the families' home during visits of the healthcare professionals. Owing to organizational issues, these visits usually take place in the morning or early afternoon. Potential young carers were therefore overlooked because they were not at home at those times, as explained by one of the coordinators:Moreover, the coordinators stated that in private homes it was the patient and not the family who was considered to be the primary client. Even coordinator trainings did not have a significant impact on this view. This further contributed to the fact that young carers were overlooked.4.54.5.1\u201c\u2026 had any contact with a young carer in his whole career.\u201dTo access affected families, much effort was devoted to working with network partners and groups of people who were potentially in contact with young carers in their work environment, in addition to schools, family practitioners and youth, health and social care organizations. According to the recruitment protocol, coordinators were to be informed by a network partner when a family or a young carer had been identified as potential participants. Many partners were willing to work together with the coordinators, or at least be vigilant. However, as the interviews revealed not a single partner informed the coordinators about a young carer or families. One coordinator (4) cited the feedback of a social worker who had not: (Coordinator 7)Understanding the phenomenon of young carers is the central precondition for facilitators in order to recognize them. This allows the conclusion that the general lack of awareness about young carers was a major recruitment barrier, despite the reasonable assumption that the network partners like general practitioners, social workers, healthcare professionals in hospitals and teachers have contact with young carers in their work environment. The implication is that these children are not recognized as such:\u201c(\u2026) give the phenomenon a human face.\u201d (Board member 2).This lack of awareness raises the need for a more general discussion of the phenomenon, both within communities and among network partners. This was further underlined by the coordinators' difficulties in addressing the topic with network partners. A more in\u2010depth discussion of the issue might have facilitated recruitment, as it would: 5price of participation. Minimizing the price should increase the likelihood of successful participant recruitment declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article."} +{"text": "Security is critical in the deployment and maintenance of novel IoT and 5G networks. The process of bootstrapping is required to establish a secure data exchange between IoT devices and data-driven platforms. It entails, among other steps, authentication, authorization, and credential management. Nevertheless, there are few efforts dedicated to providing service access authentication in the area of constrained IoT devices connected to recent wireless networks such as narrowband IoT (NB-IoT) and 5G. Therefore, this paper presents the adaptation of bootstrapping protocols to be compliant with the 3GPP specifications in order to enable the 5G feature of secondary authentication for constrained IoT devices. To allow the secondary authentication and key establishment in NB-IoT and 4G/5G environments, we have adapted two Extensible Authentication Protocol (EAP) lower layers, i.e., PANATIKI and LO-CoAP-EAP. In fact, this approach presents the evaluation of both aforementioned EAP lower layers, showing the contrast between a current EAP lower layer standard, i.e., PANA, and one specifically designed with the constraints of IoT, thus providing high flexibility and scalability in the bootstrapping process in 5G networks. The proposed solution is evaluated to prove its efficiency and feasibility, being one of the first efforts to support secure service authentication and key establishment for constrained IoT devices in 5G environments. The Internet of Things (IoT) enables advanced applications and new business opportunities in global sectors such as Industry 4.0 and Smart Cities and Precise Agriculture, among others . IndustrRecent IoT wireless technologies , LoRaWANNB-IoT is one of the technologies standardized by the 3GPP and offers a seamless integration with cellular deployments from TELCOS that give coverage and support to the technology. NB-IoT is designed to be used in wide areas and provides good penetration and coverage, which makes it an interesting alternative in smart cities and other use cases.With the introduction of the new generation of cellular technologies, known as 5G, we face the situation of having several technologies\u2014especially in the case of LPWAN, where each technology provides their own features and mode of operation. In this sense, we understand that it is paramount to provide mechanisms that unify and homogenise certain aspects of the life cycle of the IoT device, such as bootstrapping. This process involves authentication, authorization, and key management and can occur using an infrastructure that does not belong to the IoT device owner. Furthermore, the use of mechanisms to access external services (beyond the access to the network) is also important and has to be done securely. The next generation of mobile communications with 5G is already taking this into account, providing the secondary authentication schema to secure the communication with external services . In thisThe process of bootstrapping is a first step, prior to an IoT device being able to securely communicate in a wireless network. This process involves authentication, authorization, and key management operations, and it is vital to control network resources and data communications. Nevertheless, traditional bootstrapping is not adapted to the features of recent wireless technologies formed by IoT devices with limitations in computing and networking capacities to implement complex security protocols. In this sense, some of the bootstrapping protocols that are proposed do not account for constrained networks or multiprimary authentication and secondary authentication. Primary authentication always must be successfully performed before starting any secondary authentication. On the one hand, primary authentication is managed exclusively by the network operator\u2014it defines the mechanisms and steps necessary for a device to connect to the serving network, i.e., the 4G or 5G Core. On the other hand, secondary authentication is related to security domains outside of the serving network, thus not necessarily managed by the same entity as the 4G/5G Core. This allows flexibility with regard to communication technology for security domain administrators.In 3GPP cellular communication standards, bootstrapping consists of both To mitigate these challenges, this paper is focused on providing a secure and lightweight bootstrapping process for the authentication and key establishment of constrained IoT devices with a focus on scenarios of the smart industry, cities, and global sectors such as Industry 4.0. In particular, this paper provides the implementation and comparison of a secure bootstrapping solution called Low-Overhead CoAP-EAP (LO-CoAP-EAP) , with a In this paper, we introduce a set of contributions that aim to provide secure bootstrapping and enable secondary authentication envisioned in the next generation mobile networks (5G) using the EAP lower layer known as LO-CoAP-EAP. We have implemented the protocol in an experimental testbed using real hardware devices.The rest of the paper is organized as follows. LPWAN technologies do not provide a standardized or common solution for securing communications, bootstrapping, or the management of the life cycle of the device and the key material. As we can see in In this sense, interest in providing a solution that supports the secure management of a great number of devices\u2014management that is envisioned to be present in an LPWAN\u2014has been present in Internet Drafts (I-Ds) from the Internet Engineering Task Force (IETF) ,11, as wThere are some early proposals in IETF I-Ds to extend the LoRaWAN Joining Procedure with AAA infrastructures ,14, whicLightweight M2M 1.1 [In addition to the IETF, other organizations and alliances such as Open Mobile Alliance (OMA) in their white paper M2M 1.1 integrat M2M 1.1 , a recenThe Next Generation Internet (NGI) initiative by the Digital Single Market of the European Commission . NGI TRUST requests security technologies such as bootstrapping to support the development of a human-centric Internet by developing a stronger European ecosystem of researchers, innovators, and technology providers. Bootstrapping trust at the protocol level maintains a trustworthy Internet Infrastructure.The EU has also shown a clear interest in the advancement of security-related topics, through actions such as We understand from the aforementioned initiatives that the process of achieving a secure system should go through the reliance of known, tried, and tested standards, which lay the foundation for stable and trusted systems.As the landscape of a new generation of communication technologies evolves, for example with the development of 5G , we can In our early work, we also focused on providing an LPWAN with the necessary mechanisms to secure communications, being proponents of different mechanisms to secure LPWANs ,14,20.In this work, we continue that effort to confirm that providing security to LPWAN technologies is possible. In previous work, LO-CoAP-EAP was tested with a real LoRa network (a LoRaFabian network) ; in thisPANA (Protocol for Carrying Authentication for Network Access) is an EAFor the case of PANA, we show in https://sourceforge.net/projects/panatiki/) is a PANA adaptation for wireless IoT devices with limitations in terms of computing and networking. PANATIKI follows the PANA standard, although reducing the original state machine of the PaC client to create a suitable version for limited devices. This lightweight PANA version is an optimized PANA implementation and is ideal for the comparison and evaluation of the LO-CoAP-EAP proposal for constrained devices with NB-IoT wireless technology.For constrained scenarios, Moreno et al. developeLO-CoAP-EAP is a lowThe LO-CoAP-EAP solution allows for a good balance of performance, flexibility, scalability, and technology independence for enabling secure authentication and the generation of fresh keying material in limited IoT devices. The integrated techniques enable an important group of features to LO-CoAP-EAP. First, the integration of the CoAP protocol allows for the implementation of a lightweight bootstrapping procedure as a service of CoAP. This solution enables a wireless technology independence because it is runs over the UDP/IP stack. Unlike PANA-based solutions, LO-CoAP-EAP is based on a CoAP, an application protocol used by most of IoT devices for machine-to-machine (M2M) communications. This reduces the code size and complexity because it does not need a new protocol to transport the EAP IP messages to perform the bootstrapping process. Moreover, LO-CoAP-EAP employs AAA infrastructures to enable enhanced properties such as identity federation, supporting more scalable deployments with multi-domain scenarios. The inclusion of an EAP allows different IoT devices and organizations to use different authentication mechanisms depending on their requirements. Finally, as well as PANA, LO-CoAP-EAP enables the key derivation to be employed to protect the end-to-end communications, so that the IoT device can be included as a trustworthy entity in the application domain.The design of LO-CoAP-EAP considers the limitations of IoT devices in terms of computing capacities, low power consumption, and wireless communication technologies (LPWAN) such as NB-IoT. The following sections provide a detailed description of the developed solution evaluated in a real NB-IoT network and with 5G specifications.LO-CoAP-EAP represents an improvement over PANA in the realm of IoT by using CoAP. This is because CoAP is an application layer protocol that is expected to be present in most IoT stacks as a fundamental building block over IPv6/UDP . It remoThe original articles that present CoAP-EAP and LO-CoAP-EAP evaluate the protocol performance in comparison with PANA using the COOJA simulator. In this case, as we are introducing this protocol in a new technology (NB-IoT), we deliver experimental results using real devices. The experimental evaluation can be found in 3GPP Release 13, together with Long-Term Evolution (LTE) for Machines (LTE-M), and has been extended in Releases 14 and 15. The NB-IoT design requires support for a massive number of low-throughput devices connected to the same site cell. This equates to 40 devices per household in a densely populated area such as London [Narrowband IoT (NB-IoT) is a new radio interface for LPWANs standardized by the 3GPP. It is one of the most promising IoT standards defined in the s London . NB-IoT NB-IoT employs a single narrowband of 200 kHz with low baseband complexity to maintain a lower cost and a large coverage range. Technically, NB-IoT has a link budget of 164 dB, significantly better than previous standards. For instance, compared to legacy GPRS technology, NB-IoT counts with an extended coverage of 20 dB. This grants NB-IoT extended coverage ranges of up to 10 km in line-of-sight scenarios and of up to 1 km in densely populated areas. The radio modulations of NB-IoT in the Physical layer (PHY) are Binary Phase Shift Keying (BPSK) and Quadrature Phase-Shift Keying (QPSK) for the uplink and downlink, respectively. The access scheme employs Single-Carrier Frequency-Division Multiple Access (SC-FDMA) for the uplink communications and Orthogonal Frequency Division Multiplexing Access (OFDMA) for the downlink. In both cases, it employs subcarriers of 15 kHz in half-duplex communication mode.Data rates range from 160 bps to 250 kbps, depending on scenario conditions. Most IoT applications have permissive latency requirements, with a target latency of 10 s or less. It is expected that by 2025 the number of deployed NB-IoT devices will surpass 5 billion devices. As a consequence, NB-IoT devices are targeted to be marketed with prices of US$5 per unit. NB-IoT development is based on LTE and supports all the signaling needed to establish communications with a LTE base station. Thus, any LTE compatible base station can be upgraded via software to support NB-IoT. This characteristic greatly reduces the deployment time and complexity of operations. NB-IoT can be deployed in three different operation modes, namely (i) Inband, (ii) Guardband, and (iii) Standalone. In Inband mode, the device employs a resource block of 180 kHz within a LTE carrier band. In Guardband mode, the energy is irradiated in the unused guard resource blocks between two LTE carriers. In Standalone mode, it occupies one GSM channel, and not LTE radio band resources.Regarding recent forecasts of IoT use , the depTo achieve precise agriculture, the farming sector has highly benefited from Wireless Sensor Network (WSN) technologies and is expected to equally benefit from IoT technologies to addreIn particular, precise irrigation methods are rapidly developing in order to save water while improving yields and fruit quality. Although irrigation has been practised for centuries, precision irrigation is a new issue, as the sector has had to respond to societal demands for reductions in water allocation and improvements in efficiency. Irrigation strategies have been proven to successfully increase Water Use Efficiency (WUE) by reducing water use. Thus, the current trend of fertirrigation management implies (i) precision crop irrigation and fertigation, (ii) the use of crop-based information (crop indicators/descriptors), (iii) enhanced analysis, interpretation, and vaporization of the collected data, and (iv) the development of systems for growers\u2019 aid on efficient fertirrigation control.Recent IoT technologies formed by embedded devices and low-power wireless technologies are being deployed for precHowever, the lack of standardized security solutions for IoT technologies is one of the main obstacles to deploying IoT devices with wireless communication in agricultural fields. International activities are fostering vendors and users towards IoT products with high security/trustworthiness by standardization efforts and regulations . MoreoveRegarding the features of precise agriculture, this use case presents an excellent environment for showing the importance of the proposed security solution for the deployment and secure communications of IoT devices with NB-IoT technology. In this environment, IoT devices are deployed to collect sensitive agriculture data that must be sent to data-driven decision support systems so that they can perform automatic actions that optimise the efficiency of critical resources . In precise agriculture, the use of IoT technologies requires addressing security aspects related to the deployment and setup of the communications of constrained devices with low-power wireless technologies .The proposed environment is a real pilot farm located in Santomera . The environment is presented in For data gathering, IoT devices were deployed to monitor several features of agricultural crops, such as water consumption, soil moisture, and weather conditions. Each IoT device acts as a data source collecting agricultural information from the field and sends the corresponding data to the smart platform employing Internet protocols . All data from IoT devices are accessible to end users through the web interface of the smart platform located in the cloud. Nevertheless, these data may be compromised by cyber-attacks in the case that security mechanisms are not integrated.Therefore, this specific agriculture scenario is used for the demonstration of the proposed security solution integrating a group of modules to allow the secure data communication between IoT devices and the data-driven platform. To reach that, the presented use case develops our bootstrapping solution integrating the LO-CoAP-EAP protocol in NB-IoT technology for the secure authentication and credential establishment. Below, the bootstrapping procedure is described to enable the secure joining of IoT devices when they are deployed in the network and application domains. This bootstrapping generates the needed credentials between the IoT device and the IoT controller using the LO-CoAP-EAP implementation, as shown in The proposed architecture of this work is showcased in First, when powered on, the IoT device will perform the RAN attachment against a base station. Once attached to the base station, it will connect to the serving network provided by the Core. Next, in turn, the Core will activate the required mechanism that provides the functionality of sending packets to the external data network, i.e., the Internet. The IoT device will then send the user plane IP/IPv6 packets addressed to the IoT controller. These packets carry an authentication protocol data unit (PDU), which can be instantiated in either PANA or LO-CoAP-EAP. Finally, the IoT device will establish a security association with the IoT controller through the EAP. As a result of this process, the IoT device will be considered as authenticated and derive crypto key materials.For the purpose of validating the proposal, the most relevant performance parameters of NB-IoT in recent literature were considered . The reqAfter evaluating all the aforementioned works, a reduced message size and a reduced number of packets required were considered as apt performance indicators for this validation process. It has reasonably been shown that they are tightly related to the energy, scalability, and latency performance of NB-IoT communication systems.In order to validate the proposed architecture, a real-life implementation was carried out as a proof of concept. As a result, a testbed was built with real hardware while taking into consideration the constrained nature of IoT devices. Thus, the hardware was chosen with the specific purpose of replicating the characteristics of a typical scenario. An analysis of different parameters of a practical nature for the scenario are presented next.All authentication-related communications took place between two end-points, namely, the IoT controller and the IoT device. A single IoT controller handled all the authentication requests to the centralized AAA server. Both the IoT controller and the AAA server were co-located in an Intel i5-7400 CPU at 3.00 GHz with 8 GiB of system memory. Moreover, the IoT device was mainly composed of two different elements: an Arduino-compatible SmartEverything FOX board by Arrow and an NB-IoT compatible radio module. The SmartEverything board employed as an IoT device contained an ATMEL SAMD21 Ultra low-power ARM Cortex-M0+ microcontroller. The ATMEL SAMD21 had 256 kB of programmable flash storage and 32 kB of main memory. Additionally, the NB-IoT radio device employed was the Quectel BG96 Multi-mode module, configured to use LTE Cat NB1 only (NB-IoT). Due to the lack of 5G deployments in the nearby area, the employed backhaul network was a 4G system. It was composed of an NB-IoT LTE RAN and a 4G Evolved Packet Core (EPC). Without a lack of genericity, this same scenario could be achieved in a 5G system with LTE RAN or New Radio (NR) indistinctly. During all the experiments, the IoT device was static and located at approximately 575 m from the closest eNodeB.tcpdump during the whole process. These logs were later reviewed to match the data logged by the laptop connected to the IoT device. Finally, no notable adverse weather events took place during the duration of the process\u2014the field equipment remained safe and was never compromised.The service network employed during the experiments was a public 4G network managed by a mobile operator company. To gain access, the IoT device included a USIM card with a limitless data subscription plan. Additionally, during the experiments, a laptop computer was employed in the field. The laptop was connected through a serial console cable to the IoT device during the duration of all the experiments. The IoT device included a special debug code that exposed a customized command interface that controls several debug functions. Using a script, the laptop triggered the bootstrapping process from scratch by communicating with the IoT device through the command interface. This command was sent to the IoT device at random times of the day. During the bootstrapping process, the laptop logged all the serial console messages of the IoT device, including time-stamps and message sizes at the time of encode and decode. Both PANATIKI and LO-CoAP-EAP were employed in the experiments. Additionally, the IoT controller and AAA server logged all the network packets using Message Length column in NB-IoT PDU Length column considers the headers of NB-IoT, IP, and UDP, with values of 10, 20, and 8 bytes, respectively. Furthermore, the LTE RAN of NB-IoT employs a stack consisting of three different protocols, from lower to higher positions in the stack: Medium Access Control (MAC) [ol (MAC) , Radio Lol (MAC) , and Pacol (MAC) . Each ofTuino Zero 96 library (https://github.com/gimasi/TUINO_ZERO_96) was employed. Mainly, the modifications encompassed an implementation in transparent mode for the IP/UDP sockets. This library was employed in both PANA and LO-CoAP-EAP experiments. In both cases, it had a footprint of 2571 bytes of programmable memory. The libraries employed for managing the EAP authentication process were based on PANATIKI, which is a PANA implementation for the Contiki OS (https://github.com/contiki-os/contiki). In the case of LO-CoAP-EAP, the EAP libraries were adapted to work in the Arduino framework and encode/decode CoAP packets. Likewise, the PANA implementation of the testbed required some adaptation of the PANATIKI libraries in order to work with Arduino. After the adaptation process, the compiled code size was 8916 bytes in the case of LO-CoAP-EAP and 7586 bytes for PANA. In either case, the total sum of the Quectel BG96 libraries and EAP authentication libraries stayed below 11.5 KiB. Thus, this implementation was considered reasonable for a wide variety of constrained IoT devices that can manage our proposal efficiently. For instance, a similar device to the one employed in this scenario is the Arduino Nano, which also includes the SAMD21 MCU at 48 MHz and 32 KB of SRAM. As a consequence, the minimum hardware required to achieve authentication would be similar to that of a Class 1 constrained device as defined in [Quectel LTE BC95.Code footprint was also taken into consideration during the developing of the testbed. For this reason, the development employed libraries targeted at constrained devices in order to maintain a low programmable memory size. For peripheral communications with the Quectel BG96 NB-IoT modem, a modified version of the During the execution of the real-life experiments, the total elapsed time needed to perform a complete authentication process exchange was measured for each protocol. For each iteration of the experiment, all the packets detailed in Bootstrapping is critical for the secure authentication and credential establishment in NB-IoT and 4G/5G networks. This paper has proposed the adaptation of lightweight bootstrapping protocols to enable a secondary authentication feature of 5G for constrained IoT devices. In fact, this approach represents the adaptation and evaluation of both PANA and LO-CoAP-EAP, to provide high flexibility and scalability in the bootstrapping process. We adapted two EAP lower layers, i.e., (i) PANATIKI as a IETF standardized EAP lower layer and (ii) LO-CoAP-EAP. The latter is a new EAP lower layer specifically designed for IoT and provides high flexibility and scalability in the bootstrapping process. We have compared their leveraging abilities in NB-IoT and 4G/5G environments. Moreover, this paper has explained how limited IoT devices can bootstrap and establish credentials in real scenarios with a 4G/5G network and an AAA infrastructure. Furthermore, the solution allows for deriving credential keys between a limited IoT device and an external IoT controller to create secure channels for protecting M2M data exchange. To validate the proposal, we have implemented, deployed, and evaluated a pilot testbed with real limited IoT devices as part of the proposed use case of precise agriculture. In this case, real devices are connected via NB-IoT modules to a 4G core network and an AAA infrastructure. The performance results demonstrate that LO-CoAP-EAP is a feasible and efficient solution to be used in NB-IoT and future 5G networks to enable the secondary authentication and the security association of limited IoT devices.As future work, we consider the implementation of OSCORE to provide efficient secure end-to-end communication for constrained devices, as well as the inclusion of compression techniques to minimise the payload sizes and the number of messages required for the bootstrapping process. Furthermore, the proposed solution can be employed in other applications where the efficient and secure deployment of NB-IoT devices is crucial. Although we presented payload size and runtime latency as performance indicators, further experiments with different IoT device configurations and requirements shall be considered. New results could suggest the level of fitness of the proposal to each specific use case or scenario, together with modifications that would clearly improve its current design."} +{"text": "It is predicted that by 2025, all devices will be connected to the Internet, subsequently causing the number of devices connected with the Internet to rise. As indicated by Cisco, there will be 500 billion devices connected with the Internet of Things (IoT) by 2030. Furthermore, Telefonica anticipates that 90% of vehicles will be associated with the IoT by 2030 and forecasts that every person will have an average of 15 connected devices by then. However, a review in 2015 proposed that more than 250 million connected vehicles will be available globally by 2020\u2014an increase of 67%. The IoT offers numerous industrial opportunities that permit industries to create novel strategies and models to actualize their ideas. Such industrial opportunities additionally lead to effective and creative examination possibilities for researchers and specialists in multi-disciplinary research areas, thus combining research studies, engineering abilities, sciences and humanities all under one umbrella. Additionally, IoT is changing the world into a smart world in which everything is effectively accessible quickly. A few smart applications are shown in Next-generation IoT-based smart healthcare;Next-generation IoT-based smart cities;Next-generation IoT-based smart agriculture;Next-generation IoT-based data analytics;Next-generation IoT-based industrial IoT;Next-generation IoT-based multimedia;Next-generation IoT-based spectrum sharing techniques.Consequently, this Special Issue has attracted researchers from academia and industries to investigate the opportunities for next-generation IoT-based user scenarios and study their effect on the solutions of the issues and challenges discussed above and propose viable solutions. Several researchers have contributed to different areas of interest related to next-generation IoT-based applications and user scenarios, including the following:Industrial IoT: The improvement of smart industries has had a vast and enduring effect on the inevitable future of global manufacturing. Industry 4.0 has consolidated smart industry into cyber-physical technologies, making intricate advancements more proficient and effective and improving the performance, quality, controllability, management and fairness of industrial measures in developing IoT-based smart industries. Next-generation cheaper sensing advancements are basic requirements for information collection and robust execution by manufacturing industries and supply chains. Simultaneously, a great deal of research has been conducted that has focused its attention on breaking down the industrial performance and usage of facilities. Most manufacturers need a profound understanding of the contrast between customary and pioneering manufacturing plant frameworks, as well as the wide arrangement of sensor advancements related to Industry 4.0. In , the autHealthcare IoT: Some pathologies openly influence society, causing general healthcare issues. For example, pneumonic infections and chronic obstructive pulmonary disease (COPD) are presently the third leading cause of death, while tuberculosis is the ninth leading cause, with 1.7 million deaths and over 10.4 million further occurrences. Studies show that computational techniques, such as IoT-based technologies, contribute to health-related data analyses of lung pathologies by computerized tomography (CT). In , the autFog computing: Fog computing technology (FCT) recommends using computational resources that are close to the edge of the network. In , an FCT 5G technologies: Current RFID tag technology is expensive and has not been exhibited as green technology for next-generation IoT in 5G advancements; furthermore, it is not sensible for long-range data transmission in 5G systems. A recently proposed I-RFID propose Network-on-a-chip (NoC): Recently, network-on-chip (NoC) technologies have become popular in communication frameworks for heterogeneous computing technologies as they are more scalable and highly efficient. Hostile technology scaling results in these designs having both permanent and transient issues regarding the chips. In , the autNext-generation IoT-based smart healthcare: The uses of IoT in healthcare are progressively being embraced in commercial domains. Several business solutions have been provided for the healthcare of an individual. However, there is enormous potential to enhance the IoT-based healthcare frameworks as an essential medical service and to develop hospitals into secondary healthcare units. In this way, it is profoundly significant to distinguish the expected advancements to address the issues and challenges resulting from accomplishing these objectives. The future perspectives include real-time location technology for Alzheimer\u2019s patients who could wear a geospatial location sensor to allow their location to be tracked at any time. Other future technologies related to healthcare are as follows: the real-time detection of epileptic seizures and stroke, AR/VR-based tele-healthcare applications, handheld summarized healthcare records, blockchain-based secure services, remote surgery and remote interactive medical training.Next-generation IoT-based smart-cities: The IoT system has advanced to develop certain arrangements in which each application is directly or indirectly linked with the context of the smart-cities application. Such frameworks and the collaborative applications can bring significant challenges and issues for IoT-based smart-cities systems. A few of the reliability challenges that have emerged in the IoT-based smart-cities framework are due to mobility issues in the transportation system; consequently, communication with vehicles is not reliable enough. Moreover, the presence of various sensor devices will cause some reliability difficulties regarding their failure. Some predetermined scenarios need communications between a massive number of smart devices, which are possibly disseminated at distinct geographical locations. The next-generation IoT frameworks are supposed to give a suitable platform that can investigate and incorporate the information emanating from various devices. Furthermore, the security and privacy aspects of such an IoT-based smart-cities system may be user-dependent information.Next-generation IoT-based smart agriculture: Next-generation IoT-based smart agriculture is expected to assist with the advancements of farming practices and techniques to support sustainable farmers and resources. This is financially practical and sound, socially steady and performs well. This approach aims to maintain soil quality, reduce soil erosion and degradation and save water assets. A sustainable smart- agriculture system improves the biodiversity of the land and promotes a healthy and green environment. It is essential to coordinate this with the expanding interest into food and environmental change and the degradation of the biological system in the future. This has a significant role in safeguarding natural assets, reducing ozone-harming gas emissions, stopping biodiversity wastage and thinking about valued landscapes. The next generation IoT-based smart agriculture has been applied to cultivation to preserve natural resources without compromising the quality of the fundamental requirements. IoT-based smart agriculture\u2019s common practices include smart farming for sustainable agriculture and smart crop rotations that mitigate issues and challenges related to weeds, plant disease, insects and other pests.Next-generation IoT-based data analytics: With the advancement of IoT-based applications, there has been an extraordinary expansion in the number of connected devices continuously gathering data and interfacing with the environment in which they are embedded. Such smart devices empower the advancement of new data analytical techniques that are capable of making smart applications more intelligent and useful. Because of the collaboration between various applications, there is a tremendous amount of information from which helpful information can be separated. A significant issue that must be confronted is the recurrent occurrence of similar data generated by several connected smart devices. Embedding machine learning techniques, equipped with neural networks and deep learning algorithms, would help to tackle such issues and challenges. This kind of solution may require the extensive use of computing and energy resources. However, next-generation IoT-based data analytic solutions must be robust, cost-effective and use green technology.Next-generation Industrial IoT: Industrial development has advanced significantly since the industrial technological revolution and has continued to grow due to IoT-based systems\u2019 emergence. In fact, with the infusion and coordination of IoT-empowered sensors and actuators, smart manufacturing is changing customary product assembling processes, with numerous discrete industries working on the proof of concept and executing IoT advancements in their initiatives to drive benefits. However, industrialists are finding that these new IoT-based frameworks remain generally separated from their storehouses\u2019 IT infrastructure and networks. Thus, industry is confronting different difficulties: complex cycles and sequential construction systems, network issues in assembling frameworks, unreliable distant access, security dangers, transparency and information standard models. To achieve the next-generation IoT-based industry, the integration of IT and operational technology is crucial for all manufacturing processes.Next-generation IoT-based multimedia applications: Next-generation IoT-based multimedia applications also present several issues and challenges that must be carefully addressed. One of IoT\u2019s primary goals\u2014based on multimedia applications\u2014is the provision of the maximum QoE and QoS in terms of reliability, throughput and extended network life-time. These objectives can be accomplished by utilizing proficient information aggregation, interoperable and sustainable architectures, effective feature extraction, smart routing, switching and intelligent MAC layer resource allocation. Moreover, there are limitations on IoT devices requiring them to be small and consume limited energy and computational resources. Thus, next-generation IoT-based multimedia applications require a redefined architecture to offer adaptability and interchangeability for numerous interactive media heterogeneous devices while thinking about the spectrum scarcity and resource constraints assets.Next-generation IoT-based spectrum sharing techniques: It is assumed that the ever-extending IoT will create a large amount of data, leading to different prerequisites including throughput, reliability and latency. For example, a mobile device exploits existing base stations (BS) to gather information from enlisted IoT sensor devices and later forward this data to an edge server for additional information handling. Specifically, some particular IoT devices, which are at the edge of BS coverage, have rigid latency necessities on both uplink and downlink data transmission. However, due to the limited spectrum resources and the diverse nature of wireless networks, the requirements of next-generation IoT devices cannot be satisfied. Unfortunately, the current IoT technologies are not sufficient for this situation. To accomplish this, efficient AI-enabled resource sharing is required within the network. Indeed, spectrum sharing techniques between devices and networks need to be more adaptable and flexible.Next-generation IoT-based security and privacy techniques: The consideration of the usefulness of IoT-based applications relies on how well they can handle security and privacy issues for trustworthiness. The problems and challenges regarding security and privacy that come with the current IoT may be critical in holding back the full reception of IoT. It is essential to realize that security and privacy privileges are key to achieving users\u2019 confidence in IoT applications, connected devices and the related offered services. Several researchers have conducted work considering the security and privacy preservation in IoT-based systems. However, the key to the trustworthiness concerns is a direct result of the omnipresent AI-enabled frameworks, where the information handling and processing can be performed anywhere in the network. An AI-enabled network using Internet access is an essential feature that helps us to understand this issue because, unless there is an intelligent system architecture, it increases the ease of obtaining the individual user information.Next-generation IoT-based cross-layer protocols: The new advancement and strategies in IoT-based protocols show that the network clustering methods provide adequate solutions to accomplish system effectiveness by selecting optimal routing cluster-heads. However, AI-enabled IoT-based devices face challenges due to several constraints, such as the low power of devices, a high user density, long-distance communication, higher latency and data losses. Cross-layer routing protocols are usually required for energy proficiency, lower latency and lower information transmission delay. Therefore, it is also critical to present AI-enabled cross-layer protocols for the next-generation IoT-based applications. This can be achieved with the help of several learning techniques, such as Reinforcement Learning. Such protocols must differ from the state-of-the-art protocols, mainly focusing on scalability and efficiency related to energy and QoS. Besides, it is required to have a cross-layer-based evaluation of IoT devices and select optimal and stable cluster heads using AI-enabled heuristic techniques with minimum overhead.Recently, IoT has emerged as one of the most useful and fastest developing popular technologies. With the increase in the IoT\u2019s popularity, users\u2019 QoE and QoS requirements are increasing drastically. Researchers from academia and industry continuously need to improve and develop new procedures to adapt to these phenomena. Simultaneously, the IoT applications are incredibly different, incorporating smart-home, smart-healthcare, smart-industries, real-time multimedia and smart agriculture contexts. Thus, it is vital to deal with necessities, satisfaction and security issues. Utilizing AI-based solutions to make technologies robust and adaptive is another important task. Thus, in this Special Issue, we focus on presenting researchers who have investigated the potential uses of next-generation IoT-based solutions. Besides, we study the effect of next-generation IoT developments on the solutions to the challenges referenced above and propose viable solutions. This issue collects research articles covering different interest subjects that incorporate healthcare, smart-cities, smart-agriculture, IoT-based data analytics, smart-industries, IoT-based real-time multimedia applications, IoT-based spectrum sharing techniques, and IoT-based security and privacy techniques."} +{"text": "This paper proposes a modified architecture of the Long-Term Evolution (LTE) mobile network to provide services for the Internet of Things (IoT). This is achieved by allocating a narrow bandwidth and transferring the scheduling functions from the eNodeB base station to an NB-IoT controller. A method for allocating uplink and downlink resources of the LTE/NB-IoT hybrid technology is applied to ensure the Quality of Service (QoS) from end-to-end. This method considers scheduling traffic/resources on the NB-IoT controller, which allows eNodeB planning to remain unchanged. This paper also proposes a prioritization approach within the IoT traffic to provide End-to-End (E2E) QoS in the integrated LTE/NB-IoT network. Further, we develop \u201csmart queue\u201d management algorithms for the IoT traffic prioritization. To demonstrate the feasibility of our approach, we performed a number of experiments using simulations. We concluded that our proposed approach ensures high end-to-end QoS of the real-time traffic by reducing the average end-to-end transmission delay. Internet of Things along with Big Data, virtualization and fifth-generation mobile networks (5G) make one of the most promising areas of today\u2019s development technologies . IoT proWith developments of the Internet of Things, the number of connections to mobile networks is increasing steadily. According to the Ericsson forecast , by 2021An innovative IoT technology solution is the Narrowband IoT (NB-IoT) standard. This is a wireless narrowband type of global networks with a Low Power Wide Area Network (LPWAN) radio technology standard developed by 3GPP to enable a wide range of cellular devices and services, which is primarily designed for machine-to-machine applications ,5. NB-IoFurther to this, as new technologies are developed such as narrowband LoRaWAN, Long Range Wide Area Network, and NB-IoT, it is expected in the longer run 5G telecom operators will occupy new niches, offering not only the communication service but also a comprehensive solution, including system integration services and IoT service platforms ,4. This Besides the main narrowband, the integration to the mobile network is possible for functions on the LTE standards . All of these technologies have their advantages and disadvantages . HoweverThe QoS is guaranteed when using the license spectrum.It has a response delay time less than 10 ms.The flexibility of managing the QoS.The allocation of network resources is adaptive.The level of system security is high.With IPv6 addressing support, it provides high scalability.The network architecture provides the basis for the implementation of new services and providing Device-to-Device interaction.It is able to serve the growing number of devices with high transmission rates.It offers roaming support ,11.The benefits of implementing IoT based on LTE architecture are as follows:-with guaranteed GBR (Guaranteed Bit Rate);-with an unguaranteed Non-GBR (Non-Guaranteed Bit Rate) transmission rate.In 4G/5G networks, it should use a \u201csmart queue\u201d, where priority depends on the type of service ,13,14,15The GBR transmission service is used for applications when providing real-time services. Each GBR transmission service channel is associated with a given value of the QoS parameter. If the traffic transmitted by the GBR service corresponds to the value associated with the GBR service, then there are no problems associated with overload during transmission of this data and packet loss. The non-GBR service does not guarantee any specific data transmission speeds for services on the LTE network. This service is mainly used for applications such as web browsing and FTP transmission. A non-GBR channel service is highly susceptible to packet loss due to network congestion and also does not block any specific transmission resources in the LTE network.At present, the LTE network provides the required QoS for various IoT services, for example LTE-M. The main method for ensuring QoS is the use of communication channels with high bandwidth, for example in , the autWith the development of IoT services, it is natural that one of the main tasks is to adapt QoS according to the requirements of a particular type of service. Thus, the mechanisms of the traffic prioritization in 4G/5G networks for the NB-IoT systems/services is one of the most important aspects on which the development of the IoTs will depend in the future ,27,28.When a maintenance is performed at 4G/5G base stations deployed on the basis of LTE technology, a certain memory buffer is dedicated to serve as a queue. When packages are transferred, they are replaced by new ones. In order to eliminate failures, additional buffer is reserved to serve as a certain extension to the queue. This will reduce the queues or waiting time for a service. Since most of the NB-IoT data is transmitted over an uplink, the random access channel can usually become a major bottleneck for the entire system. To improve QoS parameters, an effective RACH (Random Access Channel) procedure is required to increase the success of the RACH, especially with regard to the interaction between the static properties of the physical radio channels and the dynamic properties of the queue developing in each IoT device. The authors of work in detaiWhen using allocated resources, it is important to know what data rate is set for packets\u2019 transmission. Further, speed allocation is one the necessary requirements due to the fact that some packets may require a guaranteed speed, while others do not. The other requirement is distance, especially for the delivery speed of a request to a device needed to perform certain actions. After all, the request traverses from its source to its destination visiting several intermediate nodes and creates an additional delay. It is also necessary to take into account the fact that in this case, intermediate memory buffers are required, because the loss of packets is possible anywhere along the transmission link ,31. It iWe found that NB-IoT technology is a promising technology for providing IoT services. The technology only supports non-guaranteed delivery services (non-GBR). The non-GBR service does not guarantee any specific data transmission speeds for services on the NB-IoT network. This service is mainly used for unreal time traffic, for example, consumer IoT applications. A non-GBR channel service is highly susceptible to packet loss due to network congestion and also does not block any specific transmission resources in the NB-IoT network. Therefore, the research problem is that NB-IoT has some limitations, one of them being that a GBR bearer is not created for NB-IoT RAT type and cannot guarantee end-to-end QoS requirements (E2E delay) for real time IoT traffic. That is why in our work for the future development of NB-IoT technology, we propose the idea of providing GBR to ensure ultra-low delay of IoT traffic. Thus, in addition to its existing advantages, NB-IoT will be suitable for providing services with critical levels of requirements for priority, reliability and delay for Industrial Internet of Things services, which is important for LTE-based existing 4G and future LTE-based 5G networks.The purpose of our work is to ensure the guaranteed QoS for NB-IoT services. This is achieved by developing methods of service quality management from end-to-end; namely, methods of IoT traffic prioritization , channel formation, and distribution of its resources in 4G/5G networks deployed on the basis of LTE technology.The rest of the paper is organized as follows. In this part, we illustrate the research status of development of the management algorithms and traffic prioritization mechanisms for IoT services in LTE-based 4G/5G networks. Nowadays, only one percent of the things that are around us are connected to the internet. This constitutes the current IoT which equals to around 50 billion devices, including sensors and mobile users. It is expected that this number will increase to one trillion devices by the year 2022 . TherefoIn this research paper , the autThe research discussed in providesIn , the autThe paper focuses The authors systematIn , Cheng aThe authors in have desRecently, Chen et al. in proposedRecently, the active development of network technologies has led to the emergence of new applications, such as online games, distance learning, and robots\u2019 management. The flow generated by these applications is sensitive to delays. However, compared to VoIP or video, they impose stricter restriction on the time of delivery, which is about 10 ms. For some applications , it is necessary to provide shorter delivery time to a maximum of 1 ms. A concept of a network interaction, involving a transfer of data with ultra-short delays, is called the Tactile IoT ,55.The ability to meet the specified requirements for the quality of service in LTE networks largely depends on how the base station is planning the transmission of various packets, using the resources of the wireless channel. The scheduler at the base station is responsible for planning radio resources. There are many planners/schedulers available on the market . They prThe significant increase in the number of IoT devices led to certain problems in modern mobile networks. Despite the fact that the capacity of 4G/5G networks is sufficient to meet the needs of most devices, the signal load generated by them exceeds the capabilities of the base stations. To solve this problem, we utilize the classical architecture of LTE-based 4G/5G base station interworking with the NB-IoT device, which is shown in Packet processing tier.Queue tier for data transmission over a wireless communication channel.Medium access control.Physical tier ,56.This structure consisting of four tiers that are listed below: This controller will be responsible for the mechanisms of downlink and uplink channel planning for IoT devices and will allow network operators to leave existing base stations eNodeB unchanged. The controller is a separate server machine on which the software responsible for IoT traffic resources planning (scheduler) is installed. It is possible to install this controller near the LTE base station or deployed in the cloud with the possibility of renting and increasing the performance of the server. The implementation does depend on the predicted number of IoT devices connected to the base station. Significant growth in the number of connected devices requires a powerful server machine for fast operation of the IoT controller. However, to enable IoT services, network designers need to separately allocate a narrowband spectrum of 200 KHz. There is no need for high bandwidth due to transmission of small volumes of data. If it is necessary to provide high speeds, it is proposed to transmit data in the spectrum of LTE.We propose to define priority classes where each class includes traffic of a particular NB-IoT based on the QoS Class Identifier (QCI) parameter. The QCI parameter can take one of the nine states, each of which is associated with a certain Type-of-Service (ToS), and thus with the type of the transmission channel, rate, error rate, and delay. The QCI is a label in the IPv6 \u201cChannel ID\u201d package. IoT is a label in an IPv6 package and is a part of the ToS field.For IoT services, we propose to use a prioritization method that is based on the criterion of allowable delays and the average number of service failures ,60. AccoIn most cases, IoT devices will be assigned a certain priority in advance depending on their purpose, and all messages that will be sent will have a defined priority. Each priority has its own acceptable quality of service parameters that must be ensured. In particular, the main parameter is the delay, the acceptable values of which are shown in This section describes our proposed \u201csmart queue\u201d management algorithms. Modern 4G/5G networks provide the required QoS for various services. To this end, we propose to use the \u201csmart queue\u201d concept, in which a priority depends on the type of service. Different types of services require different prioritization. As such, one of the main tasks with the development of IoT services is to adapt the QoS in accordance with the requirements of a specific type of service. Delivering services over mobile networks should consider not only the priority but also the delay, the requested speed of service execution, and a guaranteed performance. With regard to the latter, when it comes to the exchange of data between devices, it is important to agree on QoS parameters that must be ensured at both ends of the transmission. To enable priority, we suggest that certain memory buffers should be formed on the proposed IoT controller to serve as a queue. As the packets are sent, the locations of sent packets in the queue are released for the incoming packets. To avoid service denial, an additional buffer is installed. The additional buffer serves as an extension to the queue, and at the same time, decreases queuing delays for high priority packets.In this work, we propose algorithms for managing \u201csmart queue\u201d based on the proposed prioritization of IoT traffic in heterogeneous mobile network.This section provides a detailed description of our proposed IoT class L1, IoT class L2, IoT class L3, and IoT class L4, where L1 is the highest priority and L4 is the lowest priority. Our proposed algorithms are described by means of flowcharts. The purpose of the statistics collection module is to determine the traffic parameters, both general and for each specific protocol, registration of unknown traffic, or traffic analyzed with errors. A general algorithm of the analyzer\u2019s program operation is depicted in The control flow of the proposed algorithm for IoT class 2 is shown in The proposed algorithm for IoT class 3 is shown in t is calculated. The signaling data, the delay time t and the transmission request approval are sent to the IoT device (block 7). After a successful transfer, the statistical data are saved (block 8). At this point the algorithm ends (block 9). If the available resources are insufficient, then the queue analysis is carried out and the resource is allocated immediately (block 5). The base station is configured for data transmission (block 6) and the starting time of the transmission The proposed algorithm for IoT class 4 is shown in In the next section, we discuss our development of downlink/uplink modified resources.The most effective result in providing the required level of service quality in LTE is achieved by tackling the problem of frequency and time allocation in the downlink and uplink channels. Thus, we propose to allocate and distribute the resources in the downlink and uplink of IoT, using our proposed smart queue approach. Within the context of the narrowband IoT in the LTE mobile network introduction, the downlink structure and the uplink structure for the resource grid in LTE/IoT network are shown in The smallest unit of the time-frequency resource of the LTE frame is the resource block, which consists of 12 grouped frequency subcarriers. The 10 ms frame consists of 10 subframes of 1 ms each (two slots of 0.5 ms each). The channel resource is allocated to the resource block (RB), where the 180 KHz bandwidth is transmitted to 12 subcarriers with a spread between the frequencies of 15 KHz. In the time domain, 7 OFDM symbols are transmitted in each slot (14 in the subframe). The channel distribution is shown Narrowband Physical Downlink Shared Channel (NPDSCH) data transmission.Narrowband Physical Downlink Control Channel (NPDCCH) control.Narrowband Physical Broadcast Channel (NPBCH) system information transmission.When transmitting over the downlink at the physical level of the NB-IoT, the primary, secondary and NPSS synchronization channels are defined as folloEach frame starts with the transmission of the NPBCH channel, which may take zero subframe. Every 5th subframe is transmitted by NPSS signal, while the last subframe of each even numbered frame is transmitted by Narrowband Secondary Synchronization Signal (NSSS) signal. NPDSCH or NPDCCH channels are placed in the remaining free subframes. The base station in NB-IoT networks can operate with one or two antennas (R2000 and R2001 antenna ports). These ports transmit NB-IoT-specific reference signals. If the channel resource for NB-IoT is allocated in the bandwidth of the active LTE network, then the reference signals of the broadband network NRS1 and NRS2 are also transmitted to the Resource Block (RB). When placing the symbols of the NPDSCH channel, 1\u20133 OFDM symbols are reserved on the left side for transmission of the PDCCH control channel of the broadband LTE network is a physical channel used for uplink data transmission by the IoT Device. It may also carry the uplink control information. This channel is the counterpart of PDSCH channel in uplink.Narrowband Physical Uplink Control Channel (NPUCCH) is the Physical Uplink Control Channel (PUCCH) that provides the various control signaling. These signaling are known as Scheduling request, Downlink data Acknowledgement (ACK)/Negative-acknowledgement (NACK) signaling, and Channel Quality Indicator (CQI) information.The following discusses the narrowband physical uplink shared channel and narrowband physical uplink control channel shown in NB-IoT devices can transmit the responsive Hybrid Automatic Repeat Request (HARQ) feedback over a narrowband physical uplink shared channel (NPUSCH) or over a narrowband physical uplink control channel (NPUCCH). We propose different options for defining the physical structures of the NPUCCH, NPUSCH, and user multiplexing on the uplink (UL). We also propose to use a new transfer request signal for communication with the IoT controller.NB-IoT UEs (User Equipment) can transmit the responsive HARQ feedback over a narrowband physical uplink shared channel (NPUSCH) or a narrowband physical uplink control channel (NPUCCH). Options for defining the physical structures of the NPUCCH and NPUSCH and user multiplexing on the uplink (UL) are provided.In the following section, we provide simulation results performed on the \u201cSmart Queue\u201d concept of the IoT controller.To investigate the effectiveness of the proposed concept, we performed a set of simulations for the LTE/NB-IoT integrated solution. We implemented it in the form of a discrete events simulator and developed our java-based simulator for LTE/Nb-IoT. We also utilized the well-known tool named Discrete-Event Simulation and Modelling in Java DESMO-J (DESMO-J). It provides features such as queues, random number generation, and various statistical distributions. A simplified structural scheme of the simulation model is shown in The IoT device is a network endpoint with a QoS priority set under an SLA (Service-level agreement) contract according to Table.1. The main function of the IoT device is to generate a message, send a request for data transmission, send a message, receive a response message, and plan for the next data transfer procedure.The NB-IoT controller is responsible for monitoring the channel resources status at the base station. This includes message transmission, allocates necessary channel resources for specific IoT devices, the redistribution of channel resources between mobile and IoT devices, collection of data, processes and analyses of statistical data of successful and failed connections and transmissions.The eNodeB base station manager\u2019s main function is to carry out message integrity verification, and provide interaction between IoT devices, the IoT controller, and the IoT broker. eNodeB contains an array of channel resources that the IoT-controller allocates for transmission for the IoT devices.The IoT broker stores data sent by IoT devices, analyzes it, and performs certain previously defined operations, such as transmitting, processing, and storing.The main elements of the simulation model that correspond to the real components of the network are as follows:An IoT device generates an information message and sends the request for a channel resource allocation to the eNodeB base station. The request also contains the size of the message and the modulation type. At the base station, the integrity of the request is checked and then the message is redirected to the IoT controller. The IoT controller analyzes the request and the state of the channel resource array of the current base station. If the available channel resources are able to provide the required service within an allowable delay, then these resources are reserved for this IoT device. The IoT controller responds with the channel resource number to the current base station. The base station redirects the response to the IoT device, which analyzes the response and waits for its channel resource, in which it will transmit an information message through the base station to the IoT broker. At the same time, the IoT broker saves the information transmitted in the message for future use. If there are no free channel resources, then the request is served according to the above described algorithms.The number of IoT devices is set to 2000.The number of resource blocks within the narrowband of 200 KHz spectrum. The maximum number of resource blocks that can be transmitted in 1 s is 2000. In the simulation process, the number of resource blocks is a variable number which depends on the generated IoT message size and its requirement to the bandwidth.The modulation types are Binary Phase-Shift Keying (BPSK) and Quadrature Phase-Shift Keying (QPSK).The average length of a message from the IoT device depends on the modulation type used and is around 10 resource blocks.i\u03c1, where i = 1,2,3,4,5 considered for an IoT controller are \u03c11 = 0.12, \u03c12 = 0.18, \u03c13 = 0.5, \u03c14 = 0.75, \u03c15 = 1.The average loads, The ratio of IoT devices per class are R_L1 = 10%, R_L2 = 20%, R_L3 = 30%, and R_L4 = 40%.3 Permissible, D_L4 = T4 No critical.The allowable delays for each class are D_L1Types of delay are signal propagation speed over the wireless channel, packet processing time at base station, signal propagation time over wired channel, packet processing time at IoT controller, packet processing time by IoT device, and transmission awaiting time by IoT device.During our performed simulation, we set forward predefined data. This includes the following:s.p.r. t is a delay of a signal propagation over the wireless channel, p.BS (tprocessing BS)t is the delay of a signal processing time at the base station, s.p.c. t is the delay of a signal propagation over a wired channel, p.IoT.c. (tprocessing IoT controller)t is the delay of the processing time at the IoT controller, tprocessing IoT device is the delay of the processing time at the IoT device, and the delay caused by the IoT device awaiting transmission is w (twaiting for data transfer)t. A delay of the IoT data transmission end-to-end is calculated using Equation (1): The simulation model of the LTE/IoT integrated solution is depicted in To demonstrate the efficiency of the proposed concept, we performed our simulation on various loads of the IoT controller. The investigation consists of two main simulation phases:Phase I. This phase focuses on E2E QoS when processing the incoming flow of the requests using the Proportional Fair Scheduling method described in works [in works ,63,64.Phase II. This phase focuses on E2E QoS when processing the incoming flow of requests using our proposed traffic prioritization (P.IoT) method.During each simulation conducted, 100 frames of 10 ms duration are transmitted. Each frame contains 20 resource blocks of 0.5 ms duration. This means that 2000 resource blocks are transferred to one eNodeB per second. At every phase, we compare between the Prioritizing IoT traffic method and the Proportional Fair Scheduling method.In this simulation scenario, we used a 12% load. L1 and L2 represent real-time traffic, while L3 and L4 represent not real-time traffic. Thus, the proposed method is efficient for the real-time traffic. This method provides the minimum delay required for the tactile IoT messages. At 12% load, the Proportional Fair Scheduling method meets the requirements of the real-time traffic L2 (blue curve). For example, the delay of the transmission of L2 class should not exceed D_L2 = 20 ms. For L1 , it should not exceed D_L1 = 10 ms. It should be noted that for some messages of class L1 (red curve) in the Proportional Fair Scheduling, the delay exceeds the requirements of D_L1 = 10 ms. Loss of message transmission requests for classes L1 to L4 are absent. This is because the system works at low load. When using our proposed method, the delay for both L1 and L2 in real-time classes is within the desired or setup requirements. However, there are some losses of requests for non-real-time traffic of L4 class shown in In this simulation scenario, we used an 18% load. As can be seen from the simulation results presented in When using our proposed method, the delays for the real time class L2 are within the predefined requirements. For the L1 class, only 0.1% of all transmitted messages was subject to a higher delay that ranges from 1 to 5 ms. According to our proposed algorithms described in When we used a 50% load, the Proportional Fair Scheduling method did not provide the necessary delay requirements for both class L1 and class L2 real-time traffic. The losses of class messages of L1 are equal to R_L2 = 25%, and of L2 are equal to R_L2 = 12%, as shown in In this simulation scenario, we used a 75% load. In this situation, the Proportional Fair Scheduling method did not meet the necessary delay requirements for both real-time traffic class L1 and class L2. The losses of messages of class L1 are equal to R_L1 = 55%, while the losses for class L2 are equal to R_L2 = 65%, as shown in Our method provides a gain of 2.08 times better than the Proportional Fair Scheduling method, as shown in In this simulation scenario, we used a 100% load. With using the Proportional Fair Scheduling method, the results of messages loss R_L1 in class L1 is equal to 64% and in class L2 the loss R_L2 is equal to 73%, as shown in From the results of the simulation, we observe that the method of IoT traffic prioritization provided a reduction in the average E2E delay for devices transmitting data in real-time . This is due to an increase in the average transmission delay for devices that are not susceptible to delay . The efficiency of the proposed method at different loads is depicted in The prioritization method allowed the reduction in the number of service denials by 38% for class L1, and 69% for class L2, when it is compared with the Proportional Fair Scheduling method under high load conditions.Unlike the standard known NB-IoT architecture, our proposed NB-IoT architecture reduces the overload from the base station when planning radio resources. This is due to the fact that the existing LTE/NB-IoT architecture has a controller that acts as a resource scheduler for NB-IoT devices located in the eNodB LTE base station, which also includes a resource scheduler that is responsible for the allocation and planning of mobile subscriber resources. Scheduling is a process through which eNodeB decides which UEs (user equipment) should be given resource blocks (RBs), and how much resource (RBs) should be given to send or receive data. In classical NB-IoT structure, scheduling is done at per subframe basis every 10 millisecond. The entity which is governing this is known as the scheduler. Such a joint combination of controllers leads to a significant load of eNodeB due to the simultaneous connection of a large number of IoT devices, namely, it leads to an increase in CPU and RAM of the base station and as a result of the degradation of QoS parameters. Exchanging for new base stations with better computing characteristics is an expensive way, and mass deployment of the NB-IoT will require a significant number of replacement LTE base stations. To avoid this problem, we suggest separating the NB-IoT controller on a separate server, which will be responsible only for the maintenance and resource planning for IoT devices. With the significant increase in load from IoT devices, a situation may occur that will lead to a significant increase in service delay, which is not desirable for critical real-time IoT services. Our proposed solution will allow easy replacement of the server with another server with better computing characteristics, and will reduce the processing time by the controller, which directly affects the E2E delay.In our work, we developed the architecture of the integrated mobile access network LTE/NB-IoT for 4G and 5G networks. We modified the structure of NB-IoT frame, where a logical data channel is allocated to reduce the delay and communication of the NB-IoT controller. Unlike known solutions, these channels allow the allocation of one resource block to transmit a small message from the sensor IoT and provide a minimum delay of 0.5 ms in the frame. The signal channels are shown in red in the green frame in Based on the results of the simulation, we found that the proposed NB-IoT system effectively provides the acceptable value of the delay and denial count for each real time IoT service under loads of the IoT controller from 0%\u201370%. Under high load conditions (70%\u2013100%), the system performs better than the existing method, but for not all IoT critical services are the acceptable value of the delay and denial count ensured, in particular, the results of which are shown in Compared to all previous generations, LTE networks achieve lower delays in data transmission due to fewer intermediate elements. Thus, the use of LTE networks to interact with IoT elements should bring additional revenues to operators and give them an impulse to further investment growth.This paper described a proposed solution for the Tactile IoT to transfer data with ultra-short delays, based on NB-IoT technology. We discussed our performed modifications to the LTE architecture by transferring part of the functions from the base station, eNodeB, to the NB-IoT controller. This controller performed the downlink and uplink channel planning for the IoT devices, which allowed the network operators to keep existing base stations eNodeB unchanged. We modified the structure of NB-IoT frame, where a logical data channel is allocated to reduce the delay and communication of the NB-IoT controller. The proposed solution extends the functionality of IoT control in real time for LTE-based 4G/5G networks.Further in this paper, we proposed a prioritization method within the IoT traffic to provide E2E QoS in the integrated LTE/NB-IoT network. We developed an algorithm for managing a \u201csmart queue\u201d based on the IoT traffic prioritization procedures. With the number of simulations, we demonstrated that our proposed approach ensured high end-to-end QoS of the real-time traffic. This was achieved by reducing an average end-to-end transmission delay of the real-time messages from 1.17 to 2.12 times as compare to the Proportional Fair Scheduling method."} +{"text": "To the Editor\u2014We read with great interest the article by Wang et al1 and the study by Cheng et al2 that highlighted the vital role of N95 respirators for preventing SARS-CoV-2 transmission and COVID-19 among healthcare workers (HCWs). The protective role of N95 respirators in other respiratory diseases could be translated to tackle the COVID-19 pandemic.1 Preliminary results in Hong Kong demonstrated that the use of N95 respirators for triage, for medical care of suspected or confirmed cases and during aerosol-generating procedures, drastically reduced COVID-19 infection among HCWs.2 We acknowledge that the effectiveness of N95 respirators to prevent SARS-CoV-2 transmission should be confirmed and their use in clinical practice should be supported during the COVID-19 pandemic. However, we are facing a scenario of global shortage in availability of personal protective equipment (PPE), including surgical masks and N95 respirators.3 Countries are in dispute over the insufficient number of manufacturers. With unfair markets and increasing prices, low- and middle-income countries are at risk of losing their ability to acquire PPE for their HCWs. Several studies have previously reported methods for PPE decontamination4 or reuse of N95 respirators.5Globally, the discussion by health authorities regarding new approaches to managing the N95 respirator shortage is urgent. The extended use or reuse and/or implementation of decontamination methods of N95 respirators might be an alternative that can prevent SARS-CoV-2 transmission among HCWs during the COVID-19 pandemic. Therefore, we have summarized recommendations regarding the extended use or reuse of N95 respirators, and we provide an overview of published information by regulatory authorities, surveillance organizations, and ministries of health of several countries.6 Following the screening of information up to April 10, 2020, information from each country or region was collected in an electronic database. We collected the date of publication and information and excerpts from the guidance document regarding the recommendations for extended and reuse of N95 masks or filtering face pieces (FFPs). Extended use was defined as use for longer periods without removing the respirator , and reuse indicated that the respirator was removed, stored, and used at least 1 more time.7Two researchers independently scrutinized the websites of the regulatory authorities of countries or regions and of ministries of health that a members or associates of the International Coalition of Medicines Regulatory Authorities (ICMRA).Overall, 27 countries or regions were screened: 5 countries 19%) only allowed extended use ; 2 countries (7%) mentioned only reuse (Germany and Netherlands); and 3 countries (11%) recommended both strategies for rationing N95 respirators . No information was available on extended use or reuse of N95 respirators in the websites of 17 countries (63%). Some countries (Germany and Netherlands) recommended specific methods for N95 respirators decontamination, and others (Europe and United States) mentioned several options, leaving the decision to health services managers published guidance regarding extended use and limited reuse of N95 respirators. Possible methods for decontamination cited as the most promising by the CDC were vaporous hydrogen peroxide, ultraviolet germicidal irradiation, and moist heat.9 In Brazil, the National Health Surveillance Agency (ANVISA) allowed the hospital infection control commissions (CCIHs) at each health service to create protocols for reuse by the same professional: use, withdrawal, packaging, assessment of integrity, time of use, and criteria for disposal.10Emergency use authorization (EUA) by the US Food and Drug Administration (FDA) allows the use of unapproved medical products or unapproved use of approved medical products. Currently, PPE items, in vitro diagnostic tests, and ventilators are included in the FDA EUA to tackle the COVID-19 pandemic. However, the FDA still does not allow sharing or reusing N95 respirators.The impact of the COVID-19 pandemic in each country or region might be influenced by the number of cases, the proportion of patients needing hospitalization, and the infrastructure of healthcare systems. Health authorities should consider global PPE shortages and should define feasible recommendations for extended use or reuse or decontamination of N95 respirators. Regulatory agencies of few countries empowered health services managers to implement strategies for decontamination and/or reuse procedures. The Ministry of Labor and Social Affairs of Germany described the recommended decontamination method for N95 respirators in detail . On the other hand, up to 60% of the screened countries did not report any recommendations for extended use or reuse or decontamination of N95 respirators. In summary, we have provided some evidence that regulatory authorities are trending toward relaxing regulations during the PPE shortage. The extended use and reuse of N95 respirators have become the last resort because it is crucial to maintain HCW protection during the COVID-19 pandemic."} +{"text": "Objective: To compare visual outcomes and complications between manual small incision cataract surgery (MSICS) and phacoemulsification.Methods: A retrospective study was conducted in the tertiary care center. A total of 1281 cases underwent manual small incision cataract surgery and phacoemulsification from January 2014 to December 2016. The postoperative best corrected visual acuity (BCVA) along with the rates of complications were compared between both groups.Results: Five hundred and twenty-one patients (40.67%) and 760 patients (59.33%) were subjected by staff members and residents, respectively. Altogether, 689 cases (53.79%) were subjected to MSICS technique and 592 cases (46.21%) to phacoemulsification. The MSICS group had significantly harder cataract . One month postoperatively, good visual outcome (BCVA \u2265 6 /18) in the phacoemulsification group was higher than that in the MSICS group . The risk factor for poor outcome (post-operative BCVA < 6 /60 in both groups) was the presence of associated ocular pathologies. The intraoperative and perioperative complications rates were higher in the MSICS group . The most common complications were hyphema (4.35%), posterior capsule ruptures (4.21%), and prolapsed iris (3.05%). Long-term postoperative complication rates were higher in the phacoemulsification group . The most common complication was posterior capsule opacity . Pseudophakic bullous keratopathy (PBK) was similar in both groups .Conclusion: The number of patients who had experienced good visual outcomes was higher in the phacoemulsification group. However, for both groups, no significant differences were found on the long-term complication rate. The most common cause of cataracts is due to the changes caused by the aging of the lens [2]. At present, there are a variety of common cataract surgery methods as such as the following: 1) Phacoemulsification (PE) - the cataract is removed by an ultrasonic device (Phaco machine), which is considered to be the most popular surgery that is being currently used due to the small incision of approximately 3 millimeters in size, which results in a speedy recovery from postoperative wound despite the high cost; 2) Extracapsular cataract extraction (ECCE) is a surgery requiring the creation of a large surgical wound (approximately 8-10 millimeters in size) and multiple stitches, which lead to a long time for recovery and more astigmatism after surgery; and 3) Manual small incision cataract surgery (MSICS), a variety of surgical techniques currently available [3-10]. For instance, Prapokklao Hospital in Thailand has performed cataract surgery using manual small incision cataract surgery (MSICS) and has developed a surgical procedure that can be performed by using various techniques, such as Modified Blumenthal technique, Nylon loop technique, Kongsap\u2019s technique, and the Ruit\u2019s technique . The Modified Blumenthal technique has been used for small incision cataract surgery for patients at Prapokklao Hospital for over 20 years. However, at present, there is no comparative study of the post-operative visual outcomes and long-term complications from cataract surgery by manual small incision cataract surgery (MSICS) and by phacoemulsification in Thailand. Cataracts, which are the number one cause of blindness, can be treated by surgery [13]. Phacoemulsification is a better method when compared with other methods . However, in cases in which there are very hard or milky white cataracts, it has been found that manual small incision cataract surgery provides better and safer surgical results even though results emerging from the long-term surgery, including its complications over 1 year, have not yet been reported .In general, cataract surgery is safely controlled by The World Health Organization (WHO) and is carried out through a set of standardized surgical procedures, which are safe for patients with a good level of vision, by determining a good result to be at least 90 percent and a poor result to be not more than 5% [The objective of this study was to compare the level of vision of post-operative visual outcome and the long-term complications with reference to manual small incision cataract surgery (MSICS) and cataract surgery using phacoemulsification.18], fundus examination, keratometry prior to surgery, the date and time of the surgery, laterality of the eye that underwent the surgery, the surgical method, the surgeons (Staff Member or Resident), any complications that occurred during surgery and after surgery, the type of artificial intraocular lens inserted in the eye of the patient, post-operative visual acuity, and the follow-up appointment. This data was collected on the research form.This research was a retrospective comparative study and its ethical approval was reviewed by the Human Ethics Committee of Prapokklao Hospital. The data was collected from the medical records of patients, who underwent cataract surgery at Prapokklao Hospital in Chanthaburi from January 2014 to December 2016. The data in the medical records encompassed the sex, age, systemic diseases, and ocular diseases with regards to the patient. Each patient underwent an eye examination with the aid of a slit lamp under the supervision of an ophthalmologist. The examination included the measurement and assessment of visual acuity, intraocular pressure, nucleus hardness/ lens opacities .In the WHO recommendations on the outcome of cataract surgery, the visual acuity values were the following: good visual acuity (6/ 6-6/ 18), borderline visual acuity (< 6 /18-6 /60), and poor visual acuity (< 6/ 60) [18] as it follows: 1) by Nuclear sclerosis \u2264 3+; 2) Cortical and PSC were considered as \u201csoft cataracts\u201d; 3) Nuclear sclerosis \u2265 4+, and 4) mature and hyper-mature cataracts were considered as \u201chard cataracts\u201d.The classification to determine the hardness of the cataract was carried out in accordance with the lens opacities classification system III . Moreover, corneal edema (2.18%) was found one day after the surgery. This was because surgery was mostly conducted at the scleral tunnel and it was likely that trauma to the iris had occurred during lens subluxation and lens delivery. In the MSICS group, the surgeons team consisted of 58.49% residents, who had less experience and less expertise than the staff members [21]. This was likely to have caused more complications than in the Phacoemulsification group because most of the surgeons were staff members (80.07%).Regarding intraoperative/ perioperative complications, the MSICS group had more complications than the Phacoemulsification group , which was consistent with the results from the studies of Rohit C Khanna et al. and Aravind Hairpriya et al., who typically found that the complications were hyphema (4.35%), posterior capsule rupture (3.34%), and iris prolapse (3.05%) .Pseudophakic bullous keratopathy (PBK), which is a late serious postoperative complication is also another indicator of safety of each of the operative methods. In this study, patients were monitored for over 1 year (on average about 80 weeks after MSICS surgery), and it was found that there were 2 cases of PBK (0.29%) compared to 1 case of Phacoemulsification (0.17%). No significant difference was discovered, and it was found to be less than the incidence in cataract surgery (1-2%) [23].Post-operative endophthalmitis was not found in this study, and the incidence was lower than in the United States (0.04%) [Limitations in this research study1) Since this was a retrospective study, information about complications was likely to be collected less than the real numbers.2) Patient characteristics in both groups of patients were different from the beginning, which caused discrepancies between both groups of patients.3) Regarding the surgeries, residents performed 58.49% of the MSICS and 19.93% of the phacoemulsification of all patients, which resulted in a longer duration of surgery, as well as in more complications in the MSICS group than usual.Regarding the post-operative visual acuity results, the cataract patients, who underwent Phacoemulsification, had significantly better visibility than those receiving MSICS at 1 month, 3 months, and more than 1 year after the surgery. However, late postoperative complications were found more frequently in the Phacoemulsification group as compared to the MSICS group. Most of the complications dealt with the posterior capsule opacity (PCO) since hydrophilic acrylic lenses had mostly been implanted in the Phacoemulsification group. Meanwhile, no differences between the MSICS group and the Phacoemulsification group were found with respect to the long-term complication rate.Conflict of InterestAuthors state no conflict of interest.Informed Consent and Human and Animal Rights statementsInformed consent has been obtained from all individuals included in this study.Authorization for the use of human subjectsEthical approval: The research related to human use complies with all the relevant national regulations, institutional policies, is in accordance with the tenets of the Helsinki Declaration, and has been approved by the Human Ethics Committee of Prapokklao Hospital.AcknowledgementsThe article has not been presented in any meeting. Sources of FundingThis study was funded in-part by the Prapokklao Hospital research and development fund.DisclosuresNone."} +{"text": "Minimally invasive surgery (MIS) for evacuation of spontaneous intracerebral hemorrhage (ICH) has shown promise but there remains a need for intraoperative performance assessment considering the wide range of evacuation effectiveness. In this feasibility study, we analyzed the benefit of intraoperative 3-dimensional imaging during navigated endoscopy-assisted ICH evacuation by mechanical clot fragmentation and aspiration.18 patients with superficial or deep supratentorial ICH underwent MIS for clot evacuation followed by intraoperative computerized tomography (iCT) or cone-beam CT (CBCT) imaging. Eligibility for MIS required (a) availability of intraoperative iCT or CBCT, (b) spontaneous lobar or deep ICH without vascular pathology, (c) a stable ICH volume (20\u201390\u00a0ml), (d) a reduced level of consciousness (GCS 5\u201314), and (e) a premorbid mRS \u2264\u20091. Demographic, clinical, and radiographic patient data were analyzed by two independent observers.p\u2009<\u20090.0001 vs. Pre-OP). Based on the intraoperative imaging results, 1/3rd of all patients underwent an immediate re-aspiration attempt. No patient experienced hemorrhagic complications or required conversion to open craniotomy. However, routine postoperative CT imaging revealed early hematoma re-expansion with an adjusted evacuation rate of 59\u2009\u00b1\u200930% .Nine female and 9 male patients with a median age of 76\u00a0years (42\u201385) presented with an ICH score of 3 (1\u20134), GCS of 10 (5\u201314) and ICH volume of 54\u2009\u00b1\u200926\u00a0ml. Clot fragmentation and aspiration was feasible in all cases and intraoperative imaging determined an overall evacuation rate of 80\u2009\u00b1\u200919% contains supplementary material, which is available to authorized users. Intracerebral hemorrhage (ICH) accounts for up to 15% of all strokes and affects more than 2 million people annually . Of all For neurosurgical performance assessment, intraoperative imaging such as MRI , 10, 35,The study was approved by the local ethics committee of the Charit\u00e9 University Hospital in Berlin, Germany (EA1/223/19) and performed in compliance with the Health Insurance Probability and Accountability Act regulations. Informed consent was waived due to the retrospective nature of the study. Between July 2016 and July 2019, 18 consecutive patients that underwent navigated, endoscopy-assisted evacuation of spontaneous intracerebral hemorrhage using a mechanical fragmentation and aspiration device were identified. Demographic, clinical and radiographic patient data with focus on surgical workflow, pre-, intra-, and postoperative hematoma volumes, procedure-related complications, and outcome measured as the level of consciousness according to the Glasgow Coma Scale (GCS) score before and at the time of discharge and the 30-day mortality were collected and analyzed by two clinicians who were not directly involved in the patients\u2019 care .Availability of intraoperative CT or CBCT imaging.Supratentorial location without vascular pathology (excluded by computerized tomography angiography (CTA) and/or digital subtraction angiography (DSA)).Hematoma volume 20\u201390\u00a0ml.Reduced level of consciousness with a Glasgow Coma Scale (GCS) score 5\u201314.Premorbid modified Rankin Scale (mRS) score \u2264\u20091.Stable control CT after 6\u00a0h without spot sign and surgery ideally within 72\u00a0h.All patients were treated according to the guidelines of the German societies of Neurology and Neurosurgery. The indication for minimally invasive hematoma evacuation was based on previous findings , 27, 33 Positioning and navigation registrationSurgery was performed on a mobile, radiolucent, carbon fiber examination table in prone or supine position, depending on the hematoma location. The patients\u2019 head was fixed in a radiolucent carbon fiber 3-pin head clamp . For navigated hematoma evacuation based on an intraoperatively acquired image data set, the iCT or CBCT was connected to an image guidance system with infrared tracking camera and a preoperative CT scan or intraoperative CT/CBCT scan was used for automatic patient/image co-registration. The navigation reference device was fixed to the head clamp. The navigation camera was set up to allow co-registration of the navigation reference device and the registration fiducials on the iCT gantry or CBCT flat panel detector, similar to the setup for automatic patient/image co-registration in spinal navigation . The iCT2.Segmentation and aspiration trajectory planningSegmentation of the hematoma volume and planning of the aspiration trajectory was performed preoperatively or directly in the OR with image guidance software . After segmentation, the aspiration trajectory was planned corresponding to the longest axis of the hematoma under consideration of the transverse and sagittal planes or percentage, as appropriate. Statistics were calculated with GraphPad Prism for Mac . For comparison of pre-, intra-, and postoperative hematoma volumes, a repeated measure one-way analysis of variance (ANOVA) with Geisser Greenhouse correction and Tukey\u2019s multiple comparison test was performed. For comparison of the intra- and postoperative hematoma evacuation rate, a paired Demographic and clinical data are presented in Table p\u2009<\u20090.0001 vs. Intra-OP and ***p\u2009<\u20090.001 vs. Post-OP; Fig.\u00a0p\u2009<\u20090.01 Intra-OP vs. Post-OP; Fig. Data on intraoperative imaging and hematoma volume are shown in Table despite hematoma reduction was observed in 2/18 patients (11%). Postoperative re-hemorrhage beyond the initial hematoma volume and despite intraoperative reduction was observed in 1/18 patients (6%). Withdrawal-of-care decisions were eventually made in 3/18 patients due to a prolonged clinical course incompatible with the patients\u2019 living will, resulting in a 30-day mortality rate of 17%. The postoperative GCS in the surviving 15 patients declined in 1 (7%), remained equal in 2 (13%) and improved in 12 patients (80%), resulting in a median postoperative GCS of 12 (range 3\u201315) The median postoperative mRS score at discharge was determined at 4 (2\u20136) (Table A postoperative decline in the level of consciousness 6) Table .In this feasibility study, we investigated the performance of navigated, endoscopic MIS and intraoperative 3D imaging for evacuation of supratentorial ICH through mechanical clot fragmentation and aspiration. Despite lower 3D image quality than conventional CT imaging, 3D-reconstructed cranial iCT and CBCT permitted intraoperative navigation planning and reliable evacuation rate assessment with the possibility to perform an immediate re-evacuation attempt, which is important considering the noted risk of early hematoma re-expansion.The current situation for ICH appears similar to the situation during the early days of thrombectomy for ischemic stroke, where patient selection was considered one of the main reasons for the initial failure of endovascular therapy, meanwhile successfully established as the standard of care . In ICH,Many consider deep hematomas ideal for MIS procedures due to the large perihemorrhagic involvement of viable but highly vulnerable tissue. Although our study is underpowered to draw conclusions regarding the superiority of superficial versus deep MIS for ICH, technical success was achieved in both groups with comparable intraoperative evacuation rates of 79% and 80% but patients with superficial location appeared to benefit more considering a postoperative GCS improvement from 11 to 14 compared to patients with deep hematomas and an unchanged GCS of 9. Thus, our findings suggest that despite comparable effectiveness, MIS for deep-seated ICH should remain critically weighed.Interestingly, time to treatment has been shown to carry weight in ischemic stroke but it has not yet been shown to play a key role in hemorrhagic stroke although patients who undergo MIS of ICH within 24\u00a0h have a 30% greater likelihood to achieve functional independence than patients who undergo MIS within 72\u00a0h . AccordiAmong the various MIS modalities for ICH , 24, 37,For the first time, MISTIE III evidenced that reducing the absolute hematoma volume below a specific threshold appears to be the most beneficial therapeutic target . Still, Surgical performance in MIS for ICH is crucial to ensure the best possible chances of favorable recovery . This isAlthough our study inherently lacks power due to its retrospective nature, small sample size, lack of a control group and long-term follow-up, the investigated patient cohort is representative of ICH patients considered for MIS in regard to clinical presentation, demographics, hematoma volume, hematoma location, and ICH grade. Another limitation is that we did not analyze radiation exposure. Importantly, the surgeon must consider that intraoperative iCT and CBCT imaging exposes the patient to at least one additional iCT or CBCT scan for intraoperative evacuation assessment. On the other hand, we believe that the additional iCT or CBCT radiation exposure is justifiable in MIS for ICH considering the severity of the disease and impact of targeting a critical volume threshold. Although radiation exposure in iCT imaging is most likely higher than in CBCT imaging, it has been demonstrated that the effective iCT patient dose remains at an acceptable level compared to CBCT technology , 34. NevESM 1(PDF 28\u00a0kb)"} +{"text": "We aimed to compare the diagnosis performance of FAPP with conventional testing in 100 intensive care unit (ICU) patients who required mechanical ventilation, with clinically suspected HAP. A total of 237 samples , were analyzed independently by routine microbiology testing and FAPP. 58 patients had paired BALDS and ETADS. The positivity thresholds of semi-quantified bacteria were 103\u2013104 CFUs/mL or 104 copies/mL for BAL, and 105 CFUs/mL or copies/mL for ETA. Respiratory commensals were the most common pathogens. Discordant results for bacterial identification were observed in 33/76 (43.4%) BALDS and 36/82 (43.9%) ETADS, and in most cases, FAPP identified one supplemental bacteria (23/33 BALDS and 21/36 ETADS). An absence of growth, or polybacterial cultures, explained almost equally the majority of the non-detections in culture. No linear relationship was observed between bin and CFUs/mL variables. Concordant results between paired BALDS and ETADS were obtained in 46/58 (79.3%) patients with FAPP. One of the 17 resistance genes detected with FAPP (mecA/C and MREJ) was not confirmed by conventional testing. Overall, FAPP enhanced the positivity rate of diagnostic testing, with increased recognition of coinfections. Implementing this strategy may allow clinicians to make more timely and informed decisions.The FilmArray Hospital-acquired pneumonia (HAP) is the most frequent cause of nosocomial infection in intensive care unit (ICU) patients, with dramatic effects on patients\u2019 outcomes. International experts have developed guidelines to prevent and improve the management of HAP . Among sStreptococcus pneumoniae, Haemophilus influenzae). The FilmArray\u00ae Pneumonia plus Panel (FAPP) is a new panel for HAP, which offers potential advantage to detect and quantify in a single test, 27 respiratory pathogens and 7 antibiotic resistance genes.Microbiological confirmation of HAP is a crucial step for tailoring antibiotic therapy. Nevertheless, current culture methods take 48\u201372 h to obtain antimicrobial susceptibility results. Moreover, traditional techniques fail to recover pathogens in up to 30% of clinically-diagnosed HAP . RecentlThe aim of this study was to assess the performances of this new molecular test on bronchoscopy specimens [bronchoalveolar lavages (BAL) and/or endotracheal aspirates (ETA)] from 100 ICU patients with HAP requiring mechanical ventilation.The study protocol was approved by our local Ethical Committee . Patients and relatives were informed of the trial. Consent was waived according to French law.DS (forDIAGNOSIS) and ETADS, respectively). In addition, if an ETA was collected 2\u20133 days later, as part of a routine clinical care, the specimen [ETATT(forTREATMENT)] was also sent to the laboratory for microbiological analysis. A total of 237 respiratory specimens were analyzed . Both BALDS and ETADS were collected in 58 patients.The study was conducted at the Nantes University Hospital (France), in 3 ICUs located on two sites spaced 10 km apart. We recruited 100 critically ill adult patients receiving mechanical ventilation with clinically suspected HAP, between October 2018 and January 2020 . PneumonThe respiratory specimens were analyzed in parallel by routine microbiology testing and FAPP, as soon as they arrived at the microbiology laboratory. The turnaround times from samples to validated results were recorded. Results of routine microbiology testing were analyzed independently of FAPP.2 for 24\u201348 h, as necessary. Plates were examined daily for bacterial growth. Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Enterobacteriales, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and any other largely predominant pathogen were searched on the plates. In accordance with current guidelines, the positivity thresholds were 105 CFUs/mL for ETA and 104 CFUs/mL for BAL, but in BAL, potential pathogens that were present in pure culture at 103 CFUs/mL and associated with many leukocytes at Gram staining were reported as positive. Culture results were considered as negative if there was no significant growth or a normal non-pathogenic flora. Bacterial isolates were identified by mass spectrometry (BioM\u00e9rieux). Antimicrobial susceptibility testing (AST) was performed according to the CA-SFM/EUCAST guidelines , and methicillin-resistance detection (Alere\u2122 PBP2A culture colony test and BDMAX\u2122 StaphSR). Furthermore, when requested by the Clinicians, the presence in respiratory samples of Mycoplasma pneumoniae and/or respiratory viruses was investigated by real-time PCR .Gram staining and bacteriological cultures were performed for all respiratory specimens according to the French REMIC recommendations . Brieflyidelines , using V\u00ae FilmArray\u00ae Pneumonia plus Panel (bioM\u00e9rieux) was performed according to the manufacturer\u2019s instructions, with a handling time of \u223c5 min. Briefly, the respiratory sample collected with a flocked swab (\u223c 200 \u03bcL) and then mixed with a sample buffer, was injected along with an hydration solution in the reagent pouch \u201cPneumonia plus Panel,\u201d which was then inserted into the FilmArray\u00ae instrument. The test consisted of automated nucleic acid extraction, purification, amplification, detection, and analysis with each target reported as \u201cdetected\u201d or \u201cnot detected.\u201d A semi-quantitative measurement reported into bins was provided for 15 bacteria, if detected. The panel included 15 bacteria, 3 atypical bacteria, 9 viruses, and 7 antimicrobial resistance genes (The BioFirece genes . Each re4 copies/mL for semi-quantified bacteria). For ETA, in order to match the culture threshold that differentiate commensalism from pathogenicity (\u2265105 CFUs/mL), we set up a bin threshold of \u2265105 copies/mL to consider the 15 semi-quantitative bacterial targets as positive. The agreement between FAPP and culture was measured for each bacterial pathogen in the form of negative percent agreement (NPA), positive percent agreement (PPA) and overall percent agreement (OPA), and their two-sided 95 percent confidence intervals. In order to explain discrepant results, cultures were reread after routine final reports in light of results obtained with FAPP. Concordance was calculated based on the original culture reading.BAL were considered as positive with FAPP when at least one microbial target was detected (at \u2265104 bin in BAL and \u2265 105 bin in ETA for semi-quantified bacteria) in 82/100 patients. Thus, as shown in DS, and 75.6% (62/82) ETADS were positive for at least one target. Of these, more than half were positive for at least two pathogens (36/62 (58.1%) for BALDS, and 36/62 (58.1%) for ETADS), leading to the diagnosis of coinfection in 49/100 patients . Of note, if the 104 cutoff had been used for ETA, 84.1% (69/82) ETADS would have been positive, and multiple targets would have been detected in 60.9% (42/69) of these specimens (DS and ETADS). The most common pathogens detected at diagnosis were H. influenzae, S. aureus, E. coli, S. pneumoniae, and K. pneumoniae, which were found in 40 (40%), 33 (33%), 19 (19%), 17 (17%), and 10 (10%) patients, respectively ETADS). In most cases, it corresponded to viral-bacterial co-infections and S. pneumoniae) (M. pneumoniae) was detected with other bacteria in one patient. The positivity rate of ETATT obtained during follow-up was 69.6% (55/79), and 38 bacteria were below the 105 cutoff in 29 ETATT (mecA/C and MREJ (one patient), and the CTX-M ESBL (7 patients), either alone (5 patients) or combined with a carbapenemase . The median turnaround time (from sample collection to results) was 4 h 15 min (BALDS or ETADS).At the time of HAP diagnosis, FAPP yielded positive results with significant levels . An atyp29 ETATT , 2. FourDS and 53/82 (64.6%) ETADS), and respiratory viruses were detected in 8/35 (22.9%) patients who benefited from a Fast Track multiplex PCR routinely ordered by clinicians, yielding an overall positive detection in 78/100 patients. Two or more bacterial pathogens were identified and reported in 32/100 patients, in a higher proportion of BALDS than ETADS , certainly because BAL are more distal than ETA and are normally not contaminated. Indeed, this property might have encouraged microbiologists to identify and report any bacteria found in these distal specimens rather than concluding to \u201cpolymicrobial flora.\u201d Thus, only 25.0% (6/24) of culture-negative BALDS had results reported as \u201cmixed bacterial flora\u201d vs. 41.4% (12/29) of culture-negative ETADS , 26 (26%), 17 (17%), 13 (13%), and 8 (8%) patients, respectively , while in the 4 others cases, high-level cephalosporinases were confirmed with additional tests (Mastdiscs\u2122 D68C), in strains of E. cloacae complex (2 patients), S. marcescens (1 patient), and E. coli (1 patient). Two ESBL-producing K. pneumoniae that were resistant to ertapenem \u00b1 imipenem, were also confirmed to be carbapenemase (NDM or OXA-48 like) producers, by means of an immuno-chromatographic test (CORIS BioConcept RESIST-3 O.K.N.) performed 2 days after specimen collection. All strains of P. aeruginosa detected in 4 patients were susceptible to ceftazidime. The mean turnaround time from sample collection to results validation was 70 h for BALDS, and 64 h for ETADS.At HAP diagnosis, culture identified one or more bacteria in 73/100 patients (52/76 (68.4%) BALve ETADS . The mosectively . Culture, 76.1%) . RegardiDS and 46/82 (56.1%) ETADS) (DS and 21/36 (58.3%) ETADS), FAPP identified one supplemental bacterial pathogen, which was most often confirmed by FAPP in the paired respiratory sample and/or in the ETATT collected 2\u20133 days later (DS and 6/82 (7.3%) ETADS], culture yielded bacteria that were not targeted by FAPP , and two FAPP false-negative results were observed: K. oxytoca (one BALDS with a pure culture at 103 CFUs/mL), and H. influenzae BAL) ETADS) . In mostys later . By rereys later . In the nvasion) . FurtherCFUs/mL) . The atyTT obtained under treatment, 45.6% (36/79) had discordant results between both methods (4 in ETADS) in a few cases .4 in ETA were not reported in culture (23/23 (100%) in ETADS, and 36/38 (94.7%) in ETATT). On the other hand, for patients with ETA at diagnosis and 2\u20133 days later, 38.9% (7/18) of the detections with a bin value of 104 in ETADS were positive again in ETATT with a higher bin value (\u2265 105 copies/mL). No linear relationship was observed between the bin and CFUs/mL variables (10 ranges above positive thresholds (105 CFUs/mL for ETA and 104 CFUs/mL for BAL).Regarding FAPP semi-quantitative results, most bacteria with bin results of 10ariables . HowevermecA/C and MREJ, 13 blaCTX\u2013M, 2 blaNDM, and 1 blaOXA\u201348\u2013like) (S. aureus (MRSA) detected with FAPP, one found at 104 bin in ETATT did not grow in culture. The other corresponded to a false-positive mecA/C and MREJ result since a methicillin-susceptible S. aureus (MSSA) was found in culture. This result was repeatable after retesting with FAPP, but none of the comparator methods found a MRSA. No additional cases of methicillin-resistance, ESBL, or carbapenemase production were found with routine microbiology testing.Eighteen resistance markers were detected with FAPP in 15 samples (2 48\u2013like) . All ESBP. aeruginosa, A. baumannii, E. cloacae complex, K. aerogenes, and S. marcescens) and/or resistance markers of the panel. However, this antibiotic would have not been optimal in 7/83 (8.4%) patients. In fact, in those cases, culture brought to light bacterial strains with acquired resistance to amoxicillin-clavulanate , or pathogens not targeted by FAPP and naturally resistant to amoxicillin-clavulanate . A medico-economic evaluation is ongoing to determine what impacts FAPP results would have had on care and antibiotics prescribing .Lastly, based on FAPP results, an initial antibiotic therapy by amoxicillin-clavulanate could have been proposed in 83/100 patients, whose results ruled out pathogens with chromosomally-encoded cephalosporinase had the same pathogen(s) (or no pathogen) identified in both samples with FAPP. Of the 12 discrepancies observed, 5 were due to detection of one more pathogen in ETADS , 4 to detection of one additional bacteria in BALDS . In the 3 latter cases, the difference relied on two pathogens. If bacteria with a 104 bin had been considered as positive in ETADS, the agreement between both types of specimens would have been less satisfactory, with 38/58 (65.5%) concordant results were confirmed with both methods (similar results between FAPP and culture) (Among the 58 patients with paired BAL results . Regardiculture) .\u00ae Unyvero Hospitalized Pneumonia Panel, are the first two, FDA approved and CE marked, commercially available platforms which target a large number of lower respiratory tract pathogens and resistance genes from aspirates or BAL fluids , enabling identification of additional bacteria in 39.5% BALDS and 37.8% ETADS. The most common pathogens detected were consistently the same across both methods . This pathogen distribution, which mostly corresponded to bacterial species that are part of the normal throat flora, was not really different from that described in community-acquired pneumonia. According to the latest European surveillance report on healthcare-associated infections acquired in ICU in 2017, P. aeruginosa was the most common microorganism associated with pneumonia (19.9%), followed by S. aureus (18.5%), Klebsiella spp. (15.2%), and E. coli (13.5%). In the majority of cases, pneumonia was associated with intubation, and HAP episodes occurred after an average length of ICU stay of 7.3\u201312.1 days, depending on the country . In line with our data, the majority of discrepancies previously reported between FAPP and culture, concerned the same fastidious bacteria, and were explained by the higher sensitivity of the molecular test and/or antibiotics consumption before sampling , were not always explained by no bacterial growth. As noted previously, these findings pointed the limits of bacterial cultures, which are subject to interpretation and based on selection of dominant species assigned to play a pathogenic role, the minority species being not considered for ETADS, and 28/36 (77.8%) for ETATT]. A quater (25/100) of the patients enrolled in the study had received antibiotics before sampling at the time of HAP diagnosis, while ETATT were collected under antibiotic treatment. Thus, in our view, these culture-negative detections most likely corresponded to pathogens present at low abundances or to remnant DNA from non-viable bacteria, notably in supplemental ETATT, rather than non-specific amplifications. In fact, FAPP results from ETATT and/or paired BALDS or ETADS allowed to verify a lot of FAPP-positive results for bacteria that had been undetected by culture. As a result, FAPP may prove useful to guide treatment in situations of diagnostic uncertainty where patients have received antibiotics before sampling, and/or have unfavorable treatment outcomes after obtaining culture, because the higher sensitivity of this method decreases the likelihood to miss out on pathogens of the panel.As expected, implementation of FAPP shortened the delay in getting results . In accordance with recent evaluations , FAPP in country . In our \u20136 days) . The mossampling . Here, ipathogen . Howevernsidered . These rnsidered . MoreoveDS or ETADS), compared to 32/100 patients by culture. These data corroborate other published results, and outline that the true incidence of polymicrobial HAP is probably underestimated with conventional techniques . However, a small proportion (38.9%) of targets quantified as 104 copies/mL in ETADS, were recovered later with higher bin values in ETATT. Thus, we show that in those potentially contaminated samples, targets quantified as 104 copies/mL by FAPP, can be reported as negative to provide results concordant with those routinely reported by culture, in accordance with current guidelines (\u2265 105 CFU/mL) . NonetheDS and ETADS were collected, and could be compared. Latest European and American guidelines for the management of HAP provide divergent recommendations on sampling techniques to prioritize for diagnosis of HAP. While scientific societies from North America place a high value on non-invasive sampling with semiquantitative cultures , European guidelines suggest obtaining distal quantitative samples with invasive techniques to improve the accuracy of results, and reduce overutilization of antibiotics .An originality of this work lies on the inclusion of 58 patients from whom both BALibiotics . In factH. alvei, M. morganii, and S. maltophilia) in 3/100 patients, were missed by the panel. On the other hand, the molecular test led to an increased identification of respiratory pathogens, and to the rapid detection of some genotypic markers of resistance in 8 patients. Thus, in total, for covering FAPP findings, the narrow-spectrum antibiotic amoxicillin-clavulanate could have been a therapeutic option in the majority of patients (83%). Nonetheless, natural or acquired resistances to amoxicillin-clavulanate would have gone unnoticed in 8.4% of them. All carbapenemase and/or ESBL-producing strains were correctly detected with the multiplex panel (AST agreed with FAPP). However, it should be noted that the overall prevalence of antimicrobial resistance was low in our study, and it should also be kept in mind that a lack of detection of resistance genes does not necessarily means susceptibility to antibiotics as there are resistance mechanisms that are not detected by FAPP . Regarding methicillin resistance, consistent with previous observations, we noticed the false-positive detection of mecA/C and MREJ genes in one specimen containing a MSSA in culture .In conclusion, our study demonstrates that FAPP provides results at a speed and sensitivity never possible before, and may allow clinicians to make more informed decisions about antibiotics use and isolation of patients. There is still room for improvements in terms of breadth , resistance (MRSA), and cost, but this culture-independent technique may achieve more reliable identification of causative agents than culture. There will be a learning curve for physicians to establish how best to use FAPP results in the management of ICU patients with HAP. To achieve maximum benefit from this new molecular test, nuances in results interpretation might be applied on the basis of clinical presentation, timing of initial antimicrobial therapy (fresh vs. post-treatment samples), sampling type (BAL vs. ETA), and local bacterial ecology and resistance patterns. We are currently assessing the impact of this platform on antibiotic use and patients outcome in our hospital, and are evaluating if an algorithm-based treatment plan guided by FAPP would be of great benefit.All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by the Groupe Nantais d\u2019Ethique dans le Domaine de la Sant\u00e9 (GNEDS). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.LC, AR, and SG were involved in all the aspects of the study and were the guarantors for the data. BG, MB, KL, BR, and AR performed the clinical procedures. LC, TD, EP, and SG performed the laboratory procedures. LC, TD, FG, CP, M-AV, and SG analyzed the data. LC and AR wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Our World in Data) to these GCM parameters. Our analysis showed that with time, the parameters were influenced by different factors; for example, the parameter related to the maximum number of predicted cumulative confirmed cases was greatly influenced by the total population size, as expected. The other parameter related to the rate of spread of COVID-19 was influenced by aging index, cardiovascular death rate, extreme poverty, median age, percentage of population aged 65 or 70 and older, and so forth. We hope that with their consideration of a country\u2019s resources and population dynamics that our results will help in making informed decisions with the most impact against similar infectious diseases.The outbreak of the novel COVID-19, declared a global pandemic by WHO, is the most serious public health threat seen in terms of respiratory viruses since the 1918 H1N1 influenza pandemic. It is surprising that the total number of COVID-19 confirmed cases and the number of deaths has varied greatly across countries. Such great variations are caused by age population, health conditions, travel, economy, and environmental factors. Here, we investigated which national factors influenced the spread of COVID-19 through systematic statistical analysis. First, we employed segmented growth curve models (GCMs) to model the cumulative confirmed cases for 134 countries from 1 January to 31 August 2020 (logistic and Gompertz). Thus, each country\u2019s COVID-19 spread pattern was summarized into three growth-curve model parameters. Secondly, we investigated the relationship of selected 31 national factors (from KOSIS and The novel coronavirus disease 2019 (COVID-19), a highly transferable viral disease, is a respiratory illness caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has person-to-person contact as the main route of transmission and causes flu-like symptoms and in severe cases death . The pubMany factors can influence the epidemiological characteristics and contribute to the increased mortality rate of COVID-19 ,7,8,9,10Furthermore, several studies have investigated the impact of weather on the COVID-19 transmission, with special attention being paid to temperature and humidity, indicating that temperature is inversely related to COVID-19 incidence . MoreoveA look at history tells us that pandemics and epidemics have consistently and significantly affected human lives, and that governments have continually tried to find ways of slowing down the spread of these diseases; for example, quarantines were employed during the Ebola outbreak in West Africa ,15. The Here, we first applied segmented growth-curve models (logistic and Gompertz models) to the cumulative confirmed cases of 213 countries. Next, we applied the segmentation algorithm to divide the cumulative curve of COVID-19 cases into several segments of time series cases corresponding to a specific segment, which can then be modeled by the conventional growth curve models into a sigmoid curve. As the spread of COVID-19 has been prolonged, several countries experienced more than one wave summarize the spread of COVID-19 into sets of three parameters \u03b1, \u03b2, and \u03b3, where \u03b1 is the maximum number of predicted cumulative confirmed cases, \u03b2 is the time when we start to see a rise in the number of confirmed cases, and \u03b3 is the rate of spread of COVID-19. Thus, each country\u2019s COVID-19 spread pattern was summarized into three GCM parameters. Then, a regression model was employed to investigate the relationship between 31 selected national factors from in Data and Kore in Data such as The COVID-19 data of daily confirmed cases and deaths can easily be downloaded from the European Centre for Disease Prevention and Control (ECDC) website ,21. ECDCp is the number of confirmed cases.Data smoothing was used to remove noise from a dataset, allowing important patterns to stand out. Thereafter, daily confirmed case data were smoothed by simple moving average (1) to reduce the effect of outliers and (2) to remove the weekly periodicity observed in the data. There were several outliers that showed greater or smaller abnormalities, which made it difficult to fit the statistical model. In addition, weekly periodicity was observed in the daily confirmed case data for many countries. Although we tried to present numerically through autocorrelation function, the trend had randomness, giving a limit to the analysis. Therefore, considering the period of 7 days, we set the window size to 7, and simple moving average (SMA) was used before model fitting as shown belowOur World in Data website [Our World in Data website provides data about research and data to make progress against the world\u2019s largest problems such as poverty, disease, hunger, climate change, war, and existential risks. It mainly focuses on the large problems that continue to confront us for centuries or much longer, as well as the long-lasting forceful changes that gradually reshape our world. From this website, we obtained 15 time-independent social and economic factors assumed to be related to COVID-19 in the literature, such as population, population density, median age, being aged 65 or over, being aged 70 or over, GDP per capita, extreme poverty, cardiovascular death rate, diabetes prevalence, female smoker, male smoker, handwashing facilities, hospital beds per thousand people, life expectancy, and human development index [Time-independent national factor datasets website and the website . The Ournt index ,27,28,29The Korean Statistical Information Service (KOSIS) website Under this analysis, the growth curve models (GCMs) logistic model and Gompertz model were employed to model the transmission of COVID-19 using the cumulative confirmed cases for each country. These growth models are commonly used to explore risk factors and predict the probability of occurrence of a certain disease, investigate factors that control and affect growth, and extinction laws of the population . The modt is the number of days since the first case occurrence, and \u00a0t is the number of days since the first case, and \u00a0As the COVID-19 situation continues, fitting a growth curve model on daily confirmed cases over a long period of time has become impossible as it no longer takes on an s-curve (i.e. sigmoid function). To fit the above growth curve models, there is a need to divide the study period of countries experiencing more than one wave a wave . Segmentation is a method of finding peaks and breakpoints, where a peak is the timestamp at which daily new confirmed case is highest in a segment, and breakpoint is the timestamp that splits the consecutive two segments in a time series dataset. To better see trends, we smoothed out the irregular roughness of the graph of daily confirmed cases. However, daily new confirmed cases have high randomness arising from (1) the fact that daily new confirmed cases have a periodicity of seven days (due to differences in daily new confirmed cases between weekends and weekdays) and (2) measure errors of one day. Therefore, we applied the Nadaraya\u2013Watson kernel regression estimator (NWE) ,33,34 wiPeak detection fit the above-mentioned growth curve models ((1) and (2)) for each segment independently. These new models did not preserve continuity at breakpoints, but this did not matter since the objective of our analysis was to condense daily new confirmed cases into several parameters . The number of countries with three segments was very small, making the comparison analysis insignificant to use in the regression analysis. For countries with more than 2 segments, the analysis period was, therefore, cut off at the second breakpoint. For countries with 2 segments, segmented growth curve model then would produce two sets of parameters, one set from each segment. After the segmentation algorithm was applied to 134 countries, these countries were fitted to segmented logistic and Gompertz models. To filter out poorly fitted countries, we excluded countries whose MSSE was higher than 0.4, as defined below:MSSE is a more suitable measure compared with MSE (mean squared error) or MAPE (mean absolute percentage error) because the MSE does not consider scales of population among each country, while MAPE overestimates its error when the number of daily new confirmed cases, and tz model . In addition, correlation analysis for segmented logistic and Gompertz models with the log-scaled of parameters was performed to determine the similarity between parameters of the two models see .y, a measure of the spread dynamics of COVID-19 for a country.The above segmented growth curve models summarize the spread of the pandemic into three parameters did not produce consistent results between segments and models as the other parameters did, although its interpretation was the same between the models. Therefore, its results and any analysis concerning it were not a focus in our study, and its results were relegated to the Regression model was employed to investigate the relationship of selected national factors reasonabrld Bank ) and nonThe objective of our analysis was to determine whether these factors influence the spread of COVID-19. From the segments in each growth curve model, our focus was on whether (1) the differences in the size of the estimated coefficients and (2) the estimated coefficients were statistically significant between two models and two segments. We used a 5% significance level in this analysis. Statistically significant results provided evidence for the possibility of these factors influencing the spread of COVID-19.For the number of maximum predicted cumulative confirmed cases and significance of national factors was observed, whereby significant variables generally had larger coefficient values than non-significant variables . Our resThe number of maximum predicted cumulative confirmed cases is significantly influenced by only population in both the two GCMs and segments of each model B. The coThe rate of spread of COVID-19 is influenced by 16 significant variables in the Gompertz model, and 10 significant variables in the logistic model A. Age-reOne in five (20%) adults in the world smoke tobacco , being oExtreme poverty impairs rapid response of the government to newer pandemics or even other disasters, leaving its people highly susceptible to the infections. It influences a government\u2019s preparedness to deal with disasters (new pandemics included) and interferes with health system response such as drugs, protective gear, information campaign, and the inability of poor health systems to handle newer pandemics. Malnutrition increases one\u2019s susceptibility to and severity of infections and is thus a major component of illness and death from disease. The risk of death is directly correlated with the degree of malnutrition ,51,52. MNumber of international travelers and foreign visitors increases the chance of spreading and catching the SARS-CoV-2 virus among the population , mainly Our World in Data on the spread of COVID-19 in 134 countries. First, we modeled the spread of COVID-19 using segmented logistic and Gompertz models, and then we investigated the influence of national factors on the spread of COVID-19. We observed that some factors were significant in both GCMs or the two segments for each model, while others were significant in only one model or segment, which implies a change in segments. We believe that although the curves from GCMs can describe similar behavior in some phases of growth, with one of the most important differences being that the Gompertz process is asymmetric, whereas the logistic curve is a symmetric process, explaining the differences observed in the results of the two models. Therefore, using a given growth curve model can have a substantial impact on forecasting [In this study, we investigated the relationship of 31 national factors from KOSIS and ecasting . By builWe observed that the number of maximum predicted cumulative confirmed cases was significantly influenced by only one factor, while the rate of spread of COVID-19 was influenced by seven factors, in both the two GCMs and segments of each model . This mapulation B, while ariables A. This mWe saw the influence of population size on the spread of COVID-19. Among the hardest hit countries by the COVID-19 pandemic in the world, the USA, India, and Brazil are also among the countries with the largest populations in the world. Some countries with the highest number of maximum predicted cumulative confirmed cases and the highest rate of spread of COVID-19 have a large percentage of their population living in extreme poverty ,63 and iHowever, there are some limitations in our analysis. For example, a key limitation of this analysis is that although we modeled the spread of COVID-19 for 134 countries, the GCMs still produced some missing parameter values between the segments and models for some countries mainly due to failure of convergence , which mMoreover, COVID-19, which is a contact-transmissible infectious disease and is said to spread through the population via direct contact between individuals ,72,73 asIn addition, the role of host genetics interaction and COVID-19 progression has gained a large amount of interest as one of the factors being proposed to influence the spread of COVID-19 . For exaFurthermore, we also hope to repeat this analysis using number of cumulative COVID-19 death cases. The number of death cases are just as important as confirmed cases in the understanding of influential factors and epidemiological characteristics of COVID-19, as we believe that COVID-19 death cases will provide more insight as they may be more related to age distributions and health-related variables.Much is still unknown about the clinical and epidemiological characteristics of COVID-19, such as individual risk factors for contracting the virus and infections from asymptotic cases. However, from the above discussions, our findings show the relationship between age distributions, life expectancy, malnutrition, extreme poverty, cardiovascular death rate, smoking, and population size and the spread of COVID-19. We hope these studies will provide important information for policymakers and governments in making informed scientific decisions while considering a country\u2019s economy, population dynamics, climate, and health system, which would likely have the most impact in future prevention works against similar infectious diseases."} +{"text": "Foeniculum vulgare Miller (Apiaceae), collected from three localities in Montenegro to furnish a total of 12 EOs. Liquid and vapor phases of the samples were analyzed by Gas Chromatography/Mass Spectrometry and Headspace-Gas Chromatography/Mass Spectrometry techniques, and 18 compounds have been identified. Although both quantitative and qualitative differences between the samples were notable, the phenylpropanoids anethole (ANE) and estragole and the monoterpenoids \u03b1-terpineol (TER) and fenchone (FEN) could be singled out as the most abundant constituents. The EOs from Podgorica belong to the most common ANE-rich chemotype, while the predominance of the monoterpenoid fraction is characteristic of the samples from Nik\u0161i\u0107 and Kotor. The latter is particularly rich in TER (up to 56.5%), with significant amounts of FEN and ANE. This chemical profile could represent a new chemotype of fennel EO. Vapor phases contained mainly monoterpenoids, with increased amounts of FEN and TER, while the number of phenylpropanoids was significantly decreased.Previous studies relating to prolonged and fractionated distillation procedures highlighted essential oils\u2019 (EOs) chemical composition to be significantly dependent on the extraction duration and harvesting time. As a continuation, a hydrodistillation procedure was applied to ripe fruit material of fennel, As aromatic and volatile liquids obtained from different plants and their different parts, essential oils (EOs) have been regarded with great interest throughout human history . Many ofFoeniculum vulgare Miller , belonging to the Apiaceae family, is a glabrous, erect, glaucous green biennial or perennial plant that grows up to 2.5 m. Shiny and striate stems bear leaves that are more or less triangular in outline with filiform and acuminate lobes, while terminal umbels, formed by 12\u201325 tiny yellow flowers, give ovoid-oblong, sweet-tasting schizocarpic fruits up to 10 mm long [ mm long . It deve mm long . A wide mm long . The ess mm long ,12. In t mm long . FollowiSimilarly as reported , plant mRelative yield percentages calculated per weight of dried plant material for each EO fraction and cumulative yields over the entire extraction time are shown in \u03b1-terpineol and fenchone could be singled out as the main compounds , camphene (2), \u03b2-pinene (3), and \u03b2-phellandrene (7).Some of the constituents are characteristic only for the liquid phase, such as fenchyl acetate (13) and terpinen-4-ol (15) that were present in certain fractions of the EOs from each locality. However, their amounts were of low importance. On the other side, the headspace injection led to the identification of volatile compounds such as http://eo.3d-qsar.com, accessed on 23 November 2021) revealed the presence of 36 FVEO compositions. Discarding 18 compositions published in the previous article [The overall dynamic by which FV gives EO is quite different for every plant sample, although certain similarities could be observed for Nik\u0161i\u0107 and Kotor since the main part of those EOs was extracted within the first 2 h . On the contrary, the material from Podgorica formed an almost uniform yield curve until the end of the extraction process , which wPimpinella anisum L. [Anethum graveolens L. [Ocimum basilicum L. [Artemisia dracunculus L. [Illicium verum Hook.f. [ANT and its isomer EST are very common ingredients of FVEO, usually present in fruits and flowers, as reported in numerous papers ,14,15,16nisum L. ,18, Anetolens L. , Ocimum licum L. ,21, Arteculus L. ,23, and Hook.f. . ANE is Hook.f. . It is r Hook.f. ,26,27. RThuja, Lavandula, and Artemisia species [FEN is an irregular bicyclic monoterpene ketone. The same as ANE, it is one of the common constituents of FVEO ,14,15,16 species ,32.Thymus caespititius Brot. [Salvia libanotica Boiss. & Gaills [Melaleuca alternifolia (Maiden & Betche) Cheel [TER is a volatile monoterpenoid alcohol that occurs in a large number of EOs. It is one of five isomers, being the most common one found in nature, along with terpinen-4-ol. It is a very important constituent of several species, such as us Brot. , Salvia & Gaills , and Mele) Cheel . TER atte) Cheel , anticane) Cheel , anticone) Cheel , antiulce) Cheel , antihype) Cheel , anti-ine) Cheel , and ante) Cheel . It is fe) Cheel . The com\u03b1-phellandrene, \u03b1-pinene, limonene, and FEN are usually reported as the main characterizing compounds [azoricum and dulce were found to contain mostly ANE, while EST was found in prevalence in the vulgare variety [The literature survey has revealed plenty of data. As a well-known industrial aromatic plant with different food and pharmaceutical applications, FV is thoroughly investigated. Thus, much research has been conducted so far to investigate its chemical composition. The results differ greatly depending on harvest time, region, and plant part, among other factors . Some auompounds ,16,43,44ompounds ,16,45. Compounds ,46. The variety . All theChemical profiles of the EO samples from Nik\u0161i\u0107 (F5-F8) and Kotor (F9\u2013F12), however, cannot be related to any of those already reported ; this isIntraspecific chemical polymorphism is quite common in aromatic plants. It depends on a combination of factors related to the environment and genetics, as well as the anatomical and physiological characteristics of plants. These factors are difficult to verify, so the existence of different chemotypes is often not clearly related to the possible causes . Keeping\u03b1-phellandrene , limonene , 1,8-cineole , and \u03b3-terpinene . Whereas the samples from Podgorica were still abundant in phenylpropanoids , the ones from Nik\u0161i\u0107 and Kotor were significantly deprived of this fraction.The study presented herein also included the vapor phase analysis. The samples were characterized by an increase in the monoterpene fraction, mainly represented by FEN and TER. The percentages of FEN were particularly higher: Up to 4 times than the ones reported in the liquid phases, even 5 times in some fractions of FVEO from Podgorica (F3HS). However, some other minor compounds enhanced their amounts with vaporizing, such as azoricum variety included bulbs and aerial parts material from Egypt, Spain, and Holland. The analysis showed that monoterpenes prevailed in most headspace matrices, with an abundance of 6 (up to 61.54%) followed by a significant amount of ANE, particularly in the Egyptian samples [vulgare variety. Additionally, an azoricum variety accession from Austria was rich in EST and FEN as well [A search of the literature available has revealed little data regarding this type of analysis. However, while FVEO\u2019s vapor phase has not been analyzed, the headspace aroma of certain FV organs has. Thus, an analysis on the samples . Further as well .1, 2, 3, and 7) not found with the liquid phase analysis, thus highlighting the capability of this technique of minor compounds detection. To the best of the authors\u2019 knowledge, this analysis has been the pioneer for FVEO.The volatile analysis of the FVEO samples presented herein revealed chemical profiles quite different from the corresponding ones in the liquid phases, generally with much higher amounts of low-boiling components and smaller amounts of the heaviest ones . MoreoveBearing in mind the great influence of the chemical composition on the biological properties, as well as the effects of synergism and/or antagonism between the main and/or minor compounds, various further investigations can be suggested. In that sense, FVEO samples from Nik\u0161i\u0107 and Kotor abundant in TER have a priority of importance due to the numerous biological applications of this monoterpenoid.Ripe fruits of wild growing FV were collected from 3 localities in Montenegro covering the Mediterranean and sub-mediterranean regions: Doclea , a suburban archeological site about 3 km from Podgorica city, Uzdomir hill in the rural area of Nik\u0161i\u0107, and the foothill of the St. John\u2019s Fortress in Kotor , approximately 700 m from the seacoast. The FV material was collected in the early autumn period of 2019. Air-drying of the collected material was performed in a shady place for approximately 20 days. Voucher specimens have been deposited in the Department of Biology at the University of Montenegro . Taxonomic identification of the species was conducted according to the official European flora [2SO4), filtered, and deprived of the solvent in vacuo to furnish EOs. Thus, 4 fractions from each FV sample were obtained, and the prepared EOs were stored in tightly closed dark vials until further analysis. EOs have been isolated by hydrodistillation using a Clevenger-type apparatus with the extraction method previously reported ,4. DriedA chemical analysis of FVEO samples was performed for both liquid and vapor phases. The analyses were carried out twice, showing reproducible results.m/z, the ion source temperature of 200 \u00b0C, and a scan time of 0.2 s.For the chemical quali-quantitative analysis of the EO samples, a gas chromatograph (GC) directly coupled to a mass spectrometer (MS) Perkin Elmer Clarus 500 model was used. The GC was equipped with two columns, one of which was a Restek Stabilwax (fused-silica) polar capillary column, and the other was a Varian (VF-1ms) apolar column. Helium was used as the carrier gas (1.0 mL/min). The column temperature was programmed as follows: from 60 \u00b0C to 220 \u00b0C at a rate of 5 \u00b0C/min, and held for 10 min. The MS parameters were ionization voltage taken at 70 eV, the mass range was from 40 to 500 The identification of the components separated by GC/MS was performed first by comparing the mass spectra for each compound with that reported in the MS libraries database (Wiley and Nist 02) and then by comparison of Linear Retention Indices (LRI) of each compound calculated using a mixture of n-alkanes , with available retention indices in the literature. GC-FID (flame-ionization detector) analysis was performed under the same experimental conditions using the polar column as described for the GC-MS measurements. Relative percentages for quantification of the components were calculated by electronic integration of the GC-FID peak areas using the normalization method without using corrections factors (RRFs).To describe the vapor phase chemical profile, a Perkin-Elmer Headspace Turbomatrix 40 autosampler connected to a Clarus 500 GC-MS was used. About 2 mL of the sample was placed in a 20 mL vial sealed with headspace PTFE-coated silicone rubber septa and caps. The headspace parameters applied were the following: Needle temperature was 90 \u00b0C; 3.5 min pressurization time; and 0.3 min of injection time. The gas phase of the sealed vials was equilibrated for 10 min at 60 \u00b0C and was followed immediately by compound desorption into the GC injector in splitless mode. Quantification of the compounds was performed by GC-FID under the same conditions described above.In line with the previous studies ,4, a fraThe extraction method applied gave FVEO fractions that differ greatly in their chemical compositions. Although the main characterizing constituents are always present, variations in their amount are particularly evident between the first three fractions (up to 3 h of extraction) and the last one (the last 3 h of extraction). Whereas the material from Podgorica gave EOs belonging to the well-known ANE-rich chemotype, the ones from Nik\u0161i\u0107 and Kotor were characterized by the predominance of a monoterpenoid fraction. This chemotype is particularly rich in TER, also containing significant amounts of FEN and ANE, the common FVEO ingredients. Regarding the individual fractions of the FVEO samples from each locality, the results obtained are in compliance with the previous work , indicatEO vapor phases were more enriched in monoterpenoids, with FEN being the most abundant one accompanied by a significant amount of TER. A markedly decreased amount of phenylpropanoids was observed in all of the samples as well. Therefore, low-boiling terpenes are more abundant when using headspace injection. The use of both analytical techniques represented valid applicability for better identification of the FVEO components."} +{"text": "Mitochondrial DNA depletion syndrome (MDS) is a group of severe inherited disorders caused by mutations in genes, such as deoxyribonucleoside kinase (DGUOK). A great majority of DGUOK mutant MDS patients develop iron overload progressing to severe liver failure. However, the pathological mechanisms connecting iron overload and hepatic damage remains uncovered. Here, two patients\u2019 skin fibroblasts are reprogrammed to induced pluripotent stem cells (iPSCs) and then corrected by CRISPR/Cas9. Patient\u2010specific iPSCs and corrected iPSCs\u2010derived high purity hepatocyte organoids (iHep\u2010Orgs) and hepatocyte\u2010like cells (iHep) are generated as cellular models for studying hepatic pathology. DGUOK mutant iHep and iHep\u2010Orgs, but not control and corrected one, are more sensitive to iron overload\u2010induced ferroptosis, which can be rescued by N\u2010Acetylcysteine (NAC). Mechanically, this ferroptosis is a process mediated by nuclear receptor co\u2010activator 4 (NCOA4)\u2010dependent degradation of ferritin in lysosome and cellular labile iron release. This study reveals the underlying pathological mechanisms and the viable therapeutic strategies of this syndrome, and is the first pure iHep\u2010Orgs model in hereditary liver diseases. Iron overload is an important feature in deoxyribonucleoside kinase mutant mitochondrial DNA depletion syndrome. A combined model of patient\u2010specific induced pluripotent stem cells\u2010derived liver organoids and hepatocytes reveals a sensitivity to iron overload\u2010induced ferroptosis in patients. This ferroptosis is a process by NCOA4\u2010dependent degradation of ferritin in lysosome and cellular labile iron release. DGUOK mediates the phosphorylation of deoxyguanosine and deoxyadenosinepurine into the corresponding nucleotides in mitochondria. DGUOK mutation unbalance the mitochondrial deoxyribonucleoside triphosphate (dNTP) pool which can cause mtDNA synthesis breakdown, eventually cause MDS. The significant clinical presentation of DGUOK mutant MDS patients is liver impairment, or accompanied with epilepsy, hypotonia, ataxia, and nystagmus. Patients usually died early due to severe liver failure before 2 years\u2019 old. There are no effective therapies for this disease, expect liver transplantation, leading to poor prognosis in the majority of patients. Therefore, finding effective therapeutic targets of pathological processes of the liver failure is brook no delay.Mitochondrial DNA depletion syndrome (MDS) is a group of severe, phenotypically heterogeneous, recessively inherited disorders characterized by marked reduction of the mtDNA content in affected tissues and organs.1 DeoxDGUOK mutant patients developed iron overload progressing to liver failure. The pathological mechanism connecting iron overload and liver failure in DGUOK mutant MDS, however, remains to be uncovered.Some research efforts have been made focusing on the dNTPs pools using fibroblasts or myoblasts of patients, but they are insufficient to uncover mechanisms underlying the severe liver failure. On the basis of clinical case studies, almost all failure.5, 6 Alcoholic liver disease, nonalcoholic fatty liver disease, acetaminophen overdose\u2010induced acute liver failure, and viral hepatitis are all related to liver iron loading. Excess iron\u2010induced oxidative stress is thought to give rise to these diseases. In recent years, an iron\u2010dependent form of cell death, known as ferroptosis, has been widely investigated. During the process of ferroptosis, the redox\u2010active ability of iron produce free radicals, leading to lipid peroxidation and initiation of signaling pathways crucial for cell death. Inhibition of system Xc\u2212, glutathione (GSH) or cysteine depletion, and glutaminolysis can cause ferroptosis. Glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1, as key components in elimination lipid peroxides, can protect cell from ferroptosis. Previous studies revealed that ferroptosis is also an autophagic process with degradation of ferritin to release iron to trigger ferroptosis. Ferroptosis is an important factor in many diseases, like hemochromatosis, liver toxicity, liver fibrosis, cardiomyopathy, acute renal failure, and neurodegenerative diseases. While iron overload in liver is a characteristic clinical feature of DGUOK mutant patients, directly relevant research is not yet reported.Liver is a major site of iron storage and plays a central role in systemic iron homeostasis. This makes liver a preferential target of iron overload toxicity.7 Alco iPSCs derived 2D hepatocyte like cells and advances in 3D cell culture represents a promising approach whereby human iPSCs can provide a renewable source of hepatocytes and liver organoids which carry the whole genetic background of a patient. iPSCs\u2010derived 2D hepatocyte\u2010like cells (iHep) is a good model. Many studies used iHep to model liver diseases and proved its efficacy in exploring the pathological mechanisms of a broad range of diseases. A good example is the work in the first toxicity iHep model in a genetic disease of valproic acid induced hepatotoxicity in Alpers syndrome. Another advance technology of in vitro model is the generation of organoids. The 3D organoids system provide the advantage in maintaining the cell\u2010to\u2010cell contacts and the 3D spatial cellular organization of tissues or organs. Recently, a long\u2010term expansion of functional 3D liver organoids was established with the advantage of pure hepatocyte\u2010organoids with a low quantity of cholangiocyte\u2010like cells. This 3D liver organoids system was used in some liver disease like alcoholic liver injury, hepatocellular carcinomas, but not be used in genetic liver disease yet. Moreover, human iPSCs can be genetically edited by the CRISPR/Cas9 system to supply isogenic controls.It is critical to establish a powerful model system mimicking the liver of DGUOK mutant MDS enabling the studies of intrinsic mechanism, as well as drug discovery for clinical therapeutics. It has been difficult to acquire liver samples from DGUOK mutant patients for research, while either fibroblasts from patients or animal models exist different features with human biology. The breakthrough in the generation of induced pluripotent stem cells (iPSCs),24 iPSHere, we establish an in vitro liver disease model of liver organoids and hepatocytes developed from iPSCs of DGUOK mutant patients, along with associated controls and isogenic cell lines corrected by the CRISPR/Cas9 system. Using this model, we show that mtDNA depletion and respiration dysfunction in the patient iPSCs\u2010derived hepatocyte\u2010like cells and both iHep and iHep\u2010generated organoids (iHep\u2010Orgs) are more sensitive to iron overload\u2010induced ferroptosis which could recused by the GSH precursor, N\u2010acetylcysteine (NAC). This ferroptosis is a process mediated by nuclear receptor co\u2010activator 4 (NCOA4)\u2010dependent degradation of ferritin in lysosome. Our study provides the first hereditary liver disease model using pure hepatocyte organoids, giving critical mechanistic insight into liver failure of DGUOK mutant MDS.22.1DGUOK were reprogrammed to iPSCs. As the diagram in Figure\u00a0 In order to correct the DGUOK gene, gRNAs were designed to target exon 6 of P1 and intron 1 of P2. Two ssODNs were designed for each patient as repair templates: one normal sequence, the other containing synonymous mutations to avoid the repaired allele being targeted and cut by the CRISPR system . These iHep also showed periodic acid\u2010Schiff (PAS) staining indicative of glycogen accumulation and indocyanine green (ICG) uptake and release indicative of hepatic excretory function . To generate liver organoids, iHep were seeded in Matrigel. About 2 weeks after seeding, iPSCs\u2010derived liver organoids (iHep\u2010Orgs) came out and fetal hepatic protein: fetoprotein (AFP) Figure\u00a02A. Funct2.3 To assess mtDNA quantity, we performed immunofluorescence using an anti\u2010DNA and TOMM20 antibody in control, patient and corrected iHep. We showed that number of mitochondria without mtDNA per cell was much greater in patient iHep than in control and corrected iHep staining. Patient iHep displayed decreased \u0394\u03a8m compared with control and corrected iHep . We found a higher level of ROS in the patient iHep than control and corrected iHep in iron metabolism in control, patient and corrected iHep and iHep\u2010Orgs. We showed that expression of transferrin (TF) increased in both patient iHep and iHep\u2010Orgs . TF, a plasma iron carrier, expresses in liver and binds to plasma iron for transport.Iron overload was regularly detected in hepatocytes of DGUOK mutant patients by liver histological examination.4 Thus\u22123\u00a0m FAC . Thus, iron in our organoid model should be comparable to that in livers from patients. We also treated iHep with indicated concentrations of FAC for 48 h, and the similar result was observed that the percentage of surviving cells was less in patient iHep compared with control and DGUOK corrected iHep. .To determine the pathological mechanism of iron overload and lethal liver failure in DGUOK mutant MDS, we used ferric ammonium citrate (FAC) to induce iron overload. iHep\u2010Orgs were treated with FAC at the indicated concentrations for 72 h, and a significant cell death was observed in patient iHep\u2010Orgs compared with control and corrected iHep\u2010Orgs at the concentration of 5\u00a0\u00d7\u00a010C Figure\u00a04A. We meo severe,32 and. Figure\u00a0. These r we measured lipid peroxidation in control, patient and corrected iHep treated with FAC. We found that lipid peroxidation in patient iHep was higher than in control and corrected iHep. Additionally, this iron overload\u2010induced lipid peroxidation was significantly reversed by DFO and Fer\u20101 . We then examined GSH, which serves as a reducing co\u2010substrate for GPX4, and found that patient iHep displayed a reduction in GSH level compared with control and corrected iHep released from ferritin degradation is required for lipid peroxidation upon ferroptosis.36 We p Figure\u00a0. we examined the colocalizations of ferritin and NCOA4 by immunofluorescence. The result showed that the colocalization ratio was higher in patient iHep than in control or corrected iHep , streptomycin (50\u00a0\u00b5g\u00a0mL\u22121) and penicillin (50 U mL\u22121). Human fibroblasts were reprogrammed into iPSCs as previously described. Briefly, fibroblasts were infected with retroviruses encoding human SOX2, KLF4, OCT4, and c\u2010MYC (Addgene). After around 25 days, ESC\u2010like colonies were picked and cultured on Matrigel (BD) in mTeSR medium (STEMCELL Technologies).The fibroblasts were cultured in Dulbecco's modified Eagles medium (DMEM)/high glucose (HyClone) containing 10% fetal bovine serum (FBS) (Hyclone) with 1% nonessential amino acids (NEAA) (Gibco), 1% GlutaMAX (Gibco), 200\u00a0\u00d7\u00a010escribed.27 BriGuide RNAs were designed to target DGUOK exon 6 (patient1) and intron 1 (patient2) using the Zhang lab CRISPR design tool (crispr.mit.edu). The annealed oligos which are complementary to each other containing the gRNA sequence were ligated to Cas9 plasmids pX330 which were already digested with BbsI by T4 DNA ligase (Life Technologies). Two ssODNs were designed for each patient as repair templates. One was the normal sequence, the other was the sequence containing synonymous mutations to avoid the DNA double\u2010strand continuously being cut. Guide RNAs and ssODNs sequences are listed in Table S1 (Supporting Information). 4\u00a0\u00b5g Cas9 and 2\u00a0\u00b5g ssODNs were transfected into DGUOK iPSCs using Nucleofector Kits for Human Stem Cells (Lonza). After transfection, cells were cultured in mTeSR1 with Rock inhibitor Y\u201027632 (Selleck). After about 5 days, cells were plated in mTeSR1 with Y\u201027632 at the density of 500 cells per 1 well of six\u2010well plate. After 2 weeks, individual colonies were expanded enough to identify the target sequence by PCR. Potential off\u2010target sequences for CRISPR/Cas9 are listed in Table S2 (Supporting Information). Activin A, BMP2, FGF4, HGF, KGF, and Oncostatin M were purchased from Peprotech. RPMI 1640, and B27 were purchased from Invitrogen. The iHep cells were maintained in hepatocyte culture media (HCM) (Lonza) within one week.The protocol of differentiation of iPSCs derived hepatocyte has been described previously.27 Act We generated iHep\u2010organoids by seeding 50\u202f000 iHep cells mixed with Matrigel into a well of a 24 well plate. After Matrigel solidification, 500\u00a0\u00b5L organoids generation medium was added per well. Organoids generation medium: AdDMEM/F12 (Thermo Scientific), 0.5% Penicillin\u2010Streptomycin, 1% GlutaMAX, 10\u00a0\u00d7\u00a010\u22123\u00a0m HEPES, 1% B27 minus vitamin A, 15% R\u2010spodin1\u2010conditioned medium, 3\u00a0\u00d7\u00a010\u22126\u00a0m ChIR99021 (Sigma), 10\u00a0\u00d7\u00a010\u22123\u00a0m nicotinamide (Sigma),10\u00a0\u00d7\u00a010\u22129\u00a0m gastrin (Sigma), 50\u00a0ng mL\u22121 EGF (Peprotech), 20\u00a0ng mL\u22121 TGF\u2010\u03b1 (Peprotech), 100\u00a0ng mL\u22121 FGF7 (Peprotech), 100\u00a0ng mL\u22121 FGF\u201010 (Peprotech), 50\u00a0ng mL\u22121 HGF (Peprotech), 2\u00a0\u00d7\u00a010\u22123\u00a0m A83\u201001(Tocris), 10\u00a0\u00d7\u00a010\u22126\u00a0m Y\u201027632, 1\u00a0\u00d7\u00a010\u22126\u00a0m dexamethasone (Sigma), 10\u00a0ng mL\u22121 Oncostatin M. During generation, the medium was changed every 3 days. After 14 days generation, organoids were mechanically fragmented with pipette tips blowing and separated by centrifugation for 200\u00a0g 10\u00a0min. The pellets was re\u2010seed into Matrigel with a split ratio of 1:4 into a well of a 24 well plate. Then, the medium was changed every 3 days and the organoids could passaged every 10 days with a split ratio of 1:4.The protocol of generation of liver organoids has been described previously.26 We \u22126\u00a0m LysoTracker Deep Red for 1 h in cell incubator. Then, immunofluorescence of ferritin was executed as described above.Cells were fixed with 4% paraformaldehyde (PFA) for 30\u00a0min, and permeabilized in 0.5% Triton X\u2010100 for 15\u00a0min. Then cells were blocked with 0.05% Triton X\u2010100 and 10% FBS for 30\u00a0min at room temperature. Then cells were incubated with primary antibodies for 2 h at room temperature or overnight at 4\u00a0\u00b0C. After washing, cells were incubated with corresponding secondary antibody for 1 h in dark. Then, cells were incubated with DAPI (Sigma) for 5\u00a0min in dark. Cells were imaged using Zeiss LSM 710 confocal microscope. The following primary antibodies were used: Anti\u2010DNA antibody , TOMM20 antibody , ferritin antibody , NCOA4 antibody , SOX17 antibody , AFP antibody , ALB antibody . For the colocation detection of ferritin and lysosome, lysosome was stained by LysoTracker Deep Red (Invitrogen). Before fixed, cells were incubated with 0.1\u00a0\u00d7\u00a010 Before fixed, the organoids were extracted from Matrigel in ice\u2010cold recovery solution for 1\u00a0h. Then, organoids were fixed in 4% PFA in 4\u00a0\u00b0C for 1\u00a0h, then permeablilized in PBS supplemented with 0.1% Tween and blocked with 2% FBS for 1\u00a0h at room temperature. ALB and AFP antibodies were incubated at 4\u00a0\u00b0C overnight. After washing, cells were incubated with corresponding secondary antibody 4\u00a0\u00b0C overnight in dark. Then, cells were incubated with DAPI for 30\u00a0min in dark. Before imaged, organoids were cleared in a glycerol based clearing solution for 10\u00a0min. Organoids were imaged using Zeiss LSM 710 confocal microscope. 3D image analysis of organoids was performed using the Imaris software.The protocol of immunofluorescence of organoids has been described previously.46 BefTotal mtDNA was extracted using a TIANamp Genomic DNA kit (TianGen). Total RNA was extracted according to the manufacturer's instruction. cDNA was synthesized by reverse transcription of 2\u00a0\u00b5g total RNA per sample using the ReverTra Ace kit (Toyobo). QPCR was performed using a CFX\u201096 real\u2010time PCR detection system (BioRad) in conjunction with SsoAdvanced Universal SYBR Green Supermix (BioRad) using the following conditions: an initial denaturation step of 95\u00a0\u00b0C for 30 s, followed by 40 cycles of denaturation at 95\u00a0\u00b0C for 5 s and annealing\u2010elongation at 60\u00a0\u00b0C for 20 s. Primer sequences are listed in Table S3 (Supporting Information).\u03b2\u2010actin antibody . The secondary antibodies were goat antirabbit with HRP and goat antimouse with HRP .Cells were lysed in radio immunoprecipitation assay (RIPA) buffer (Beyotime) with protease inhibitor PMSF (Beyotime) and cocktail (Roche) on ice for 30\u00a0min. Protein concentrations were determined by BCA Protein Assay Kit (Sigma). Protein was separated by 15% polyacrylamide/sodium dodecyl sulfate polyacrylamide gel electrophoresi (SDS\u2010PAGE), and electro\u2010transferred onto polyvinylidene difluoride (PVDF) membrane (Merck Millipore). The membrane was blocked in 5% dry nonfat milk in Tris\u2010buffered saline plus 0.1% Tween 20 (TBST) for 2 h at room temperature, followed by incubation with primary antibody overnight at 4 \u00b0C. Secondary antibodies were goat antirabbit and goat antimouse with (horseradish peroxidase) HRP, which were applied in block for 1 h at room temperature followed by chemiluminescence imaging using the Electro\u2010Chemi\u2010Luminescence (ECL) (Merck Millipore). Relative ratio of respective density of each proteins band was quantified using Image J software. The following primary antibodies were used: MT\u2010ATP6 antibody , MT\u2010ATP8 antibody , GPX4 antibody , FTH1 antibody , NCOA4 antibody , \u22121 DiI\u2010Ac\u2010LDL for 2 h in cell incubator, then the organoids were extracted, fixed, incubated with DAPI and imaged by fluorescence microscopy as mentioned above. ICG uptake and release assay was performed by incubating 1\u00a0mg\u00a0mL\u22121 ICG (Cayman) for 2 h in cell incubator. Images of ICG uptake were captured using a microscope, then the organoids were washed with PBS and refilled with organoids medium for 6 h in cell incubator. Then, images of ICG release were captured using a microscope. For albumin secretion detection, the supernatant of organoids was collected after medium changed for 48 h and determined using a Human Albumin ELISA kit (Bethyl Laboratory), according to the manufacturer's protocol. CYP3A4 activity was measured using a P450\u2010Glo Assay Kit (Promega), according to the manufacturer's protocol. In brief, the organoids were at first treated with 20\u00a0\u00b5M rifampicin for 48 h to induce CYP3A4 activity. Then the organoids were incubated with luminogenic substrate for 2 h and the supernatant was measured using a luminometer.To assess glycogen storage, the organoids were stained by PAS staining kit (Polysciences), according to the manufacturer's protocol. LDL uptake was detected with DiI\u2010Ac\u2010LDL (Alpha Diagnostic). The organoids were treated with 10\u00a0\u00b5g\u00a0mLm using Potentiometric dye TMRM (Invitrogen) has been described previously. Living cells were treated with 25\u00a0\u00d7 10\u22129m TMRM in cell incubator for 30\u00a0min, and then replaced with 5\u00a0\u00d7 10\u22129m TMRM for imaging using a Zeiss LSM 710 confocal microscope (Carl Zeiss) at 37\u00a0\u00b0C under 5% CO2. All image analysis was performed using the image J software.Measurement of \u0394\u03a8eviously.47 Liv OCR was measured using the XF24 extracellular flux analyzer (Seahorse Biosciences). iHep were seeded on Matrigel\u2010coated XF24 plates at 50\u202f000 cells per well allowed to attachment overnight. The next day, cells media were replaced with XF base assay medium supplemented with 25\u00a0\u00d7 10\u22123m glucose, 1\u00a0\u00d7 10\u22123m sodium pyruvate, and 2\u00a0\u00d7 10\u22123m L\u2010glutamine in a non\u2010CO2 incubator for 1 h. OCR was were measured in XF media in response to oligomycin (1\u00a0\u00d7 10\u22123m), carbonyl cyanide 4\u2010(trifluoromethoxy) phenylhydrazone and rotenone (1\u00a0\u00d7 10\u22126m) and antimycin A (1\u00a0\u00d7 10\u22126m). All plotted values were normalized by counting cell number after measurement.Measurement of OCR using XF24 extracellular flux analyzer (Seahorse Biosciences) has been described previously.48 OCR Cell extraction was performed with 2.5% trichloroacetic acid, then neutralized and diluted in 10\u00a0\u00d7 10\u22123m Tris\u2010acetate (pH 7.75). ATP levels were measured using the Luciferase/Luciferin reagent according to the manufacturer's protocol. For determination of mitochondrial ATP levels, cells were treated with 10\u00a0\u00d7 10\u22126m oligomycin for 15\u00a0min before measurement.Measurement of ATP using using the ENLITEN ATP Assay System (Promega) has been described previously.47 Cel6 cells were collected then divided equally into 2 parts. One was for determination of lactate, and the other was for pyruvate. Mitochondrial complex I and complex V activity assays were determined using mitochondrial complex I activity assay kit (Abcam) and mitochondrial complex V (ATP synthase) activity assay kit (Novagen) following manufacturer's instruction.Lactate and pyruvate were measured using the lactate assay kit and pyruvate assay kit (BioVision) according to the manufacturer's protocol. 1 \u00d7 10\u22129m SYTOX Green for 20\u00a0min and examined by a fluorescence microscope. The ratio of dead cells (SYTOX Green\u2010positive) to total cells was quantified using image J software.For organoids, cell death was measured using SYTOX Green (Invitrogen) according to the manufacturer's instructions. In brief, organoids were stained 30\u00a0\u00d7 10For iHep, cell viability was measured using the cell counting kit\u20108 (CCK\u20108) viability assay (Beyotime) according to the manufacturer's instructions. Briefly, iHep were plated in a 96\u2010well plates and treated with various concentrations of FAC and drugs for determined times. The 10\u00a0\u00b5L CCK\u20108 reagents were added to each well and incubated for 4 h. Then the plates were measured by a microplate reader at 450\u00a0nm.\u22126m H2DCFDA and 2\u00a0\u00d7 10\u22126m C11\u2010BODIPY(581/591) for 30\u00a0min at 37\u00a0\u00b0C, respectively, followed by using a flow cytometer .Total ROS and lipid ROS were measured by flow analysis with H2DCFDA (Sigma) and C11\u2010BODIPY (581/591) (Invitrogen) staining according to the manufacturer's protocol, respectively. To analyze total ROS and lipid ROS, cells were stained with 25\u00a0\u00d7 10 Cells were incubated with 5\u00a0\u00d7 10\u22126m of Phen Green SK for 15\u00a0min in cell incubator. Then, cells were harvested and analyzed with a flow cytometer.Measurement of cellular LIP using the fluorescent probe Phen Green SK (Invitrogen) has been described previously.49 Cel6 cells were collected then divided equally into 2 parts. One was for determination of reduced form of GSH, and the other was for total GSH.Intracellular GSH levels were measured using Glutathione Colorimetric Assay Kit (Bivison) according to the manufacturer's protocol. 1 \u00d7 10\u22126m LysoSensor for 1 h in cell incubator, then washed with PBS and incubated in fresh hepatocyte medium for 30\u00a0min. The images were captured using a fluorescence microscopy. The relative lysosomal activity was quantified using Image J software.Lysosomal activity was measured using LysoSensor Green DND\u2010189 (Thermo Fisher), as described previously. Briefly, cells were cultured with 1\u00a0\u00d7 10\u22121) and penicillin (50 U mL\u22121). Medium was changed 12 h after transfection and the supernatant was collected 48 h after transfection. The supernatant was filtered through a 0.45\u00a0\u00b5m filter and centrifuged at 50\u202f000\u00d7g for 2.5 h. Then the viruses were used to infect iHep in the presence of polybrene (Sigma). shNCOA4 target sequences: 5\u2019\u2010CCCAGGAAGTATTACTTAATT\u20103\u2019. FAC , DFO , Ferrostatin\u20101 , Z\u2010VAD\u2010FMK , necrostatin\u20101 , NAC , Baf A1 .Lentiviral vectors encoding shRNA targeting human NCOA4 were produced by the co\u2010transfection of the lentiviral vector pLKO.1\u2010puro with packaging plasmids PMD2.G and PSPAX2 into 293T cells using polyfectine (PEI). 293T cells were grown in DMEM/high supplemented with 10% FBS, streptomycin (50\u00a0\u00b5g mLAll statistical analyses were performed using GraphPad Prism 6 software. The data used in this study are presented as mean \u00b1 S.D, from three independent experiments. P values were determined by one\u2010way ANOVA and two\u2010way ANOVA followed by post\u2010hoc Holm\u2013Sidak test, as indicated in the figure legends. P values of less than 0.05 were considered as statistically significant. More detailed information has been provided in each figure legend.For further methods, please see the Supporting Information.The authors declare no conflict of interest.X.L. conceived, designed and supervised the project. J.G. designed and carried out most experiments. J.G. carried out most data analysis. L.D. and X.H. participated in iHep\u2010Org generation, western blot experiments, and data analysis. G.X., L.Y., and F.B. participated in mitochondrial respiration experiments. S.L. supplied control iPSCs materials and helpful suggestions. H.S., M.G., and L.Z. participated in CRISPR editing. H.H. provided the 293T\u2010HA\u2010Rspon1\u2010Fc cells. X.L. and J.G. wrote the manuscript. Y.W. participated in manuscript edit. All authors commented on the manuscript and declared no conflicts of interest.Supporting InformationClick here for additional data file."} +{"text": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is accountable for the protracted COVID-19 pandemic. Its high transmission rate and pathogenicity led to health emergencies and economic crisis. Recent studies pertaining to the understanding of the molecular pathogenesis of SARS-CoV-2 infection exhibited the indispensable role of ion channels in viral infection inside the host. Moreover, machine learning (ML)-based algorithms are providing a higher accuracy for host-SARS-CoV-2 protein\u2013protein interactions (PPIs). In this study, PPIs of SARS-CoV-2 proteins with human ion channels (HICs) were trained on the PPI-MetaGO algorithm. PPI networks (PPINs) and a signaling pathway map of HICs with SARS-CoV-2 proteins were generated. Additionally, various U.S. food and drug administration (FDA)-approved drugs interacting with the potential HICs were identified. The PPIs were predicted with 82.71% accuracy, 84.09% precision, 84.09% sensitivity, 0.89 AUC-ROC, 65.17% Matthews correlation coefficient score (MCC) and 84.09% F1 score. Several host pathways were found to be altered, including calcium signaling and taste transduction pathway. Potential HICs could serve as an initial set to the experimentalists for further validation. The study also reinforces the drug repurposing approach for the development of host directed antiviral drugs that may provide a better therapeutic management strategy for infection caused by SARS-CoV-2. The COVID-19 pandemic rapidly spread to more than 175 countries within the first three months of its outbreak . The dea2+ channels [2+ channel inhibitor, in the treatment of infections caused by filoviruses further implicates the importance of HICs in viral survival [Ebola and influenza viruses exploit HICs to enter the host by utilizing Cachannels ,6. Bunyachannels . The usesurvival . Most lisurvival . The entsurvival ,10,11. Tsurvival ,13,14. Asurvival . Additiosurvival ,16,17. Msurvival .The current study focuses on understanding the interactome of SARS-CoV-2 proteins with HICs through in silico approaches. PPIs of SARS-CoV-2 proteins with HICs were trained on the PPI-MetaGO algorithm to associate the proteins based on its features. Later, PPI networks (PPINs) of SARS-CoV-2 proteins with HICs were generated. Thereafter, a biological pathway analysis of HICs interacting with SARS-CoV-2 proteins was performed and a pathway map depicting the role of HICs upon interactions with SARS-CoV-2 proteins was generated. Furthermore, FDA-approved drugs interacting with potential HICs were identified. The study may provide an insight to better understand the interactome of SARS-CoV-2 proteins with HICs and may unravel the development of future therapeutic strategies against SARS-CoV-2 infection. The study also underlines the potential significance of repurposing of drugs.A total of 181 interactions of HICs with SARS-CoV-2 proteins and 21 interactions among SARS-CoV-2 proteins were obtained from the BioGRID database (release 4.92.192) . Thus, 4SARS-CoV-2 and HIC interactions were predicted with an accuracy of 84.09% and AUC of 0.89 . ConfusiOf the 328 HICs, 40 were found to interact with 15 SARS-CoV-2 proteins. The functions of 40 HICs interacting with SARS-CoV-2 proteins are listed in PPI maps of HICs-SARS-CoV-2 proteins are depicted in The HICs were found to interact with multiple SARS-CoV-2 proteins.ITPRs are the major receptors involved in the release of calcium from the endoplasmic reticulum (ER) ,20,21,22LRRCs are involved in the development and functions of immune cells . E, M, O2+ ions from the mitochondrial membrane [VDACs control the flow of Camembrane . M, ORF3GJ allow the intercellular flow of Ca2+ ions and enable communication between adjacent cells . M, nsp4ANO are calcium-dependent chloride channel proteins. SARS-CoV-2 E, S, M, ORF7b, ORF7a, nsp6, nsp4 and ORF8 A\u2013D,G\u2013J iProteins belonging to TRP family have a role in signal transductions ,27,28, iThe Intra Protein Interactions among the 40 HICs Were Generated as PPINs Using STRING Database (v11) to IdentSeveral identified HICs were reported in various cellular processes that aid in viral replication and survival. These processes could be the outcome of interactions among the HICs along with the interactions between HICs and SARS-CoV-2 proteins.2+ storage using ITPRs that in turn promoted viral replication [Viruses exploiting ITPRs were reported to affect the host by increasing metabolic stress and enterotoxicity . Additiolication . The intLRRCs played important roles in T-cell/ B-cell and lymphocyte function and development . Thus, tVDACs were reported for their involvement in the transportation of metabolites from mitochondria to ER during viral replication and interacted with the structural and non-structural proteins (nsps) of dengue virus .Host cell junctions were reported to be destroyed by viruses during invasion . Another2+ ions during viral entry [+ to pass as per the electrochemical gradient, channels at synapses and cation channels for inwardly rectifying activity, respectively [Other important HICs found were the potassium voltage gated channels that were known to act as modulators of potassium flow into the cell and also had roles in the reactivation of na\u00efve T cells ,52,53,54al entry ,24,55. Tal entry ,57,58,59al entry ,64,65,66al entry , was alsectively ,72,73,74ectively . The GABectively .Pathway analysis of 40 HICs was performed using the STRING database to gather functional insights about the processes regulated by the HICs. Forty-six biological pathways corresponding to the KEGG PATHWAY database were identified. Inflammatory mediator regulation of TRP channels, insulin secretion, renin secretion, pathways involving gap junction, taste transduction, calcium signaling pathway, apelin signaling pathway and GnRH secretion were selected as significant pathways .+2 ions [+2 concentrations. Renin also catalyzed the conversion of AGT to angiotensin I. The release of interleukins may be further stimulated by angiotensin receptors activated upon the conversion of angiotensin I to angiotensin II catalyzed by ACE protein [+ channels and caused voltage-dependent Ca+2 influx [+2 ions and thus change in the level of Ca+2 ions may act as stimuli for communication by GJA1. Various L-type calcium channels mediate the influx of intracellular Ca+2 ions. Intracellular Ca+2 induced the Raf/MEK/ERK pathway that regulated the transcription of COX2 and led to inflammation reactions [+2 from endoplasmic reticulum was regulated by ITPRs and from mitochondria by VDACs. Additionally, calcium was released from Ca+2 acidic stores through TPCNs and MCOLNs. Na+ and Ca+2 ions influx was facilitated by TRP channels and ASIC1. TRP channels may lead to pain, hyperinflammation, and oxidative stress [+ ions by KCN and HCN channels was stimulated by Na+. The PLCB2/ITP3 signaling cascade mediated the transduction of bitter, sweet and umami taste by opening of TRPM5 followed by depolarization of plasma membrane that allowed the influx of Na+ ions. Furthermore, Na+ influx prevailed due to the SCN membrane channels. SCN channels interacted with CALHM1 and contributed in the mechanism related to taste inception. Thus, the loss of taste occurring in COVID-19 patients may be linked to the deregulation of these channels.The advent of omics technologies led to an enormous progress in the generation of signaling pathways . The pat+2 ions . The int protein . The orc protein . Glucose2 influx . The red2 influx may be aeactions . Additioeactions . The mobe stress . The infHICs interacting with viral proteins could be potential drug targets for drug repurposing. Additionally, the traditional drug development method is considerably expensive and time consuming. Drug repurposing is an efficacious process by which effective drugs can be identified. A list of FDA-approved drugs interacting with HICs and SARS-CoV-2 protein is listed in Studies pertaining to the development of novel therapeutic strategies are of utmost importance to combat the heterogeneity in the infection caused by SARS-CoV-2. The current study aimed to understand the interactome of SARS-CoV-2 proteins and HICs through various bioinformatic analyses. It is known that the coronavirus family uses the E protein to induce intracellular membrane remodelling, generating new membrane vesicles that serve as a viral replication site responsible for the depolarisation of membranes . FurtherACE2 and TMPRSS2 expression has been reported in the salivary gland cells of the tongue and tonsils. It might allow the virus to fuse its membrane with the host cells as compared to other oral tissues [Several host pathways were found to be altered due to interactions between SARS-CoV-2 proteins with HICs. Inflammatory mediator regulations of TRP channels pathway include TRP channels . TRP channels that respond to temperature are known as thermo-TRPs. Among them, TRPA1, TRPM8, and TRPV1-4 are found in the nerve endings and play a major role in pain perception. These proteins are modulated indirectly by inflammatory mediators such as proinflammatory cytokines . The act tissues . SARS-CoACE2 expression increased considerably in human pancreatic beta cells in response to inflammatory cytokines, thus rendering the beta cells more susceptible to SARS-CoV-2 infections [Glucose-induced insulin secretion is the main principle of insulin release . Deteriofections . Hence, 2+ levels act as stimuli to the gap junctions. ITPRs play a crucial role in maintaining the intracellular Ca2+as they act on the ER for the regulation of cytoplasmic calcium concentration [Gap junctions contain the intercellular channels that allow a direct communication between the cellular compartments. These channels permit the transfer of ions, amino acids, secondary messengers and other metabolites between adjacent cells. Changes in the intracellular Cantration .2+-concentration-dependent metalloproteinase domain-containing protein (ADAM10). Moreover, the increase in Ca2+ concentration could further be attributed to viral proteins interacting with ITPR3 [The renin-angiotensin-aldosterone system (RAAS) is an essential system for electrolyte homeostasis and blood pressure management through the ACE2 axis. The deregulation of RAAS homeostasis results in the development of distress in lungs, induced apoptosis, vasoconstriction, increased oxidative stress and edema . ACE2 acth ITPR3 . Additioth ITPR3 . Experimth ITPR3 .HICs interacting with viral proteins could be potential drug targets for drug repurposing. The identification of drugs that could be targeted against potential HICs was performed. However, drugs targeted against HICs can be toxic in some case . TherefoAn overview of the methodology followed to study the PPIs between SARS-CoV-2 and HICs is described in One of the most crucial steps while building any model based on a ML algorithm is the extraction and enhancement of a good dataset. Primarily, a list of 28 unique SARS-CoV-2 proteins were downloaded from the RefSeq database and 328 HICs were retrieved from HGNC database . InteracThe PPI-MetaGO algorithm was applTo evaluate the performance of PPI-MetaGO, a 10-fold cross-validation was performed . AdditioThe PPIs among HICs-SARS-CoV-2 proteins and the PPI maps were visualized using Cytoscape-3.8 . Additiop-values for further analysis.The KEGG PATHWAY analysis of HICs interacting with SARS-CoV-2 proteins was performed using the STRING database (v11). Significant pathways were selected based on statistical measures including strength and false discovery rate (FDR) score provided by An extensive literature search was performed using PubMed to annotate reactions such as protein\u2013protein interactions, translocations, activations and inhibitions that describe HICs in association with SARS-CoV-2 proteins ,108. SevFDA-approved drugs interacting with HICs were parsed using the Drug\u2013Gene Interaction database (DGIdb) ,111. FurSeveral computational approaches, including ML-based algorithms, were applied to study the interactome of SARS-CoV-2 proteins with HICs. Biological insights of HICs interacting with SARS-CoV-2 proteins were gained using pathway analysis. TRPM4 and KCNN4 were found to a play role in insulin secretion. TRPA1 was found as an important molecule in heat, pain, and taste sensitivity inside the host. GJA1 has a crucial role in pathways involving gap junctions and ASIC1 was found to be a part of inflammatory mediator regulation of TRP channels. ITPR1 was found to be involved in six predicted pathways, including inflammatory mediator regulation of TRP channels, gap junction, renin secretion and apelin signaling pathways. Moreover, FDA-approved drugs interacting with potential HICs were identified that can be repurposed. Most likely, our analyses showed promising results that further require experimental validation. HICs could be further explored as a potential class of targets for the better management of the infection caused by SARS-CoV-2."} +{"text": "In addition cytotoxicity and hemolytic activity of these bacteriocins were investigated on human epithelial colorectal adenocarcinoma cells (Caco-2) and rat erythrocytes, respectively. Pediocin PA-1, bactofencin A, and nisin were observed to lose their stability while passing through the gastrointestinal tract, while microcin J25 is only partially degraded. Besides, selected bacteriocins were not toxic to Caco-2 cells, and integrity of cell membrane was observed to remain unaffected in presence of these bacteriocins at concentrations up to 400 \u03bcg/mL. In hemolysis study, pediocin PA-1, bactofencin A, and nisin were observed to lyse rat erythrocytes at concentrations higher than 50 \u03bcg/mL, while microcin J25 showed no effect on these cells. According to data indicating gastrointestinal degradation and the absence of toxicity of pediocin PA-1, bactofencin A, and microcin J25 they could potentially be used in food or clinical applications.Bacteriocins are receiving increased attention as potent candidates in food preservation and medicine. Although the inhibitory activity of bacteriocins has been studied widely, little is known about their gastrointestinal stability and toxicity toward normal human cell lines. The aim of this study was to evaluate the gastrointestinal stability and activity of microcin J25, pediocin PA-1, bactofencin A and nisin using Bacteriocins have a broad diversity in terms of sizes, structures, and mechanisms of action . Our teais study . Rapid iis study or therais study . Bacteriis study . Bacteriis study . In addiis study . But, deis study . There aAlthough bacteriocins were mainly studied as food additives, they have exhibited desirable properties for clinical applications as well. Interestingly, several bacteriocins were found to be effective against multi-drug-resistant bacterial strains; hence, they can be used in human and animals for treating local or systemic infections caused by antibiotic-resistant bacteria . In factHowever, although bacteriocins have shown great potential for clinical applications, only a few bacteriocins have been progressed to be used in clinical trials. Those include microbisporicin , mutacin 1140 and duramycin .Salmonella in poultry and improve growth performance in chicken are commercially available for mastitis treatment.In the veterinary sector, due to the increased emergence of antibiotic-resistant strains, the systemic use of antibiotics as growth promoters has been banned in many countries. Consequently, bacteriocins or their producing strains can be considered as promising safe alternatives in order to target pathogens and improve animal health. Microcin J25 is a Gram-negative bacteriocin and it has been used to control chicken and pig chicken . Additioin vitro studies on eukaryotic cells followed by in vivo studies on animal models status , the usel models . Althougl models , there il models , while ml models . It shoul models . On the In this study we examined the GI physicochemical stability and toxicity of four highly purified bacteriocins representing different classes of bacteriocins and having different structures and mechanisms of action. Namely, two ribosomally synthesized and posttranslationally modified peptides (RiPPs), nisin and microcin J25 (lasso peptide), and two non-modified bacteriocins pediocin PA-1 (class IIa bacteriocin) and bactofencin A (class IId bacteriocin) were selected. In addition to their comportment in conditions miming those of the GI tract, their cytotoxicity on human colonic adenocarcinoma cells and hemolytic activity were evaluated.Escherichia coli MC4100 carrying the plasmid PTUC 202 was used as indicator strain for nisin and pediocin PA-1, while Staphylococcus aureus ATCC 6538 (ATCC) and Salmonella enterica subsp. enterica serovar Newport ATCC 6962 were used as indicator strains for bactofencin A and microcin J25, respectively. L. ivanovii HPB28 and S. aureus were cultured in Tryptic Soy supplemented with 0.6% yeast extract (TSBYE), at 30 and 37\u00b0C, respectively. S. Newport was cultured at 37\u00b0C overnight in Luria-Bertani (LB) . All strains were maintained at \u201380\u00b0C as stock cultures in their corresponding culture media supplemented with 20% glycerol, and propagated twice at 24 h intervals before use.Microcin J25 was produced by PTUC 202 . For the2PO4 (3 g/L), K2HPO4 (7 g/L), (NH4)2HPO4 (2 g/L), and casamino acid (1 g/L) was supplemented with 1 mL/L of 20% MgSO4, 10 mL/L of 20% glucose, and 1 mL/L of 1 g/L thiamine, inoculated with an overnight culture of E. coli MC4100 pTUC202 (2% v/v in LB broth), followed by overnight incubation at 37\u00b0C in rotary shaking at 250 rpm. Bacterial cells were separated by centrifugation at 8,000 g for 20 min at 4\u00b0C. The supernatant was pre-purified by solid phase extraction (Sep-Pak C18) at 4\u00b0C at a flow rate of 2 mL/min, followed by reversed-phase high-performance liquid chromatography on a preparative C18 column at a flow rate of 10 mL/min. Microcin J25 was quantified by RP-HPLC using an analytical C18 column (Production and purification of microcin J25 were carried out as described previously . Briefly States) .Pediococcus acidilactici, while its stability is improved by the lack of Met31which is sensitive to oxidation. Briefly, pediocin PA-1(M31L) was synthesized as described previously and UV detection at 220 and 254 nm using 0.1% AcOH/H2O (A) and 0.1% AcOH/CH3CN (B), at 14 mL/min flow rate.Pediocin PA-1 carrying the Met31Leu substitution [pediocin PA-1(M31L)], was selected for the study, as the antimicrobial activity of this linear analog is similar to the wild-type bacteriocin naturally produced by eviously by standLactobacillus salivarius, while its stability was enhanced were mixed with simulated salivary fluid in 1:1 (v/v) ratio, and the final solution was incubated for 2 min at 37\u00b0C.Gastric conditions: The sample from the oral condition was diluted in 1:1 (v/v) ratio with simulated gastric fluid containing pepsin (2000 U/mL in the gastric mixture), and lipase (60 U/mL in the gastric mixture). The final solution was then adjusted to pH 3 with HCl 5 M, followed by incubation under shaking for 90 min at 37\u00b0C.Small intestine conditions: Samples from gastric conditions were diluted in 1:1 (v/v) ratio with 20 mL of simulated intestinal fluid containing bile salt and pancreatin to acquire a final volume of 40 mL. The final solution was adjusted to pH 7 using NaOH 5 M, followed by incubation for a further 2 h at 37\u00b0C. The digestion mixtures were heat-treated at 80\u00b0C for 10 min and centrifuged at 8,000 g for 10 min at 4\u00b0C for further analysis. Bacteriocins in the different samples were analyzed by analytical HPLC-UV and liquid-chromatography mass spectrometry (LC-MS/MS). The antimicrobial activity of the samples was also estimated by microtitration assays and agar well diffusion assays.The inhibitory activity of the different bacteriocins was determined qualitatively by the agar well diffusion assay, as described by 5 CFU/mL, from which 50 \u03bcL were added to each well. The microtiter plates were incubated for 24 h under appropriate conditions, and optical densities were recorded at 595 nm . Control wells contained untreated culture and appropriate medium (blanks). The number of inhibition wells was noted and inhibition activities were calculated in \u03bcg/mL.Quantitative determination of inhibitory activity of bacteriocins was carried out using the broth microdilution method as described by The digestion mixtures were analyzed by LC-MS/MS on an ultra-high-performance LC system connected to high-resolution electrospray ionization \u2013 quadrupole \u2013 time of flight (ESI-Q-TOF) mass spectrometer . Separations were achieved on an Acclaim RSLC Polar Advantage II column at a flow rate of 300 \u03bcL/min, using the following gradient of solvent A (ultra-pure water/0.1% formic acid) and solvent B (HPLC-MS grade acetonitrile/0.08% formic acid) over a total run time of 17.5 min: linear increase from 10% B to 60% B for 12 min, linear increase to 100% B for 0.2 min, decrease to 10% B for 0.5 min. The ESI-Q-TOF instrument was externally calibrated before each run using a sodium formate solution consisting of 10 mM sodium hydroxide in isopropanol/0.2% formic acid . Data-dependent LC-MS/MS data were acquired in positive ion mode in the mass range m/z 250\u20132500, using collision induced dissociation with collision energy calculated from m/z and charge states. The LC-MS/MS data were treated with Data Analysis 4.4 .2 (The liquid-chromatography mass spectrometry (LC-MS/MS) data were converted into mgf files and subjected to the online GNPS workflow2 , using t2 at 37\u00b0C. For the LDH release assay, cells were seeded in 96 well flat bottom culture plates (1 \u00d7 104 cells/well). Caco-2 cells, which have been frequently used for permeability studies across the intestinal epithelium, were selected in this study for evaluating the cytotoxicity of bacteriocins and their interaction with intestinal epithelium during GI transit to determine their cytotoxicity.The human colonic adenocarcinoma (Caco-2) cells were purchased from ATCC. Cells were cultured in DMEM medium in a humidified atmosphere containing 5% CO2. Lysis solution (Promega) was added in a 1:1 ratio to selected control wells to induce the maximal release of LDH and the plate was incubated for 5 min. Then 100 \u03bcL of medium were transferred to another 96-well plate for LDH release measurement and CytoTox-ONE\u2122 reagent was added to each well and incubated for 10 min at room temperature. Finally, stop solution was then added to each well, and absorbance was measured using a spectrophotometer at excitation/emission wavelengths of 560/590 nm. Each compound-treated value was blanked with the values of the control-treated cells and cytotoxicity was expressed as percentage of the maximal LDH release (lysis solution treated cells), calculated by the following formula:The CytoTox-ONE\u2122 cytotoxicity assay kit was used to measure the LDH release. The growing Caco-2 cells (in DMEM + 10% FBS) were treated with 100 \u03bcL of bacteriocins at different concentrations and incubated at 37\u00b0C for 48 h with 5% COg for 10 min to pellet red blood cells. The supernatant was then transferred into clear 96-well plates and the absorbance was read at 540 nm (hemoglobin). The percentage of hemolysis was calculated as follow, with RBC standing for red blood cells:The experiment was performed after prior approval from the local ethics committee by TransBIOTech laboratory. Hemolytic potential of bacteriocins was evaluated according to Data were expressed as mean \u00b1 standard deviation (SD) for at least three independent experiments. Dose-response curves were generated using GraphPad Prism version 8.2 GraphPad Software .Highly pure pediocin PA-1(M31L), nisin Z, bactofencin AM14L, M18L) and microcin J25 were produced . The MS L, M18L ain vitro GI stability and toxicity studies. The antimicrobial activity of the bacteriocins was confirmed by agar diffusion assays, which showed significant inhibition against target bacteria.Such a high purity level is a prerequisite for The physiochemical and biological stability of the bacteriocins in GI tract conditions was assessed using RP-HPLC, microdilution, and agar well diffusion assay . GastroiThe stability of the three bacteriocins in the different GI conditions was further studied using LC-MS . AnalysiMolecular networking derived from LC-MS/MS analysis of the digestion solutions allowed to assess the bacteriocin degradome in each condition \u20137. As a For nisin Z, a few degradation products were identified . They reThe effect of different bacteriocins on the membrane integrity of Caco-2 cells was evaluated by LDH release assay. The dose-response curves for LDH release in Caco-2 cells treated with different concentrations of bacteriocins was plotted . The resThe hemolytic activity of microcin J25, nisin Z, pediocin PA-1(M31L) and bactofencin A was evaluated using rat erythrocytes as described previously . ResultsThe resulting dose-response curves are shown in in vitro efficacy of bacteriocins against clinically important pathogens, such as vancomycin-resistant Enterococcus, methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Salmonella and E. coli have been determined using various well-accepted conventional enzymes .in vitro model, pediocin PA-1 was observed to be stable in the stomach, but completely degraded in the small intestine (in vitro model (static) while There are limited number of studies regarding the stability of bacteriocins in the GI tract. It has been shown that bacteriocins can be degraded by proteolytic enzymes such as pepsin, trypsin and chymotrypsin in the stomach or intestine . Notablyntestine . Howeverin vitro. The MIC values of nisin Z and pediocin PA-1(M31L) against L. ivanovii was shown to be 1.65 and 0.09 \u03bcg/mL, respectively. While that of microcin J25 was observed to be 0.0356 \u03bcg/mL against S. Newport, and that of bactofencin A was 5 \u03bcg/mL against S. aureus. In another study, Apart from the bioavailability assessment of bacteriocins, their possible interaction with epithelial cells (Caco-2 cells) was evaluated. To evaluate the cytotoxicity effect of bacteriocins in Caco-2 cells, LDH release assay was carried out to assess the membrane integrity of the cells upon exposure to different bacteriocins. The results of this study indicated that membrane integrity remained unaltered in presence of all tested bacteriocins at concentrations up to 400 \u03bcg/mL. It should be noted that this concentration is significantly higher than that required to target pathogenic/spoilage bacteria 50 values: since these samples are not pure and all contain salts and various contaminating compounds from culture conditions, which may affect concentrations and possibly activity, leading to high discrepancy in the results. In addition commercial nisin samples, contain different nisin analogs which should be taken into consideration. Using MTT assay, 50 value of commercial nisin A (Nutrition 21) to be 385.7 and 301.5 \u03bcg/mL in Caco-2 and HT29 , respectively (\u00ae) was shown to reduce the viability of Vero cells, and MCF-7 and HepG2 cells to 50% at 45.21 and 352 \u03bcg/mL, respectively in Vero cells was determined to be 0.62 \u03bcg/mL using LDH cytotoxicity assay. The inconsistency of results between the earlier studies and the current one could be due to impurities and salts in the substances tested. In this study, the purity of nisin and other bacteriocins used was more than 95%, while in the most of the other studies, crude preparation of nisin with high concentrations of salt were used, which can explain the different results. In addition, other factors such as the type of analog used, assay type, cell line, and exposure time can affect the outcome of different investigations.To the best of our knowledge, this is the first study that evaluates the cytotoxicity of several bacteriocins with different structures, mechanisms of actions and spectra of inhibitory activity. In a previous report, pediocin PA-1 and nisin (Sigma-Aldrich sample) were shown to be cytotoxic at high concentrations against Vero and SV40 cells using trypan blue staining viability assay. In fact, SV40 was more sensitive than Vero cells, and at 700 AU/mL (approximately 10\u201320 mg/mL), pediocin PA-1 and nisin were observed to reduce cell viability to 36 and 50%, respectively; therefore cytotoxicity of pediocin PA-1 was determined to be higher than that of nisin . Differeectively . In anotectively . In a st\u00ae) was shown to cause 6.6% hemolysis in sheep blood cells (\u00ae) at 3.35 \u03bcg/mL concentration was reported to be 6% in human red blood cells. In another study by Hemolytic activity is used for initial toxicity assessment and estimation of therapeutic index. Nisin remains to be the most studied bacteriocin, while there are very few data available for the other bacteriocins. At 33.75 \u03bcM (113 \u03bcg/mL) concentration, nisin C and bactofencin A in the GI tract, suggesting that these bacteriocins can be safely used in food preservation. Although microcin J25 showed high stability in the GI tract, it did not exert any toxic effect. The data from this study indicate that bacteriocins were non-toxic against eukaryotic cell lines and hemolysis was demonstrated to occur at concentrations significantly higher than their MICs. However, further in vivo studies are required to confirm these data and to evaluate the effect of long-term exposure to bacteriocins.Ultimately, this study provides unique scientific data on GI behavior and toxicity of several well-known bacteriocins, which differ in their structural characteristics and mechanisms of action. Using different The original contributions presented in the study are included in the article/SS, SZ, and IF participated in the experimental design, and responsible for the laboratory analysis, data analysis, and writing. SR participated in writing. FC, YB, and EB participated in experimental design, laboratory analysis, and data analysis. MS participated in experimental design and laboratory analysis. IF obtained the financial support for this study. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "When Service Provision Assessment (SPA) surveys on primary health service delivery are combined with the nationally representative household survey\u2014Demographic and Health Survey (DHS), they can provide key information on the access, utilization, and equity of health service availability in low- and middle-income countries. However, existing linkage methods have been established only at aggregate levels due to known limitations of the survey datasets.For the linkage of two data sets at a disaggregated level, we developed a geostatistical approach where SPA limitations are explicitly accounted for by identifying the sites where health facilities might be present but not included in SPA surveys. Using the knowledge gained from SPA surveys related to the contextual information around facilities and their spatial structure, we made an inference on the service environment of unsampled health facilities. The geostatistical linkage results on the availability of health service were validated using two criteria\u2014prediction accuracy and classification error. We also assessed the effect of displacement of DHS clusters on the linkage results using simulation.The performance evaluation of the geostatistical linkage method, demonstrated using information on the general service readiness of sampled health facilities in Tanzania, showed that the proposed methods exceeded the performance of the existing methods in terms of both prediction accuracy and classification error. We also found that the geostatistical linkage methods are more robust than existing methods with respect to the displacement of DHS clusters.The proposed geospatial approach minimizes the methodological issues and has potential to be used in various public health research applications where facility and population-based data need to be combined at fine spatial scale. As a part of the Demographic and Health Surveys (DHS) project, Service Provision Assessments (SPA) provide a comprehensive overview of health service delivery in low- and middle-income countries (LMICs). When these national level surveys of health facilities from SPA are combined with individual- or household-level surveys on population, fertility, family planning, reproductive health, child health, and nutrition , they caSeveral studies have proposed analytical methods to establish a link between survey respondents in household data (clusters from DHS) and individual facilities from SPA based on the Global Positioning System (GPS)-referenced location information of each dataset. For example, Hong et al. used a nHowever, existing methods were recommended for analyses at regional levels , 18, 28 To address the incompleteness of SPA in their linkage to DHS surveys at disaggregated levels, one can consider a geospatial approach based on spatial interpolation. Spatial interpolation has long been used in geographic information science (GIScience) to estimate the magnitude of the features at locations without data using known values at a number of selected locations , 26. AmoSpecifically, spatial dependence in Kriging embodies two types of effects . The firThe effect of geographic displacement of DHS cluster data on their linkage of health facilities in SPA has been assessed primarily focusing on geospatial proximity, which frequently imposes over-simplified assumptions. For example, the administrative boundary method links the two data sets assuming that the boundary dominates a choice of health facility. Except for in a few counties where the health care system was built upon administrative boundaries, however, administrative boundaries are delineated for a purpose of governing and not for delivering efficient health services. Similarly, kernel density or road networks are purely based only on the location of health facilities regardless of the physical and social/political environments surrounding DHS clusters, such as the presence of water bodies or mountains. Further investigation of the effect of geographic displacement of DHS clusters is needed.In the present study, we developed a novel geostatistical approach to link health facility survey data with geocoded DHS clusters at a disaggregated level . The proposed approach accounts for both the geographic configuration of DHS clusters and SPA facilities, as well as the local environmental conditions of the sampled health facilities in SPAs. We demonstrated the effectiveness of the proposed approach by estimating the general service readiness (SR) of each health facility as an exThere are 20 regions in mainland Tanzania and we focused on the two regions: Iringa and Njombe. These were selected because two of the authors have extensive contextual knowledge of these regions. The two regions, located in the Southern Highlands in the southwestern part of the country, are comprised of multiple districts and town councils as shown in Fig.\u00a0In mainland Tanzania, the public health care system is organized as follows. Primary health care services are the most common and comprise community-based health activities , village health services , dispensaries and health centers. Dispensaries are generally run by a clinical assistant and can provide preventive and curative services, while health centers can admit patients and provide some surgical services planning . For exaData used in the present study came from multiple and publicly available sources. The 2014\u20132015 Tanzania SPA Provision Assessment (TSPA) provides information on availability of basic and essential health care services and readiness to provide quality services [For the 2015\u20132016 Tanzania Demographic and Health Survey and Malaria Indicator Survey (TDHS-MIS), National Bureau of Statistics (NBS) carried out and supervised fieldwork activities, while ICF International provided technical assistance. Sampling was carried out in two stages. In the first stage, 608 clusters corresponding to enumeration areas delineated for the 2012 census were selected. For the second stage, a complete listing of households in all 608 selected clusters was carried out, and then 22 households were randomly selected from each cluster, yielding a representative probability sample of 13,376 [https://www.moh.go.tz/hfrportal). These data include facility name, type, ownership, and GPS coordinates. Data were obtained from the website in 2019 as an up-to-date census of existing facilities. Census data were unavailable for the same year as the TSPA was sampled, although the yearly rate of change in health facility placement is usually very low.Census data on all health facilities in Tanzania were obtained from the Ministry of Health, Community Development, Gender, Elderly and Children (MoHCDEC) website (www.worldpop.org). The road network data were obtained from Humanitarian Data Exchange (https://data.humdata.org/). We classified this dataset originally derived from OpenStreetMap into one of the following classes: primary, secondary tertiary roads, and tracks. We collected the elevation information from Shuttle Radar Topography Mission (SRTM) at the resolution of 3-arc sec, which was produced by National Aeronautics and Space Administration (NASA). We downloaded the digital elevation model (DEM) dataset from Jet Propulsion Laboratory (https://www2.jpl.nasa.gov/srtm/). The land use product was obtained from AFRICOVER project , which we downloaded from Food and Agriculture Organization of the United Nations (http://www.fao.org/geonetwork/srv/en/main.home). The land cover data originally derived from Landsat satellite images were regrouped into ten types of land uses: built area, woodland, forest, tree savannah, shrub land, sparse herbaceous vegetation, herbaceous crops, shrub crop, flooded vegetation, and water bodies.We sourced the gridded population data for Iringa and Njombe regions at 100 m spatial resolution projected for years of 2014 and 2015 from WorldPop database , we replaced the mean m by known varying means i-th health facility in the SPA survey. The varying means Z. The KED estimator isKriging with an external drift (KED) is similar to kriging with the simple kriging with varying local means in Eq. in that Statistical Calibration: In both the local trend estimation and kriging models, some model predictions exceeded the range of general SR scores. Although it is possible that some dispensaries may have a lower or higher general SR score than measured values in the SPA facility survey, any model prediction outside the range of 0 and 100 is unacceptable. To avoid these spurious results and rectify a systematic bias if present, we performed a stochastic calibration using a subset of the census data, which hereafter referred to as testing sites. Note that the census data contain the locational information of complete health facilities in the study area, although their SR scores are unavailable except at SPA facility sites. However, the information on the facility type\u2014Hospital, Health Center, Dispensary, and Clinic\u2014was known at all health facilities registered in the census data. We created testing sites as a subset of the census data by applying a criterion based on the geographic proximity to the same type of facilities in SPA sample. Here, we assumed that two adjacent health facilities that were classified as the same type of health facility likely shared equivalent or similar general SR scores.To operationalize this concept, we first examined each facility in the census if there was a SPA facility of the same type located within 5 km. If so, the facility in the census data would be included in the \u2018testing sites\u2019 and we assigned the general SR score of the nearest SPA facility to the testing site. Here we limited the search radius of the nearest same type SPA facility within 5 km based on a sensitivity analysis , we could not directly use the census data in the evaluation. Instead, we used the information on the facility type as a proxy variable and quantified two evaluation metrics: (1) the prediction error at a subset of the census health facilities, i.e., testing sites identified in the calibration; (2) the classification error per facility type over the census of health facilities.To calculate prediction error of the three algorithms of a generalized linear regression model, SKLM, and KED, we fitted the models with SPA data and obtained the predicted SR scores at testing sites. We also estimated general SR scores at testing sites using three commonly used linkage methods: administrative boundary link, Euclidean buffer link, and kernel density estimation methods. The details of the three linkage methods can be found in Skiles et al. , but we For the classification error, we assessed if the predicted general SR score at each health facility is within the facility type specific range of general SR scores inferred from SPA. For each health facility in the census, we compared the general SR score predictions obtained from different linkage methods with the corresponding lower and upper bounds specific to the facility type. The comparison results were quantified as classification error across all the 563 health facility sites. If a model predicts a general SR score that is within the range of lower and upper bounds per facility type in Table\u00a0Based on the proposed geostatistical linkage approach, we estimated the general SR at 40 DHS clusters. Under the consideration of the displacement each DHS cluster, we generated 100 sets of alternative and equally probable cluster locations. We simulated locations that are within 5 km for urban clusters and 10 km for rural clusters . At eachglm for a trend estimation, gstat package for variogram modeling and spatial prediction, and ks package for kernel density estimation.All statistical analysis was conducted with R software (version 4.0.2) using n = 77) of these health facilities were selected, including 18 Hospitals (23.4%), 25 Health centers (32.5%), 2 Clinics (2.6%), and 32 Dispensaries (41.6%). As shown in Table\u00a0A total of 563 health facilities operated in the study region in 2019 that consist of 20 Hospitals (3.6%), 62 Health centers (11.0%), 10 Clinics (1.8%), and 471 Dispensaries (83.6%). In the SPA facility survey of 2014\u20132015, about 13.7% A total of 98 facilities were included in the testing sites that have at least one SPA facility of the same type located within 5 km. The testing sites consist of 17 Hospitals, 29 Health centers, 2 Clinics, and 50 Dispensaries. Their reference values for general SR scores were in the range of 29.15 and 92.73 with the mean of 60.01 and standard deviation 16.15. By design, 77 facilities in the testing sites are from SPA.We developed a multivariate linear regression model for a local trend estimation and the regression results are summarized in Table\u00a0The spatial correlations between general SR scores were modeled using the variogram of residuals obtained at the SPA sites. The resulting experimental variogram and its optimal model fit are presented in Fig.\u00a0Based on the variogram model, we generated surfaces of predicted SR scores using two geostatistical prediction models. In addition, we created a surface of local trend that is solely based on a regression model as a baseline. The resulting prediction surfaces are shown in Fig.\u00a0Fig.\u00a0In terms of classification error, SKLM yielded the highest success rate of 79% (classification error of 0.21), closely followed by the regression model (78%) and KED model of 77%. Among the three existing methods, their performance varies substantially with the administrative boundary model having a 69% of success rate, kernel density estimation 47 % and buffering method 31%, respectively.The effect of the cluster displacement on the estimation of general SR scores varies per DHS cluster, with the averages for SKLM and KED were 51.97 and 51.12, respectively. Meanwhile, the variability of these estimated scores, quantified by standard deviation, ranges from 2.99 to 13.44 for SKLM and 3.54 to 13.52 for KED. More substantial differences were found in comparison to the existing methods of administrative boundary, buffering, and KDE. We presented the variability of general SR scores at each DHS cluster obtained from two geostatistical methods (SKLM and KED) and administrative boundary method using a boxplot in Fig.\u00a0We developed a geostatistical approach to link SPA and DHS datasets at a disaggregated level , and presented the application of the proposed linkage methods using data from Tanzania. The proposed geostatistical linkage methods utilize the information on the spatial configuration of DHS clusters and SPA facilities similar to existing geospatial linkage methods, such as administrative boundary, buffering distance, and kernel density estimation, but our method is an improvement upon these. Specifically, the proposed linkage method explicitly accounts for the geographic, socio-economic, and demographic conditions of and around sampled health facilities in SPA as well as their spatial autocorrelations. They are critical to reducing the misclassification error introduced when SPA survey is linked to DHS clusters, given the SPA is typically conducted as a nationally representative sample of facilities except in a small number of countries where they have conducted a census of all facilities . The knoIn our investigation of the effect of geographic displacement of DHS cluster data on the linkage, we concluded that geographic displacement may lead to a biased link between the health service environment and population, as shown in the wide range of box lengths across DHS clusters of Fig.\u00a0It is worth noting that proposed linkage methods are flexible and can be replicated with other service readiness or health indicators and in different settings. As key determinants for optimal health facility sites likely differ per region or country, it is necessary to modify the proposed multi-step geostatistical model to accommodate both the regional characteristics and the data availability. Many countries are at risk of not achieving the targets of the Sustainable Development Goal (SDG) 3 , because they continue to face challenges related to health services delivery and access. In order to improve service delivery and simultaneously, health outcomes and equity, a better understanding of how the health service delivery environment influences service utilization is needed. A first step in this involves linking health-sector and individual-level data for further analysis. As demonstrated using the data from two regions in Tanzania, our methods can contribute to better understanding of service utilization from an ecological perspective, but at a more local level, helping countries identify factors associated with the provision and utilization of services and address gaps to achieve SDG 3.However, the proposed geostatistical approaches should be applied with caution when the SPA consists of small number of facilities. Empirical variograms constructed from small samples likely are unreliable and incapable of inferring underlying spatial structures of the process, which could consequently yield biased and inaccurate predictions. To avoid spurious results, we recommend to linking DHS data to SPA surveys at a disaggregated level only when a sufficient number of facilities are included in the SPA. Similarly, caution is required when interpreting the performance evaluation reported in the present study. Due to the absence of validation data, we created testing sites that include all SPA facilities and utilized their general SR scores in both model fitting and validation. Consequently, the linkage method based on buffering showed the best performance in the prediction error but the worst in classification error. This inconsistent finding suggested that the model performance evaluation was sensitive to the choice of validation data.The current study has several strengths, including use of representative data; applicability of linkage methods to disaggregated levels unlike existing studies; use of rich data on population density, road networks, land use, and quality of services offered in health facilities; and potential for replication with other data nationally and in other settings. Nevertheless, the study has some limitations. First, we lack information on service readiness for facilities in the census. The SPA survey takes about 14% (77 sites out of 563) of the health facilities present in the study area. Potentially, the small sample size may have biased the inference of the spatial structure of the underlying process. Second, when the proposed approach is applied to a large study area, the stationarity assumption imposed on the relatively small study region has to be reevaluated. If the assumptions cannot be met, an alternative approach, including the division of study area to homogeneous regions, should be considered. Similarly, the spatial resolution of the prediction may affect the performance of the model. It should be also noted that the four covariates used for the present study are not exhaustive and may need revision when the proposed methods are applied to other regions. Finally, our SR information is based on general services. The readiness and availability of services may vary within a facility based on type of services being offered . In this general exercise, we had limited our analysis to general SR to demonstrate the feasibility of our method.In future research, the proposed linkage methods can be further improved by adopting sophisticated statistical methods and data mining techniques in combination with geostatistics. We also expect that a hybrid approach that involves both machine learning and geostatistics is applicable for different health service environments at regional and national levels. Lastly, we expect that the information on the access, utilization, and quality of health service estimated at DHS clusters will be used to assess the population health outcomes, including health status, health care-seeking behaviors, and policy questions about program impact and targeting related to population health.We developed a flexible approach for linking SPA and DHS data at a disaggregated level , improving upon the limitations of existing linkage methods which are only applicable at regional scales. These linked data can be used to improve understanding of how the service delivery environment influences utilization of services, and subsequently health outcomes, at the individual-level for myriad health domains . This approach can be adapted with other health services indicators and in other settings. Thus, the contribution of the current study can help identify gaps in service provision to inform programming and policy in various settings. This improved knowledge can help countries improve health outcomes for their populations, improve equity, and achieve SDG 3."} +{"text": "Candida albicans. This study aimed to determine how much the resistance profile of non-biofilm microorganisms can change.Clinical strains of microorganisms, including pathogenic yeast-like fungi (YLF), are resistant to currently used antifungal agents. Thus, it is relevant to study the combinations of existing antimicrobial drugs and a medicinal extract of plant origin (farnesol). In previous studies, farnesol showed a relatively strong anti-biofilm effect against C. albicans and one reference strain were used to study the interaction of farnesol with the most used antimycotics. To determine the sensitivity of YLF to antimycotic drugs, such as nystatin (50 \u03bcg), amphotericin B (10 \u03bcg), ketoconazole (10 \u03bcg), clotrimazole (10 \u03bcg), voriconazole (10 \u03bcg), fluconazole (25 \u03bcg), miconazole (10 \u03bcg), and intraconazole (10 \u03bcg), the classic disk diffusion method was used. In the second stage, one of the six strains was used to simulate candidiasis of the gastrointestinal tract in an in vivo quail model. As an unusual experimental design, this study investigated the effects of pretreated C. albicans in quails, not the in vivo pathogenicity of C. albicans, after treatment with farnesol.Six clinical isolates of Candida strains did not improve with farnesol in all strains. All concentrations of farnesol demonstrated a fungistatic effect in 23 of 56 (7\u00d78) cases (41%). The remaining 54% demonstrated no changes in the resistance to antifungal drugs or deterioration of the indicators in rare cases (5%). At 100 \u03bcM farnesol, sensitivity improved in 33 of 56 cases (59%). Candidiasis or the severity of clinical disease of the quail digestive tract developed to a lesser extent if fungi were treated with farnesol.The resistance profiles of in vivo model with biofilms, farnesol can be considered a new antimycotic drug or an additive to existing antimycotics.Farnesol does not always show a positive result on single cells without biofilm in the laboratory. However, in a biofilm or an Candida albicans is a conditionally pathogenic yeast-like fungus (YLF) that threatens a human\u2019s weakened immune system [C. albicans, and the main mechanism was probably associated with the suppression of hyphal development [e system ,2. Becauelopment -5.In biological communities, microorganisms use various mechanisms for their communication. Depending on the cell density, bacteria and YLF can produce quorum sensing (QS) signaling molecules involved in regulating gene expression and coordinating collective behavior in their natural niche. These secondary metabolites play a major role in the adhesion and colonization of host tissues and surfaces, morphogenesis, and biofilm development ,7. In yeFarnesol is continuously produced in biofilms at temperatures from 23\u00b0C to 43\u00b0C and in quantities approximately proportional to colony-forming units per milliliter. Chemically, farnesol is acyclic sesquiterpene alcohol endogenously synthesized by the ergosterol pathway. Its synthesis depends on neither the type of carbon or nitrogen source nor the chemical nature of the nutrient medium, and it is a thermally stable molecule not exposed to extreme pH values .et al. [Escherichia coli. They found that phosphatidate phosphatase (PgpB) and undecaprenyl diphosphatase (YbjG), two integral membrane phosphatases of E. coli, can hydrolyze farnesyl diphosphate into farnesol [Farnesol is obtained in natural and synthetic ways. Farnesol can be isolated from linden-colored essential oils, musk grains, neroli, and pettigrain. In yeast, farnesol is a by-product of the ergosterol biosynthesis pathway formed due to the enzymatic dephosphorylation of farnesyl pyrophosphate. The enzymes of this pathway are encoded by ergosterol genes. In bacteria, phytoene/squalene synthase (YisP) acts as a phosphatase, catalyzing the formation of farnesyl diphosphate. Wang et al. describefarnesol . Large-sCandida species. In these cases, studying whether farnesol affects changes in the sensitivity of clinical strains of Candida to antifungal drugs is especially interesting.The question is why study the QS farnesol molecule? The list of antifungal agents is huge, and new drugs appear every year. However, the sensitivity of microorganisms to new drugs is weak, variable, and unequal. In addition, tests for sensitivity to antimycotics, unfortunately, are not considered a routine procedure, not always available, and usually not considered a standard technique in patient management. As practice shows, resistance studies are prescribed by doctors only in cases of deep mycoses, mainly caused by non-albicans C. albicans pathogenicity in quail model experiments.This study aimed to prove that farnesol can increase the antifungal activity of some antimycotics and can reduce The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Peoples\u2019 Friendship University of Russia (RUDN University) ethical committee ..The study was conducted from May to July 2021. The samples were processed at the Department of Microbiology and Virology, Medical Institute, Peoples\u2019 Friendship University of Russia, Moscow, RussiaC. albicans isolates (C1-C6) were isolated from women with vulvovaginal candidiasis. The identification of microorganisms was carried out using the matrix-activated laser desorption/ionization technology \u201cBruker Daltonik MALDI Biotyper\u201d . A score of >2000 was considered reliable. The reference strain (C7) was C. albicans ATCC 10231. Colonies of diurnal cultures of C. albicans from Saburo agar were washed off with physiological solution . The YLF concentration was 0.5 according to McFarland, corresponding to 1.5\u00d7108 cells/mL [Clinical strains of six cells/mL -16.Farnesol (trans-farnesol) was purchased from Sigma-Aldrich (Germany): Molar mass 222.37 g/mol, mass of the substance 0.886 g/mL, Ns=The amount of the substance in moles = 0.886:222.37=0.004 M/mL or 4,000 \u03bcM/mL. Three concentrations of farnesol were used, and PhS (pH 7.0) was used as the control.To determine the sensitivity of YLF to antimycotic drugs , nystatin , amphotericin B , ketoconazole , clotrimazole , voriconazole , fluconazole , miconazole , and intraconazole , the classic disk diffusion method was used.(a) 25 \u03bcL PhS+farnesol were applied to sterile paper disks, corresponding to a farnesol concentration of 100 \u03bcM; (b) 25 \u03bcL PhS was applied to sterile paper disks; (c) 25 \u03bcL PhS was applied to the disk with the antimycotic; (d) 25 \u03bcL farnesol (100 \u03bcM) was applied to the disk with an antimycotic; (e) 25 \u03bcL farnesol (50 \u03bcM) was applied to the disk with an antimycotic; and (f) 25 \u03bcL farnesol (25 \u03bcM) was applied to the disk with an antimycotic.The diameter of the growth suppression zone of the culture to the antimycotic was measured in accordance with the NCCLS M-44 protocol.ad libitum and were manipulated in accordance with the Local Ethics Committee for Animal Experimentation, Peoples\u2019 Friendship University of Russia, Moscow, Russia .Female Texas white broiler quails , 21 days old, 30 heads, body weight 350-370 g, were used. Birds passed veterinary control and had all documentation. Quails were quarantined for 7 days before the experiment under the supervision of a veterinarian. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals . Quails C. albicans to cause infection, the daily culture of YLF was washed 3 times with PhS, and the YLF concentration was 0.5 according to McFarland. Then, 25 \u03bcL of 100 \u03bcM farnesol was added to 1 mL microbial suspension (experiment), and 25 \u03bcL PhS (control) was added to another test tube. The tubes were incubated for 1 h at 37\u00b0C under constant shaking. After interaction with farnesol, YLF was washed three times with PhS [C. albicans+farnesol, and the control group was infected with C. albicans without farnesol.To identify the effects of farnesol on the ability of with PhS ,19. The C. albicans (1 mL) was given to quails through a digestive probe once daily for 5 days. The birds were killed every 5 days for five heads. The experiment lasted 20 days: 5 days for infection and 15 days for the course of the experiment. All animals injected with C. albicans were examined histopathologically [Statistical Package for the Social Sciences 20.0 was used for statistical analyses, and a significance level of 5% was adopted. Experiments with antimycotic disks were run with three replicates per condition. Data in the table on sensitivity to antimycotics are given without \u00b1 for better table perception, and \u00b1 did not exceed 0.4.in vitro. Especially, the clinical strain C1 showed resistance to most drugs. In the other six strains, the resistance levels differed among the groups of drugs and varied within the groups. The synergistic effect of farnesol with polyene antimycotics was as follows: farnesol (100 and 50 \u03bcM) increased sensitivity to NS in six strains, and farnesol 25 (\u03bcM) increased sensitivity to NS in four strains; farnesol (100 \u03bcM) increased sensitivity to AP (50 \u03bcM) in six strains, farnesol (50 \u03bcM) increased sensitivity to AP in four strains; and farnesol (100 \u03bcM) increased sensitivity to AP (25 \u03bcM) in six strains were effective. The resistance of one clinical isolate was quite high, and farnesol even slightly worsened the indicators. Only in one strain of YLF, sensitivity to FU increased with farnesol at all three concentrations. Another culture increased sensitivity with farnesol (100 and 50 mM). In other cases, farnesol did not affect the sensitivity to FU. Sensitivity to MIC increased in three YLF cultures with farnesol . The growth retardation zone with IT in three of seven cases was higher than the control with the addition of farnesol at all three concentrations .th day after infection, curd overlays were more distinct, with rounded foci. On the 15th day before the slaughter, clinical signs of the disease were observed in the form of depression, drowsiness, and poor appetite. One of the five quails remaining died. On opening, the mucous membrane of the goiter was bumpier due to the different intensities of the overlays, which in sections took the form of a solid yellow-white film, and the serous membrane became folded. Delicate, loose, and yellow-white overlays were noted on the mucous membrane of the oral cavity and tongue, in which multiple budding and pseudomycelial forms of Candida were detected. On the 15th day, the remaining four quails killed presented with typical, well-marked candidiasis lesions in the anterior part of the digestive tract. In the histological sections, multiple filaments of the fungus were found growing perpendicular to the thickness of the mucous membrane.In the control group of birds killed within the first 5 days after infection, swelling, weak hyperemia of the goiter mucosa, thick viscous mucus and a delicate whitish plaque containing an abundance of budding cells, and pseudomycelia were observed. In the histological sections of the goiter, oral cavity, and esophagus, the start of fungal growth into the epithelial cover of the mucous membrane was detected. On the 10th day after infection on the mucous membranes of the digestive tract, a delicate whitish plaque containing a small number of budding cells was observed. On the 15th day before slaughter, clinical signs of the disease in the form of depression, drowsiness, and poor appetite were not observed. Not a single quail died. On autopsy, the mucous membrane of the goiter was covered with a delicate white biofilm, in which mainly C. albicans budding cells were found.In the experimental group of quails, where YLF was treated with farnesol, hyperemia of the goiter mucosa was not noticed in the first 5 days after infection. In the histological sections of the goiter, oral cavity, and esophagus, the start of fungal growth into the epithelial cover of the mucous membrane was detected but to a lesser extent than in control. On the 10C. albicans whether in the form of a biofilm or individual cells not connected by a matrix. In the previous experiments, where Candida was in the form of a biofilm, farnesol worked perfectly. In living models, microorganisms also form biofilms. Therefore, farnesol reduces the pathogenicity of Candida, and the infection does not develop so rapidly and strongly. Thus, this experiment showed that C. albicans treated with farnesol becomes less pathogenic for animals. YLF are less able to form a biofilm under the influence of farnesol.This study demonstrated a significant difference in Candida spp. is a justification for the preliminary determination of their sensitivity to antifungal drugs and continuous mycological monitoring of the spread of these strains. Identifying such resistant strains significantly limits chemotherapy possibilities for candidiasis [The increasing dominance of polyresistant strains of didiasis ,6,21. TrCandida, including filamentation, biofilm formation, drug susceptibility, and apoptosis [C. albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida kefyr, Candida krusei, and Candida glabrata. Whether farnesol can affect the resistance of the strain to modern drugs remains an open question. Because this process of stability or sensitivity will always be dynamic for different isolates and change over time, it will always be relevant.As a QS molecule, farnesol participates in regulating various physiological processes in poptosis ,4,16,22.poptosis . The secCandida biofilm and the concentration used. However, the effect of farnesol on its synergy or antagonism with known antifungal drugs has been studied to a lesser extent. This study showed that farnesol certainly affects the degree of YLF resistance, indicating its potential role as an antimicrobial agent [Candida strains are not improved by farnesol in all strains. All concentrations of farnesol demonstrated a fungistatic effect in 23 of 56 (7\u00d78) cases (41%). The remaining 54% demonstrated no changes in the resistance to antifungal drugs or deterioration of the indicators in rare cases (5%). At 100 \u03bcM farnesol, sensitivity improved in 33 of 56 cases (59%).Farnesol and its derivatives exhibit antibiofilm, anticancer, antitumor, and fungicidal properties ,18,24. Tal agent ,4,25,26.in vivo model. Candidiasis of the digestive tract developed to a lesser extent if fungi were treated with farnesol. In combination with increased sensitivity to existing drugs, the effect of reducing the pathogenicity and reducing biofilm formation is a good result. We believe that the effect of farnesol on the sensitivity of C. albicans to antifungal drugs is successful. Our plans are to check the change in sensitivity to other drugs for this purpose. Of course, this experience will be useful for a deeper understanding of the potential antifungal mechanism of QS molecules and a continuing search for new drugs against Candida and other YLF.In the previous studies ,4, farneThe supplementary data can be available from the authors on reasonable request.AK, NS and EV: Conception and designed the study. NS, AS, and OK: Collected the samples and data analysis. IP, AI, NZ, and MM: Drafted the manuscript. All authors read and approved the final manuscript."} +{"text": "OR) of 2.319 after adjustment for confounders. Ninety-nine mediator CpGs were selected from SIS, and then, seven candidate CpGs were obtained by de-sparsifying the LASSO regression. Four of these CpGs showed statistical significance, and the average causal mediation effects (ACME) were attenuated from 0.066 to 0.126. Notably, three significant mediator CpGs had absolute sensitivity parameters of 0.40, indicating that these mediation effects were robust even when the assumptions were slightly violated. Genes (BCL3 and FKBP5) harboring these four CpGs were related to CD. These findings suggest that changes in methylation are involved in the mechanism by which smoking increases risk of CD.Epigenome-wide mediation analysis aims to identify high-dimensional DNA methylation at cytosine\u2013phosphate\u2013guanine (CpG) sites that mediate the causal effect of linking smoking with Crohn\u2019s disease (CD) outcome. Studies have shown that smoking has significant detrimental effects on the course of CD. So we assessed whether DNA methylation mediates the association between smoking and CD. Among 103 CD cases and 174 controls, we estimated whether the effects of smoking on CD are mediated through DNA methylation CpG sites, which we referred to as causal mediation effect. Based on the causal diagram, we first implemented sure independence screening (SIS) to reduce the pool of potential mediator CpGs from a very large to a moderate number; then, we implemented variable selection with de-sparsifying the LASSO regression. Finally, we carried out a comprehensive mediation analysis and conducted sensitivity analysis, which was adjusted for potential confounders of age, sex, and blood cell type proportions to estimate the mediation effects. Smoking was significantly associated with CD under odds ratio ( Inflammatory bowel disease (IBD) is a complex etiology comprising Crohn\u2019s disease (CD) and ulcerative colitis (UC) . PreviouDNA methylation has a role in the immune dysfunction phenotype associated with IBD, as it is influenced by certain smoking known toDNA methylation is immensely cell-type specific, and several studies have demonstrated the impacts of cellular heterogeneity on the DNA methylation status , which mIn this study, we identify the mediating effect of the association between smoking and CD through methylation mechanisms at CpG sites. We applied the multiple-mediator causal model framework to estimate and test unbiased mediation effects in high-dimensional epigenetic studies, in particular the existence of omitted variables or confounders. In the primary analysis, we first reduced the pool of potential mediator CpGs using the SIS method aDatasets were obtained from a case\u2013control study of DNA methylation and IBD. The genome-wide DNA methylation data using the Illumina 450K methylation array are available at the Gene Expression Omnibus (GEO) website under accession GSE87648 to identify IBD-associated epigenetic analysis . In our p-value (default \u22650.01) and samples with a mean p-value of all probes greater than 0.05 were filtered out; a total of 28,931 probes containing SNPs (MAF\u00a0\u2265\u00a00.05) in their sequences were also removed from the final data; and all probes located in chromosomes X and Y were filtered out. Meanwhile, for normalization of methylation data, a quantile normalization algorithm and outcome (CD), respectively. M\u00a0=\u00a0 denotes high-dimensional mediators (CpG sites) that we are interested in for their effects independent of the pathway from exposure to outcome. Suppose that there are multiple causally unrelated mediators and that one is interested in estimating the causal mediation effects with respect to each of them. Let C denote some set of comprehensive confounders cells, B cells, monocytes, and granulocytes) that may affect the mediator and outcome.The causal graph model in This analysis was conducted using baseline information on smoking, CD, and confounders . The folp componentwise magnitudes of the vector \u03c9 in decreasing order and define a submodel n] denotes the integer part of \u03b3n and [\u03b3n]\u00a0<\u00a0n. The SIS method was used for a rough dimension reduction to reduce the ultra-high-dimensional model to d (n (n\u00a0=\u00a0277). It was crucial that the SIS of a fast and efficient method reduce dimensionality from a large or huge scale to a relatively large scale. Therefore, to identify important mediators with the largest effects for the response, SIS identifies mediators of the top First, for the purpose of dimension reduction analysis, a high dimension may lead to false associations between covariates and response variables. We implemented SIS to reduc/log(n) instead p-value for each mediator. De-sparsifying the LASSO procedure has been implemented in R package hdi of 5% (<0.05).Second, after dimensionality reduction of SIS, variable selection was carried out next. On account of the LASSO estimates being biased and without the testing to asymptotic normality property, previous studies proposed the asymptotic normality for the de-sparsified estimates for high-dimensional data . Dezeurekage hdi . In the \u03c1 between the residuals of the mediator and outcome regressions and the average causal mediation effects (ACME) of the mediator based on the mediation package in R . To asseressions . For eacM) as the outcome and smoking (X) as a predictor, adjusting for the confounders sex, age, and estimated cell-type proportions; and (2) the outcome model, with CD (Y) as the outcome and smoking (X) as a predictor, adjusting for the mediator, i.e., the CpG site (M), and the covariates from the first model.In mediation analysis, for each candidate CpG mediator, we fitted the following statistical models: (1) the mediator model, the CpG site , NK cells, B cells, monocytes, and granulocytes. + T cells, CD4+ T cells, NK cell, B cell, monocyte, and granulocyte) for each of the samples were estimated using the estimateCellCounts function implemented in a flexible and comprehensive bioconductor \u201cMinfi\u201d , 16.266) years, which is older than that of the controls by 3\u00a0years. There was no significant statistical difference between the age and sex. On average, controls had a higher proportion of CD8OR) of 2.319 , adjusting for sex, age, CD8+ T cells, CD4+ T cells, NK cells, B cells, monocytes, and granulocytes or negative , but the absolute effect value was greater than 2. The standard error from de-sparsifying the LASSO method was relatively small, varying from 0.6791 to 1.253.A total of Then, we also analyzed the relationship between smoking and the above CpGs , focusing on positive and negative effect sizes. As shown in + T cells, CD4+ T cells, NK cells, B cells, monocytes, and granulocytes under statistical significance mediation effects , which were shown in p-values ranging from 0.001 to 0.012 from the above seven candidate CpGs in the mediation models, adjusting for sex, age, CD8to 0.012 . In Tablto 0.012 . The DNAIn sensitivity analyses of the mediated effect estimates, the SI assumption might be violated for residual correlations of the mediator and outcome regressions far from the observed estimated mediated effects on the above four CpGs. And then, we also conducted a sensitivity analysis on the above four CpGs to assess the robustness of our mediation analysis when the SI assumption was violated. Notably, the absolute sensitivity parameters at which ACME\u00a0=\u00a00 in the four mediator CpGs were 0.4 or 0.5, indicating that these mediation effects were robust even when the assumptions were slightly violated . The senSmoking is an established risk factor for the development of CD. Our results suggest that smoking might play an important role in the well-established association of smoking and CD through DNA methylation variability. Our results also highlight the need to consider various confounding factors in epigenetic studies as a relevant biological and statistical model.In epigenetic studies, it is crucial that DNA methylation plays a mediator role in the etiology of human diseases . Methyla+ T cells isolated from patients with CD and UC, underlining a role for BCL3 in the pathogenesis of IBD , were affected between IBD cases and controls. And the study found drastically elevated expression levels of BCL3 in CD4s of IBD . Anothers of IBD . FKBP5 rs of IBD . In addis of IBD and glucs of IBD . In addis of IBD . Furthers of IBD . Besidess of IBD . We specAn important strength of our causal diagram is the implementation of the counterfactual framework in mediation analysis to estimate the effects in the presence of confounders as a relevant biological model for epigenetic epidemiology. We applied screening criteria (SIS) and dimension reduction (de-sparsifying the LASSO) to select CpGs with the top largest effects and the bias-corrected regression coefficient for the outcome. Then, we conducted mediation analyses of smoking (exposure) on CD (outcome) through DNA methylation (mediator).One limitation of the study is that our sample size for the mediation analyses is small. Using epigenome-wide significant CpG sites as candidate mediators may show stronger signals in a future study with a larger sample size. In particular, the presence of unmeasured confounders may make it impossible to distinguish causal from consequential methylation events based on observational data alone . TherefoIn conclusion, this study was based on epigenetic DNA methylation data and elucidated the mechanisms related to environmental factors involved in susceptibility to IBD. Furthermore, it makes more biological sense to identify the high-dimensional mediation effect of the whole gene rather than to focus on individual methylation sites when performing a mediation analysis in an epigenetic study. Nevertheless, a statistical mediation approach might not accurately reflect the underlying causal biological mechanism. The study found that several biologically meaningful DNA methylation sites mediated the effect of smoking on CD. In future studies, the highly plausible biological mechanisms on how smoking influences CD outcome are revealed by these DNA methylation sites."} +{"text": "Both the kinase MET and the WNT signaling pathway are attractive targets in cancer therapy, and synergistic effects have previously been observed in animal models upon simultaneous inhibition. A strategy towards a designed multiple ligand of MET and WNT signaling is pursued based on the two hetero biaryl systems present in both the MET inhibitor tepotinib and WNT signaling inhibitor TC-E 5001. Initial screening was conducted to find the most suitable ring systems for further optimization, whereas a second screen explored modifications towards pyridazinones and triazolo pyridazines. Up to 54% reduction of WNT signaling activity at 10 \u03bcM concentration was achieved, however, only low affinities towards MET were observed. Overall, the thiophene substituted pyridazinone 40 was the best dual MET and WNT signaling inhibitor, with a 17% and 19% reduction of activity, respectively. Although further optimizations are needed to achieve more potent dual inhibitors, the strategy presented herein can be valuable towards the development of a dual inhibitor of MET and WNT signaling. Dual inhibitors of MET and WNT signaling may have synergistic effects, and the design, synthesis and evaluation of such first-in-class small-molecules are reported. Therefore, small-molecule WNT signaling inhibitors are predicted to be effective anti-tumor drugs in humans.5 Importantly, both MET and WNT signaling pathways hold a central role in the development of metastasis, and a strong biochemical connection between the two have been documented.6,7 Hence, simultaneous inhibition have demonstrated synergistic effects in breast cancer models,8 and overcoming of resistance in lung cancer9 and melanoma cell lines.10 In addition, a bispecific antibody inhibiting both MET and WNT signaling has been developed, which synergistically reduced tumor growth in a lung cancer model in vivo.11The tyrosine kinase MET and the WNT/\u03b2-catenin signaling pathway have both received considerable interest as targets in cancer therapy due their involvement in carcinogenesis.12,13 Well-designed DMLs can reduce drug\u2013drug interactions and side effects, and simplify drug pharmacokinetics.14,15 In search of novel treatment strategies for cancer, we aim to develop dual inhibitors of MET and WNT signaling.While the prevalent strategy in drug discovery is \u201cone drug, one target\u201d, the realization that many drugs exerts their effect through more than one target have led to the increased interest in designed multiple ligands (DML).16 and TNKS1/2 inhibitors are promising in the development of WNT signaling modulators. TNKS1/2 poly(ADP-ribosyl)ate AXIN proteins to earmark them for degradation by the ubiquitin\u2013proteasomal system. Inhibition of TNKS1/2 can therefore lead to stabilization of AXIN proteins and hence the \u03b2-catenin degradosomes that in turn enhances the degradation of \u03b2-catenin.17 The TNKS1/2 inhibitors can attain three different binding modes in the active site. This, combined with the fact that many of the known inhibitors are discovered through high-throughput screening leads to structures spanning a wide chemical space.18 Some examples of TNKS1/219\u201321 and MET inhibitors22\u201326 are shown in The proteins tankyrase 1 and 2 (TNKS1/2) are central regulators of WNT signaling,27 One example of a MET inhibitor deviating from this pattern is tepotinib (8), consisting of two unfused hetero biaryl systems. As such, tepotinib creates a link from the traditional MET inhibitors to the TNKS1/2 inhibitor TC-E 5001 (2). This similarity is elaborated in The selective MET inhibitors have traditionally two fused heterocycles connected with a one or two atom linker, as exemplified with structures 4\u20137. Several different heterocyclic moieties have been explored, and the linker enables the ligands to adopt a U-shape in the active site of MET.N2 disconnection and commercial starting materials, a selection of structural moieties from known TNKS1/2 and MET inhibitors could be combined efficiently to the target scaffold 9. In Initially, the aim was to evaluate which heteroaromatic ring systems that best could be applied in the target structure 9. By utilizing a Sin silico docking was performed. Tepotinib and TC-E 5001 were docked into the TNKS1 and MET active site, respectively, and a good overlap with the experimental structures were observed in both proteins regardless of whether an unfused oxadiazole (21) or a fused oxazolo pyridine (22) was present. The large structural similarity between 19 and TC-E 5001 (2) and the modest inhibition of WNT signaling activity with 19 should be noted, indicating a relatively sensitive pharmacophore. MET inhibition did not reach the anticipated level for neither of the analyzed compounds.With two fused heterocycles, 18 shares common chemical characteristics with known MET inhibitors, and should allow for improvement of the MET affinity. The pyridazinone in 21 and 22 can also serve as a foundation for optimization, and further modifications were therefore based on 18, 21 and 22. With the syntheses shown in 2 gave the corresponding triazolo pyridazines 31. To obtain the target structures, the pyridazinones and triazolo pyridazines were reacted with electrophilic chlorides. To potentially increase the affinity for MET, the oxadiazole pyrimidine 33 was included due to reported interactions between the pyrimidine ring and the active site in the crystal structure of tepotinib in MET.26 The novel analogs were evaluated in MET and WNT signaling assays, and the results are presented in Both pyridazinones and triazolo pyridazines could be synthesized starting from 3,6-dichloropyridazine (28). Suzuki\u2013Miyaura coupling gave the intermediate chloro pyridazines 29, which could be hydrolyzed with AcOH to substituted pyridazinones 32. Nucleophilic attack on 29 by hydrazine hydrate and subsequent cyclization with CSe.g.35 or 38 with tepotinib, the main difference is the oxadiazole in place for the phenyl group. This modification can alter the orientation of the ligand, although successful modifications have been made at this position earlier.28,29 Neither of the triazolo pyridazines 44 and 45 were able to inhibit MET, despite large similarities with e.g. AMG-208 (4). An increase from 25% to 54% in WNT signaling inhibition was obtained when moving from 21 and 22 to 35, however, low WNT signaling inhibition was observed for the remaining pyridazinones. Optimization of 18 to the structures 44 and 45 led to a similar or somewhat reduced WNT signaling inhibition. For this triazolo pyridazine series, the chlorine substituent in 42 and 43 reduced the WNT inhibition. The best combined inhibition of MET and WNT signaling, albeit modest, was achieved with the thiophene analog 40, with 17% and 19% reduction, respectively.Overall, the performed modifications did not increase the affinity for MET. Comparing N2 reactions would furnish the final products directly, facilitating screening of the most favorable structural cores. In silico docking revealed a potential good overlap of tepotinib and TC-E 5001 in both active sites. The first compound screen demonstrated low MET inhibition, while WNT signaling activity could be reduced by 50% at 10 \u03bcM concentration. Further structural modifications were carried out based on the triazolo pyridine 18 and pyridazinones 21 and 22. In the second compound screen, the pyridazinone 35 inhibited WNT signaling by 54%, however with no inhibition of MET. The thiophene substituted pyridazinone 40 was overall the best dual MET and WNT signaling inhibitor, with a 17% and 19% reduction of activity, respectively. Although further structural optimizations are needed, the novel strategy provided herein could prove valuable for further development of dual MET and WNT signaling inhibitors.The design of a scaffold for potential dual inhibitors of MET and WNT signaling was performed from the MET inhibitor tepotinib (8) and the tankyrase inhibitor TC-E 5001 (2). The target scaffold 9 was designed so that facile S254 plates, and visualized using UV light at 312 nm or 365 nm, a phosphomolybdic acid solution (12 g phosphomolybdic acid in 250 mL EtOH) or a potassium permanganate solution for detection. Column chromatography was performed with silica gel . 1H and 13C NMR spectra were obtained on a Bruker AVIII HD 400 instrument (400/101 MHz) or Bruker AVII 600 (600/151 MHz). Chemical shifts (\u03b4) are reported in parts per million (ppm), and coupling constants are reported in hertz (Hz). The residual proton solvent resonance in 1H NMR and the residual carbon solvent resonance in 13C NMR (CDCl3 at \u03b4 77.16 ppm and DMSO-d6 at \u03b4 39.52) are used as reference. Accurate mass determination (HRMS) in positive or negative mode was performed on a Waters Prospec Q instrument, ionized by electrospray (ESI). LC-MS was performed on a Thermo Finnigan LCQ Deca XP Plus with a gradient from 10% to 90% acetonitrile in water over 10 minutes.All chemicals were purchased from Sigma-Aldrich or Fluorochem and used without further purification. Air and/or moisture sensitive reactions were performed under argon atmosphere with dried solvents and reagents. TLC was performed on Merck silica gel 60 F2CO3 were dissolved in DMF (2 mL). After 5 minutes, the electrophile dissolved in DMF (1 mL) was added dropwise, and the reaction was stirred at ambient temperature for 15 hours (70 \u00b0C where noted). Work-up and purification: method A; The reaction mixture was diluted with EtOAc (2 mL) and water (2 mL), and the organic phase was separated. The aqueous phase was extracted with EtOAc (2 \u00d7 5 mL), and the combined organic phases were washed with water (4 \u00d7 5 mL) and brine (5 mL), dried over MgSO4 and reduced on a rotary evaporator. The crude product was purified by column chromatography (Hep\u2009:\u2009EtOAc (1\u2009:\u20091) \u2192 Hep\u2009:\u2009EtOAc\u2009:\u2009MeOH (10\u2009:\u200910\u2009:\u20092)). Method B: The reaction mixture was reduced on a rotary evaporator, and purified by column chromatography (2\u20135% MeOH in DCM).The nucleophile and Csa]pyridine-3-thiol (14) and 5-(chloromethyl)-3-(p-tolyl)-1,2,4-oxadiazole (10) in the presence of Cs2CO3 . Work-up and purification were performed according to method A and the title compound was obtained as a white solid . 1H NMR : \u03b4 8.10 , 7.81 , 7.73 , 7-32-7-28 , 7.22 , 6.85 , 4.45 , 2.39 . 13C NMR \u03b4 175.27, 168.77, 151.60, 141.95, 138.64, 129.63, 128.18, 127.36, 123.35, 123.23, 116.69, 114.61, 30.28, 21.67. HRMS (ESI+) m/z calcd for C16H13N5NaOS [MNa]+: 346.0733, found 346.0733.Prepared according to the general procedure by reacting triazolo+: 243.0199, found 243.0198.3,6-Dichloropyridazine (28) (301 mg 2.02 mmol), (5-acetylthiophen-2-yl)boronic acid , K2CO3 and Pd(dppf)Cl2\u00b7CH2Cl2 in 1,4-dioxane (2 mL) and H2O (0.5 mL) were reacted according to the general protocol. The intermediate 3-chloro-6-pyridazine (29d) was hydrolyzed in AcOH (3 mL). The title compound was obtained as a light grey solid . 1H NMR \u03b4 13.35 , 7.76\u20137.70 , 7.42 , 7.23 , 6.99 . 13C NMR \u03b4 162.7 , 160.0, 159.6 , 140.3 , 133.8 , 131.3 , 129.8, 119.9 , 112.3 , 104.8 . HRMS (ESI+) m/z calcd for C10H6F2N2NaO [M + H]+: 231.0340, found 231.0340.3,6-Dichloropyridazine (28) , 2,4-difluorophenyl boronic acid , K2O (1.5 mL). CS2 was added and the mixture was stirred at ambient temperature for 48 h, with similar addition of KOH and CS2. The reaction mixture was reduced on a rotary evaporator and purified by column chromatography (5\u201310% MeOH in DCM). The title compound was obtained as a yellow solid . The spectroscopic analysis was in agreement with earlier reported data.311H NMR \u03b4 14.92 , 8.24 , 7.48 . 13C NMR \u03b4 160.47, 148.74, 140.16, 126.67, 124.75. HRMS (ESI\u2212) m/z calcd for C5H2ClN4S [M \u2212 H]\u2212: 184.9694, found 184.9694.3,6-Dichloropyridazine (28) was suspended in EtOH (3 mL) and added hydrazine hydrate . The resultant mixture was heated to reflux for 3 hours and reduced on a rotary evaporator. The intermediate 3-chloro-6-hydrazinylpyridazine (30a) was suspended in EtOH (1.5 mL) and added a solution of KOH in H2 was added and the mixture was stirred in a closed vial at 80 \u00b0C for 15 h. The reaction mixture was reduced on a rotary evaporator and purified by column chromatography (5\u201310% MeOH in DCM). The title compound was achieved as an orange solid . 1H NMR \u03b4 14.89 , 8.82 , 8.32 , 8.06 , 8.01 . 13C NMR \u03b4 151.38, 150.67, 141.71, 141.06, 126.16, 121.96, 121.08. HRMS (ESI+) m/z calcd for C10H8N5S [M + H]+: 230.0495, found 230.0495.3-Chloro-6-(pyridin-4-yl)pyridazine (29b) was suspended in EtOH (4 mL) and added hydrazine hydrate . The resultant mixture was heated to reflux for 5 hours in a sealed tube and reduced on a rotary evaporator. The intermediate 3-hydrazinyl-6-(pyridin-4-yl)pyridazine (30b) was suspended in EtOH (5 mL) and added KOH . The reaction mixture was placed under argon and degassed for 5 minutes. CS32via the PyRX33 interface. The experimental crystal structures of tepotinib in MET and TC-E 5001 in tankyrase were downloaded from the Protein Data Bank (PDB: 4R1V and 3UDD), and prepared for docking using Autodock Tools . The ligands were build using Avogadro,34 and initial geometrical optimization was done using the same software. After docking, visualization of the conformations and binding interactions were done in PyMol . Initially, redocking of tepotinib and TC-E 5001 into the active site of MET and tankyrase, respectively, showed to reproduce the experimental results with good accuracy.Dockings were performed using Autodock VinaThe enzymatic MET assay was purchased from Cyclex and used following the manufacturer's instructions. Briefly, while placed on ice, recombinant MET was added to wells precoated with substrate, and the reaction was started by adding buffer containing the inhibitors in appropriate dilutions. The plate was incubated at 30 \u00b0C for 60 minutes. After washing with buffer, a HRP conjugated detection antibody PY-39 was added to each well, and incubated at ambient temperature for 60 minutes. The TMB substrate was added after another round of washing, and incubated at ambient temperature for 10 minutes. Stop solution was added, and absorbance was measured using a spectrophotometric plate reader (PerkinElmer VICTOR\u2122 X3).35The cellular luciferase-based WNT/\u03b2-catenin signaling reporter was performed using various compound concentrations and incubated for 24 hours as previously described.There are no conflicts to declare.RA-009-C9RA08954C-s001"} +{"text": "This study aimed to investigate the clinical characteristics and risk factors of patients with hepatocellular carcinoma (HCC) with extrahepatic metastases (EHM) and to establish an effective predictive nomogram.Clinical and pathological data from 607 patients with hepatocellular carcinoma admitted to the Affiliated Hospital of Qinghai University between 1 January 2015 and 31 May 2018 were documented, as well as demographics, clinical pathological characteristics, and tumor-related parameters to clarify clinical risk factors for HCC EHM. These risks were selected to build an R-based clinical prediction model. The predictive accuracy and discriminating ability of the model were determined by the concordance index (C-index) and the calibration curve. The results were validated with a bootstrap resample and 151 patients from 1 June 2018 to 31 December 2019 at the same facility.In multivariate analysis, independent factors for EHM were neutrophils, prothrombin time, tumor number, and size, all of which were selected in the model. The C-index in the EHM prediction model was 0.672 and in the validation cohort was 0.694. In the training cohort and the validation cohort, the calibration curve for the probability of EHM showed good agreement between the nomogram prediction and the actual observation.The extrahepatic metastasis prediction model of hepatocellular carcinoma constructed in this study has some evaluation capability. Hepatocellular carcinoma (HCC) is the leading malignant tumor from the liver, which is the seventh most common, and has the second highest death rate . It has HCC is a kind of cancer that develops as a result of a secondary liver illness . Liver function indices are intimately linked to the occurrence and development of HCC , 4. BesiA retrospective study was conducted on patients who were diagnosed with HCC from 1 January 2015 to 31 December 2019 at the Affiliated Hospital of Qinghai University . Inclusion criteria were as follows: 1) according to the Guidelines for diagnosis and treatment of primary liver cancer, the patient was diagnosed with HCC (2021 Edition) and 2) wThe appearance of a newly detected tumor confirmed on two radiologic images, with or without an elevation of serum tumor markers, was defined as metastasis. A patient with a smoking history was defined as having smoked continuously or cumulatively for 6 months or more in the past. Drinking more than three standard glasses of alcohol per day or more than seven standard glasses of alcohol per week for 1 month or more, either continuously or cumulatively, is considered a drinking history.During the 2 years following diagnosis, all patients were seen once every 3 months. An abdominal ultrasound, blood test, and liver function test were performed at each of the follow-up visits. When a tumor recurrence or metastasis was suspected, a contrast-enhanced CT or MRI was performed, and the results were reviewed individually by two experienced doctors.U test for variables with an abnormal distribution. Categorical variables were compared using the chi-squared or Fisher exact test. In the univariate analyses, p < 0.05 was considered statistically significant. Multivariate logistic regression analysis was used to evaluate the independent risk factors of extrahepatic metastases. In the multivariate analyses, p < 0.05 was considered statistically significant.Statistical analyses to identify risk factors were performed using IBM SPSS Statistics 23.0 for Windows . Continuous variables were compared using the Mann\u2013Whitney http://www.r-project.org/). A final model selection was performed by a forward conditional selection process. The predictive performance of the nomogram was measured by concordance index (C-index) and the calibration curve. Bootstraps with 1000 resample were used for these activities , 1673 cases were identified as HCC in the Affiliated Hospital of Qinghai University. 1066 cases were excluded according to the exclusion criteria, and 607 cases were finally enrolled, including 456 patients in the training cohort from 1 January 2015 to 31 May 2018, and 151 patients in the validation cohort from 1 June 2018 to 31 December 2019 Figure\u00a01The training cohort was concentrated on 136 cases in the EHM group, of which 112 (81.4%) were male and 24 (17.6%) were female, aged 53.42 years; 320 cases in the control group, of which 255 (79.7%) were male and 65 (20.3%) were female, aged 53.83 years. Based on the results of the multivariate logistic regression analysis in the training cohort, neutrophil, prothrombin time, tumor number, and tumor size were used as variables to construct the nomogram Figure\u00a03vs. 23.1 months, p = 0.00).The 415 patients were fully monitored for 23.694 person-months , in which 308 (74.2%) died. In all 415 patients enrolled, the overall survival (OS) of patients with EHM was significantly worse than non-EHM patients or hepatitis C virus (HCV) and the consumption of aflatoxin-contaminated food. The prevalence of HCC caused by metabolic syndrome, obesity, diabetes, excessive alcohol consumption, and non-alcoholic fatty liver disease (NAFLD) is gradually increasing . Thus, aFor patients with HCC with EHM, most previous studies examined only the relationship between clinicopathological characteristics and the prognosis of patients with HCC , 9\u201311. HAccording to the univariate and multivariate analysis of EHM in our research Figure\u00a02A close relationship between HCC size and EHM seems to exist according to our results Figure\u00a02In this study, we also demonstrated that an elevated neutrophil was a significant independent predictor for EHM of HCC. Neutrophil is one of the most simple and effective markers of inflammation and is associated with poor prognosis in various cancers , 19, 20.Currently, angiopoietin-2 (Ang-2), microRNAs (miRNAs), and lncRNA BACE1-AS are available as a test for the evaluation of EHM in HCC \u201327. HoweIn previous studies , 14, 28,The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The study was censored on October 19,2020 and was approved by the Ethics Committee of The Affiliated Hospital of Qinghai University. All subjects signed an informed consent form. The patients/participants provided their written informed consent to participate in this study.LZ is responsible for writing, LR, MQ, WW and XS are responsible for document of patients data. FY, MD and HW are responsible for document. following up, ZW, HF are responsible for guiding research and revising papers. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (No. 82160466) and Research team for minimally invasive diagnosis and treatment of biliary and pancreatic diseases .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Assessing health-related quality of life (HRQoL) among persons with dementia poses several challenges due to cognitive decline and limited perception. As a result, proxy ratings by family members or health professionals are used. The EQ-5D is the most commonly used generic and preference-based HRQoL instrument. Methodological drawbacks of the three-level version (EQ-5D-3L) prompted the development of the five-level version (EQ-5D-5L) by expanding the range in the domains. However, no comparison of the psychometric properties of both versions and different proxy ratings exist so far. Therefore, the objective of this study was to compare the psychometric properties of the EQ-5D-5L and EQ-5D-3L by application of different proxy ratings in dementia.The EQ-5D-3L and -5L were completed by n\u2009=\u200952 family caregivers and one care manager at baseline and three and six months later. In total, 106 caregiver and 133 care manager proxy ratings were completed. The EQ-5D-3L and 5L were evaluated in terms of acceptability , agreement, ceiling effects, redistribution properties and inconsistency, and informativity as well as convergent and discriminative validity.Mean proxy index scores were higher for the 5L than the 3L. Missing values occurred less frequently in both proxy ratings and versions (<\u20091%). Agreement between both measures was high but higher in caregiver than care-manager ratings (ICC 0.885 vs. 0.840). The relative ceiling effect decreased from the 3L to the 5L, more intensively in the care-manager (75%) than the caregiver rating (56%). Inconsistency between both versions was low. Informativity increased from the 3L to the 5L version, nearly equally in both proxy ratings. The 5L also demonstrated a better discriminative ability and convergent validity than the 3L, especially in the caregiver rating.Compared to the EQ-5D-3L, the EQ-5D-5L had higher feasibility and acceptability and was slightly superior by a reduction of ceiling effects and an improvement in informativity, discriminative ability and convergent validity. Proxy ratings by informal caregivers overall demonstrated better psychometric properties than professional care-manager ratings.The online version contains supplementary material available at 10.1186/s12955-022-02049-y. An essential goal of primary dementia care and psychosocial interventions for people living with dementia (PwD) is to improve health-related quality of life (HRQoL) \u20134. HowevAccording to acceptability and validity, the preference-based health utility questionnaire EQ-5D performed comparably to other well-validated dementia-specific measures, e.g. the Quality of Life in Alzheimer's Diseases (QoL-AD) , 11. ThiNevertheless, proxy-ratings demonstrate external perspectives of patients' HRQoL and should, hence, be used with caution , 15, 16.Methodological drawbacks of the former version of the EQ-5D, the EQ-5D-3L, promptedHowever, no head-to-head comparison of the psychometric properties between the EQ-5D-3L and EQ-5D-5L in dementia diseases has been published, especially not comparing the commonly used proxy ratings. It is unknown whether an expansion from three to five levels in each dimension improves the deviation of proxy ratings by informal caregivers or health care professionals. Thus, this study aimed to compare the psychometric properties of the EQ-5D-5L with those of the 3L classification system in cognitively impaired PwD with patients' proxy ratings by family caregivers and care manager, both with a close patient-relationship.The EQ-5D-3L, the EQ-5D-5L, the EQ-visual analogue scale (VAS) and the Quality of Life in Alzheimer's Diseases (QoL-AD) were completed as proxy ratings by family caregivers and care manager. Both versions of the EQ-5D were compared in terms of acceptability, agreement, ceiling effects, redistribution properties, inconsistency, informativity, and convergent and discriminative validity.This study used data collected from the ongoing interventional study DCM:IMPact (Dementia Care Management: Implementation into different Care Settings), an implementation study, which builds on the DelpHi-trial . The mixHealth care professionals assessed patients' eligibility . If patients were eligible, the professionals provided written and oral information about the study and asked for patient and caregiver written informed consent (IC). This study was approved by the local ethics committee at the University Medicine Greifswald (BB 01/2019).This analysis was based on preliminary data, including n\u2009=\u200977 patients, n\u2009=\u200952 caregivers and one dementia care manager, who had provided collaborative dementia care management for six months. Data were assessed at baseline and three and six months after the baseline assessment.The EQ-5D-3L, the EQ-5D-5L, the EQ-VAS , 27, 28,The informal caregivers and the care manager first completed the EQ-5D-3L with the EQ-VAS, followed by the completion of the EQ-5D-5L and the QoL-AD. Thus, for the caregiver, the \"Interviewer Administered Proxy version 1\" were used where the interviewer asked the caregiver (proxy) to rate the patient's health-related quality of life in their (the proxy's) opinion. For the care manager, we used the \"Proxy version 1\", where the care manager (the proxy) was asked to rate the patient's health-related quality of life in their (the proxy's) opinion. Interviews of the caregivers conducted by care manager were done first before the care manager themself completed the EQ-5D-3L and 5L.The EQ-5D is a generic HRQoL instrument containing three or five levels for the following five dimensions: mobility, self-care, pain/discomfort, usual activities, and anxiety/depression. The responses to the EQ-5D-3L were converted to health utilities, the preference-based single index measure for HRQoL anchored at 0 for death and 1 for full health , 27, 28.The following sociodemographic and clinical data were assessed: cognitive impairment measured with the Mini-Mental State Examination (MMSE) , comorbiThe responses to the EQ-5D-3L and EQ-5D-5L were converted to health utilities with the European and German value set , 38, resThe number of missing values, the score ranges (observed vs. possible range), and the floor (% with lowest possible score) and ceiling effects (% with highest possible score) were used to compare the acceptability of both instruments. Additionally, absolute and relative changes in the ceiling effect of EQ-5D-3L versus EQ-5D-5L were calculated.The agreement between both versions was assessed with intraclass correlations (ICC) and presented with Bland\u2013Altman plots. The ICC represents the proportion of variance from both index scores attributable to differences between individuals instead of the differences between the EQ-5D-3L and 5L. The higher the ICC, the higher agreement between the two versions. ICC higher (lower) than 0.7 indicates an acceptable (poor) agreement.Inconsistency was assessed as suggested by previous studies , 39, 40,The informativity of both measures was assessed with Shannon indices (i.e. Shannon\u2013Weaver index (H') and Shannon's evenness index (J')), which are appropriate measures to determine the discriminatory ability in health state classification in the comparison of the EQ-5D-3L and EQ-5D-5L. The higher the Shannon H' index, the more absolute information is captured by the measures. The Shannon Evenness index (J') captures the relative informativity of the distribution measure, regardless of the number of categories , 41. If The discriminative ability, defined as the ability to distinguish between different health and diseases stages, was assessed by different stages of functional impairment (Bayer Activities of daily living), cognitive impairment , depression as well as general physical and mental health status. Cut-off values used for this analysis were established and validated within the development of each measure. Linear trends were assessed with the nonparametric Jonckheere trend test (>\u20093 groups) or Mann\u2013Whitney test (2 groups).Convergent validity was assessed with Spearman's Correlation Coefficient between the EQ-5D-3L and EQ-5D-5L with the QoL-AD. Due to some overlap of dimensions , we assumed there should be a moderate correlation between these measures. A correlation coefficient higher than 0.3 and 0.5 was determined as a moderate and strong correlation, respectively . There sThe sample characteristics are presented in Table\u00a0There were 106 EQ-5D-3L and 5L proxy ratings by family caregivers , and 133 EQ-5D-3L and 5L proxy ratings by the care manager were included in the analyses. The mean proxy-rating score of caregivers was 0.48 (SD 0.26) and 0.50 (SD 0.32) for the 3L and 5L, respectively. The mean health status caregivers stated in the VAS was 50.0 (SD 19.7). The care manager reported a higher mean utility score of 0.52 (SD 0.22) and 0.61 (SD 0.25) for the EQ-5D-3L and 5L. The mean value in the VAS by the care manager was 49.0 (SD 18.9), slightly lower as compared to the caregiver rating. The density plots and histograms of both measures and proxy ratings are demonstrated in Additional file Missing values occurred comparably and less frequently in both versions (3L vs 5L). There was also a similar occurrence of missing values within proxy ratings (0.9% for caregiver ratings and 0.8% for care manager ratings), as demonstrated in Table Ceiling effects were for all domains smaller for the 5L compared to the 3L. The relative ceiling effect of the index decreased by 56% and 75% in the caregiver proxy rating and the case manager proxy rating, respectively. Ceiling effects were highest for the dimension \"pain/discomfort\" and \"anxiety/depression\". However, ceiling effects (8.5% vs. 3.0%) were higher and ceiling effect reduction lower (55.5% vs. 75.0%) in caregiver ratings than care manager ratings. Ceiling effects are depicted in Table p\u2009<\u20090.001) than in care manager ratings . The density plot, presented in Additional file The agreement between both versions was good, but slightly higher among caregivers . At least one inconsistent pair occurred in 14 13%) out of 106 caregiver proxy assessments and 12 (9%) out of 133 care manager proxy assessments. The highest inconsistency was found for the dimension \"pain/discomfort\", with an average inconsistency size of 1.17 and 1.09, respectively. The redistribution properties and level of inconsistency are demonstrated in Table % out of Absolute and relative informativity increased in the 5L compared to the 3L for both proxies, which demonstrated an average increase of the absolute and relative informativity . The relative and absolute informativity increase was highest for the domain \"mobility\". Absolute and relative informativity are presented in Table The EQ-5D-3L and EQ-5D-5L were equally able to discriminate between general physical and mental health stages, functional impairment, and patient hospitalizations. The five-level version of the EQ-5D better distinguishes between stages of depression and pain, demonstrating the superiority of the 5L over the 3L for caregiver ratings. Contrary to this, the EQ-5D-3L care manager rating better discriminates between stages of general physical and mental health, functional and cognitive impairment, and pain than the 5L. The discriminative ability is represented in Table Both proxy ratings demonstrated that the EQ-5D-5L had a better convergent validity with most of the measures, which revealed the superiority of the 5L version. However, the convergent validity was better for caregiver ratings than care manager ratings, demonstrated by larger correlation coefficients. The convergent validity of both measures is presented in Table To the best of our knowledge, this is the first analysis comparing the psychometric properties of the EQ-5D-5L compared to the EQ-5D-3L in PwD using proxy ratings given by informal family caregivers and health professionals. Generally, the EQ-5D-5L reveals higher index scores than the 3L. Both EQ-5D-5L proxy ratings improve psychometric properties by reducing ceiling effects and improving informativity and convergent validity. As demonstrated by the ICC, the agreement between the three- and five-level EQ-5D was excellent but slightly higher in the caregiver proxy rating than the care manager rating. Also, caregiver proxy ratings demonstrated a better convergent validity than the care manager proxy ratings. Thus, the EQ-5D-5L shows its superiority over the 3L version as a proxy rating used in dementia, primarily when family caregivers assess patients' health status.Both EQ-5D-5L proxy measures demonstrated similar feasibility, acceptability , informativity, and consistency. The agreement between both proxy-rating measures was excellent (ICC 0.885 for caregiver rating and 0.840 for care manager rating) and in line with the reported agreement in previous studies, revealing ICC higher than 0.85 , 45. MisSeveral validation studies found ceiling effects of both measures in different settings between 15 and 50%, with a decrease of up to 10% from the 3L to the 5L version , 47\u201350. In line with previously published studies, this analysis demonstrated a significant gain in absolute informativity and an improvement in relative informativity for all dimensions in the EQ-5D-5L , 45, 51.The extension from three to five levels could be more challenging, causing inconsistent valuations. However, informal caregiver and care manager proxy ratings revealed a very low inconsistency (<\u20093%), in line with previous studies (1\u20135%) , 48, 53.A systematic review by Hounsome et al. summarizBoth proxy-rating instruments also performed well regarding the convergent validity, with evidence tendency for a slightly better convergent performance of the EQ-5D-5L. However, this superiority remains uncertain and should be confirmed in future psychometric head-to-head analyses that compare both measures in dementia diseases. Most previously published studies reported a slight to considerate superiority of the EQ-5D-5L concerning the convergent validity , 45. HowThis is the first study administrating both the three and five-level versions of the EQ-5D in multiple proxies, creating a sufficient basis for a comprehensive assessment of the psychometric performance of the EQ-5D in dementia. Furthermore, a strength of this analysis was the inclusion of several in this indication critical clinical measures, like cognitive and functional impairment, general health, depression and pain, which were needed to assess the validity of the EQ-5D instruments thoroughly.However, several limiting aspects are acknowledged. First of all, the study was based on a sample size of 106 caregiver assessments and 131 care manager proxy ratings, limiting the generalizability of the results. Especially the fact that only n\u2009=\u200952 caregivers and only one heath professional (care manager) assessed patients' health limits the robustness of the presented results. Secondly, the EQ-5D-3L was completed before the EQ-5D-5L by caregivers and the health professional (care manager). Thus, overuse of levels two and four in the 5L could be possible, as reported by Janssen et al. . FurtherContrary to this, an initial completion of the 5L could have caused the overuse of level two in the 3L version. Future studies should consider randomization of the application process to reduce potential bias. Most importantly, the care manager completed the EQ-5D-3L and 5L after interviewing the caregiver, who stated the patient's health as a proxy. Therefore, this survey sequence might have influenced the care manager in assessing the patient's health status.Finally, the comparison of the psychometric performance is affected by the different value sets used. While the European value set used for the EQ-5D-3L caused a range of utility values between \u2013\u00a00.074 and 1, the German value set led to EQ-5D-5L utility values between\u2009\u2212\u20090.661 to 1. Thus, the EQ-5D-5L basically had a wider range that could be an advantage in distinguishing groups of diseases stages and general health, and also to correlate with other measures. While the agreement between the measures (3L and 5L) was excellent and both measures performed equally in the known groups' validity, the 5L had a better convergent validity, which could also be due to the basic differences in the different value sets used. Further research is needed to reveal the impact on the psychometric properties revealed within head-to-head comparisons. Cross-walks that mapped responses of the EQ-5D-3L to the EQ-5D-5L could be helpful and a prerequisite to use the same value set for both measures.Our results provide some indications that the five-level version of the EQ-5D was slightly superior over the three-level version by improving informativity and convergent and discriminative validity and reducing ceiling effects. Our findings also indicate that family caregivers' ratings may be preferable to measure HRQoL in PwD due to a better discriminatory ability and higher convergent validity. However, further research is needed to clarify and confirm the superiority of the five-level version of the EQ-5D using larger sample size and also taking reliability and responsiveness measures into account.Additional file 1: Figure S1. Density plots of the self- and proxy-rating EQ-5D-3L and 5L index values. Figure S2. Bland-Altman plots of the EQ-5D-3L and the EQ-5D-5L index values Vertical Axis represents the difference between the EQ-5D-5L and EQ-5D-3L (5L minus 3L).Figure S3. Histograms of the self- and proxy-rating EQ-5D-3L and 5L index values."} +{"text": "The EQ-5D-5L and 15D are generic preference-accompanied health status measures with similar dimensions. In this study, we aim to compare the measurement properties of the EQ-5D-5L and 15D descriptive systems and index values in a general population sample.In August 2021, an online cross-sectional survey was conducted in a representative adult general population sample (n\u2009=\u20091887). The EQ-5D-5L and 15D descriptive systems and index values were compared in terms of ceiling and floor, informativity (Shannon\u2019s Evenness index), agreement, convergent and known-groups validity for 41 chronic physical and mental health conditions. Danish value sets were used to compute index values for both instruments. As a sensitivity analysis, index values were also estimated using the Hungarian EQ-5D-5L and Norwegian 15D value sets.\u22126%) unique profiles occurred on the EQ-5D-5L and 15D. The EQ-5D-5L dimensions (0.51\u20130.70) demonstrated better informativity than those of 15D (0.44\u20130.69). EQ-5D-5L and 15D dimensions capturing similar areas of health showed moderate or strong correlations (0.558\u20130.690). The vision, hearing, eating, speech, excretion and mental function 15D dimensions demonstrated very weak or weak correlations with all EQ-5D-5L dimensions, which may indicate potential room for EQ-5D-5L bolt-ons. The 15D index values showed lower ceiling than the EQ-5D-5L (21% vs. 36%). The mean index values were 0.86 for the Danish EQ-5D-5L, 0.87 for the Hungarian EQ-5D-5L, 0.91 for the Danish 15D and 0.81 for the Norwegian 15D. Strong correlations were found between the index values . Both instruments were able to discriminate between all chronic condition groups with moderate or large effect sizes . Compared to the 15D, effect sizes were larger for the EQ-5D-5L in 88\u201393% of chronic condition groups.Overall, 270 (8.6%) and 1030 (3.4*10This is the first study to compare the measurement properties of the EQ-5D-5L and 15D in a general population sample. Despite having 10 fewer dimensions, the EQ-5D-5L performed better than the 15D in many aspects. Our findings help to understand the differences between generic preference-accompanied measures and support resource allocation decisions.The online version contains supplementary material available at 10.1186/s12955-023-02096-z. Generic preference-accompanied measures (PAMs) are health status measures that consist of two parts: the first is a descriptive system, and the second is a value set that allows assigning utilities to health profiles defined by the descriptive system. Over the past decades, an increasing number of generic PAMs have been developed, such as the EQ-5D, Short-Form 6-Dimension (SF-6D), Assessment of Quality of Life (AQoL) and Health Utilities Index (HUI) . DespiteThe 15D is a 15-dimensional generic PAM, which was developed in Finland starting from the 1970s . CountryCompared to the EQ-5D, the descriptive system of the 15D is considerably longer, more detailed, and comprehensive. Notwithstanding, the 15D and EQ-5D-5L instruments are similar in many aspects, which offers a strong basis for comparison. Firstly, a range of corresponding dimensions can be found between the two measures with similar wording, such as mobility, usual activities, pain/discomfort, and anxiety/depression/distress. Secondly, on both instruments each dimension of health has one item with five response levels measured on a severity or capability scale. Finally, both instruments investigate the current health status of the respondent. A few studies compared the measurement properties of the EQ-5D-3L and 15D in different patient populations, such as epilepsy , HIV/AIDComparing PAMs in different populations is important to inform researchers, analysts and health policy decision-makers about their performance and support the choice of instrument for cost-utility analysis. Although the EQ-5D-5L has proved to be a valid instrument in a multitude of health conditions, it might not capture all important aspects of health, especially in sensory disorders and mentTherefore, in this study, we aim to conduct an exploratory analysis that compares the measurement properties of the EQ-5D-5L and 15D in a large general population sample in Hungary. We compare measurement properties of both the descriptive systems and the index values focusing on ceiling and floor effects, informativity, agreement, redistribution properties, convergent and known-groups validity.A cross-sectional survey was conducted with a targeted sample size of 2000 members of the Hungarian adult general population (response rate 77.8%). The broader aim of the survey was to assess the mental health of the population. Permission for conducting the study was granted by the Research Ethics Committee of the Corvinus University of Budapest (no. KRH/166/2021). Participants were recruited in August 2021 from one of the largest available online panels in Hungary by a third-party survey company. Respondents registered voluntarily to complete surveys in return for points, which could be redeemed for rewards. Respondents were included who were at least 18\u00a0years old at the time of completion, gave informed consent, and confirmed that they had understood the terms and were willing to participate. \u2018Soft\u2019 quotas were applied to ensure the representativeness of the sample for the general population by age, gender, the highest level of education, geographical region, and settlement type.A self-administered survey was designed for the study that asked questions about health-related quality of life, well-being, presence of physical and mental health conditions, resource utilization related to mental health care, and sociodemographic characteristics. The list of the physical health conditions was selected according to the 2019 Hungarian results of the European Health Interview Survey (EHIS) compleme5\u2009=\u20093125 unique health states in total [The EQ-5D-5L is a generic PAM that consists of two parts: a five-item descriptive system and a 0\u2013100 visual analogue scale (EQ VAS) . The desin total . Respondin total . The lowin total . Index v15 (more than 30 billion) possible distinct health states. The 15D asks respondents to recall their current health . The Danish value set was selected in this study as a base case. This was developed using an additive model of the multi-attribute utility theory based on valuations on a 0\u2013100 visual analogue scale (VAS). Firstly, respondents were asked to weigh the top and bottom levels of each dimension individually on a VAS, then they were asked to assign a score to each level of each dimension on VAS (\u2018within dimension tasks\u2019). Data were collected in 2001 and preferences of the non-institutionalized general population of Denmark aged 18\u201375 were assessed [The 15D is another generic PAM that covers 15 dimensions of health-related quality of life: mobility, vision, hearing, breathing, sleeping, eating, speech, excretion, usual activities, mental function, discomfort and symptoms, depression, distress, vitality, and sexual activities . Each ofassessed . The indassessed . Howeverp\u2009<\u20090.05 was considered statistically significant.Our analytical framework builds on previous studies that compared the measurement properties of other generic PAMs \u201346. As aThe proportion of participants reporting \u2018no problems\u2019 (ceiling) and \u2018extreme problems\u2019 (floor) was computed for each dimension of the descriptive systems. In addition, we calculated the ceiling and floor for the EQ-5D-5L and 15D health profiles, i.e. \u2018no problems\u2019 and \u2018extreme problems\u2019 in all dimensions, respectively. We expected a higher overall ceiling in the EQ-5D-5L than the 15D at an instrument level since the descriptive system of the latter is more detailed .pi is the proportion of observations in the ith level , and L is the number of levels in a dimension of the descriptive system. The greatest amount of information can be gathered if the responses are equally used across the levels. The Shannon Evenness index (J\u2032) measures the evenness of distribution and was calculated asThe informativity of EQ-5D-5L and 15D dimensions, index values, and health state profiles was examined by Shannon\u2019s and Shannon\u2019s Evenness indices , 48. The2L, and J\u2032 ranges from 0 to 1, where a higher value indicates better informativity.Thus, H\u2032 ranges from 0 to logWe performed cross-tabulations of the corresponding EQ-5D-5L and 15D dimensions to explore how consistent the responses were. We considered an EQ-5D-5L and 15D response pair inconsistent if the 15D response was at least two levels away from the EQ-5D-5L response . The aveThe agreement between the EQ-5D-5L and 15D index values was examined using intraclass correlation coefficient (ICC) and BlanWe examined the convergent validity between the EQ-5D-5L and 15D dimensions (Spearman\u2019s correlation) and index values (Pearson\u2019s correlation). The absolute value of the correlation coefficient (r) was interpreted as follows: very weak correlation |r|\u00a0<\u20090.2, weak correlation 0.2\u2009\u2264\u00a0|r|\u00a0<\u20090.4, moderate correlation 0.4\u2009\u2264\u00a0|r|\u00a0<\u20090.6 and strong correlation 0.6\u2009\u2264\u00a0|r|\u00a0\u2264\u20091 . We expet test was used to compare the healthy and non-healthy groups. Effect size and relative efficiency (RE) were calculated. ES values were interpreted as negligible d\u2009<\u20090.2, small 0.2\u2009\u2264\u2009d\u2009<\u20090.5, medium 0.5\u2009\u2264\u2009d\u2009<\u20090.8, and large 0.8\u2009\u2264\u2009d [Known-groups validity was evaluated for self-reported physician-diagnosed health condition groups in contrast to being healthy. We hypothesized that respondents with a diagnosed physical or mental condition had significantly lower EQ-5D-5L and 15D index values. Student\u2019s 0.8\u2009\u2264\u2009d . The RE The distribution of the sample n\u2009=\u20091887) reasonably approximated that of the general population in terms of sociodemographics and 1.2% (anxiety/depression), while the ceiling ranged from 50.8% (pain/discomfort) to 87.7% (self-care) (Table EQ-5D-5L outperformed 15D regarding relative informativity (J\u2032) for all dimensions (ranging from 0.51 to 0.70 for the EQ-5D-5L and from 0.44 to 0.69 for the 15D), except for the EQ-5D-5L anxiety/depression (0.65) vs. 15D distress (0.69) Table . ConsideResponses covered all levels in both the EQ-5D-5L and 15D among the corresponding dimensions Table . The EQ-The distributions of the EQ-5D-5L and 15D index values are presented in Fig.\u00a0In the total sample, the mean index value was the highest using the Danish 15D , followed by the Hungarian EQ-5D-5L , the Danish EQ-5D-5L , and the Norwegian 15D value set . The floor was negligible for 15D and not present for the EQ-5D-5L. For the Danish EQ-5D-5L, 1.4% of the index values were in the negative range, while for the Danish 15D, the theoretical minimum is higher than 0. However, 1.2% of the Hungarian EQ-5D-5L and 0.9% of the Norwegian 15D index values were negative. When the index value range was split with a bin width of 0.05, the Norwegian 15D showed the best relative informativity (J\u2032) (0.63), followed by the Danish EQ-5D-5L (0.53), the Hungarian EQ-5D-5L (0.49), while the lowest J\u2032 was demonstrated by the Danish 15D (0.44) (Table p\u2009<\u20090.001) but a good agreement was found between the Hungarian EQ-5D-5L and Norwegian 15D index values with an ICC of 0.607 . The Bland\u2013Altman plot indicated that 93.3% of the points lay within the 95% limits of agreement between the Danish EQ-5D-5L and 15D (94.2% between the Hungarian EQ-5D-5L and Norwegian 15D). Differences between the EQ-5D-5L and 15D index values increased at lower mean values for both value set pairs , and the EQ-5D-5L index value and EQ VAS value (0.604), while a moderate correlation was found between the 15D index value with the EQ VAS 0.534). The\u00a0EQ-5D-5L index value demonstrated a strong correlation with its dimensions, except for self-care, where the correlation was moderate (\u2212\u00a00.482). By contrast, correlation coefficients between 15D dimensions and the EQ-5D-5L index value were ranging from \u2212\u00a00.596 to \u2212\u00a00.176 (eating). 15D index value correlated moderately or strongly with most of its dimensions, while only weakly with the eating dimension (\u2212\u00a00.346). Considering the EQ-5D-5L dimensions with the 15D index value, the strongest correlation was observed for the pain/discomfort dimension (\u2212\u00a00.629), while the weakest for self-care (\u2212\u00a00.369). The convergent validity results were confirmed by the sensitivity analysis (Table p\u2009<\u20090.001) for the physical health conditions subgroup, while reached 0.336 for the mental health subgroup. As for the corresponding dimensions, correlations between dimensions were, in general, higher in both subgroups than in the total sample and mental health condition subgroups.The subgroup analysis for the physical and mental health condition subgroups yielded similar results to those of the total sample. Lower ceiling was observed both in the mental 18.7%) and physical health conditions subgroups 25.5%) compared to the total sample (36.0%) for the EQ-5D-5L, while the floor was 0% in both subgroups. Similarly, for the 15D, the ceiling was reduced to a greater extent in the mental health condition subgroup (10.1%) than in the physical health condition subgroup (12.3%) against the total sample (21.0%) weak correlations with all EQ-5D-5L dimensions, such as vision, hearing, eating, speech, excretion, and mental function, which may indicate potential room for EQ-5D-5L bolt-ons. This is in line with earlier research that acknowledged these health areas as potentially not captured by the EQ-5D and proposed bolt-ons for these, including vision, hearing, speech, and cognition , 57\u201359. The following limitations should be considered. Firstly, due to the cross-sectional design of our study, we could not test the responsiveness or the test\u2013retest reliability of the instruments. Secondly, according to census data, 48.0% of the Hungarian general population reported having chronic illness , whereasIn conclusion, our findings may contribute to the discussion of which generic PAM to use in decision-making and provide useful and broad information for health economic evaluations. Despite having 10 fewer dimensions, the EQ-5D-5L performed better than the 15D in many aspects. However, certain 15D dimensions showed a relatively weak relationship with the dimensions of EQ-5D-5L, which signals room for potential EQ-5D-5L bolt-on dimensions. Future research is recommended to assess the added value of such bolt-on dimensions and compare their measurement properties to other PAMs that include these health areas among their dimensions . Additionally, longitudinal studies are needed to test the responsiveness of these instruments in relevant patient populations.Additional file 1. Supplementary materials."} +{"text": "EQ-5D is an instrument which has been utilized for a variety of purposes, including in health-economic appraisals as an input into quality-adjusted life year (QALY) calculations. Indeed, it is the most-widely applied instrument for health-economic appraisal worldwide, and is recommended for use in QALY calculations by many national Health Technology Assessment (HTA) agencies. There is also a growing body of evidence for its usefulness in a variety of settings other than economic appraisals, but such use has not been well-documented. This study addresses this issue and documents how EQ-5D has been applied in both the non-economic and economic contexts.The PubMed database was searched using the terms \u2018EQ-5D\u2019, \u2018EQ-5D AND cost\u2019, and \u2018EQ-5D AND cost AND QALY\u2019 from 1 January 1980 to 31 December 2019. We concentrated on 2019 publications for more detailed analyses. All the data collected for 2019 were downloaded and collected in EndNote. For 2019 only, we classified economic and non-economic use based on the inclusion of \u2018cost\u2019. We also checked by manual inspection whether the search terms were suitable in correctly identifying economic and non-economic use. Variants of the non-economic use of EQ-5D were classified as follows: (a) as a quality of life outcome measure; (b) as a tool for methodological research; (c) methodological issues of EQ-5D itself; (d) comparisons with other quality of life questionnaires; (e) mapping studies; (f) value sets; (g) alongside costs but no QALY calculated; and (h) other.The first publication found was from 1990. Up to and including 2019, 10,817 publications were identified, of which more than two in three did not contain any reference to costs or QALYs. In 2019, a total of 1409 manuscripts were identified, of which 239 were specifically for EQ-5D-5L. \u00a0Four hundred and seven (28.9%) included some form of \u2018costs\u2019 and 157 (11.1%) both \u2018costs\u2019 AND \u2018QALYs\u2019\u00a0terms. For EQ-5D-5L, the corresponding numbers were 104 (43.5%) and 29 (12.1%), respectively. After manually checking all the 1409 papers, three were duplicated records, which were omitted. In the remaining 1406 papers, only 40 (2.8%) contained the term \u2018cost\u2019, but not \u2018cost per QALY\u2019, and only 117 (8.3%) were identifiable as economic evaluations using the term \u2018cost per QALY\u2019. Most non-economic use of EQ-5D was as a quality-of-life outcome measure (72.8%). Other applications were: as a tool for methodological research (6.7%); comparison studies (3.7%); EQ-5D methodological issues (3.5%); containing costs but not QALYs (2.8%); mapping (1.3%); value sets (0.4%); and other papers (0.4%).The majority of the studies retrieved, covering a wide variety of research areas, reported upon the non-economic use of EQ-5D. Despite being the most-used instrument worldwide for QALY calculations, economic appraisal accounted for only a small, but important, part of published use. The EQ-5D instrument is a health-related quality-of-life (HRQoL) questionnaire which has been extensively used for a variety of purposes around the world , includiEQ-5D facilitates economic evaluation in the form of cost\u2013utility analysis, as it is a generic, instrument capable of deriving values (sometimes termed \u2018utilities\u2019) for health states which can then be used to estimate quality-adjusted life years (QALYs). EQ-5D has been widely adopted, and plays an important role in assessing the comparability, transparency, and consistency of economic evaluations in informing resource allocation in healthcare . In a reWhile this is evidence of the successful uptake of EQ-5D in the context of economic evaluation, there is a lack of knowledge regarding its use for a wide variety of other purposes. This study aimed to perform the first detailed estimation of the uses of EQ-5D for purposes other than economic evaluation. This paper: (i) calculates the relative use of EQ-5D in non-economic and health economic studies and (ii) describes and applies a classification system for the analysis of EQ-5D-related studies.To analyze the use of EQ-5D in both non-economic and economic publications over time, we performed a longitudinal exploration of publications mentioning EQ-5D. The search was restricted to the PubMed database. Included were both papers and letters to the editors that reported on original research. Commentaries on papers were excluded. Articles in languages other than English were excluded if the accompanying English abstract did not provide sufficient information for classification. The search terms \u2018EQ-5D\u2019, \u2018cost\u2019, and \u2018QALY\u2019 were used to obtain a picture of the number of publications that mentioned EQ-5D in relation to economic analyses. Articles published in the final year of the time frame employed were studied in detail: we manually examined the title and abstract of all publications identified in 2019, to investigate the non-economic use of EQ-5D. This was undertaken to determine the relative proportions of published papers that used EQ-5D for economic and non-economic purposes. In this way, we could validate the estimates obtained using search terms alone. In addition, we constructed a system to classify and tabulate the non-economic uses of EQ-5D.To determine the pool of relevant papers, we searched for EQ-5D and related terms: \u2018EQ-5D\u2019, \u2018EQ5D\u2019 and \u2018EuroQol\u2019. To allow for compound words, we added a \u2018wildcard\u2019 after the search terms: \u2018EQ5D*\u2019. This made the first query: (EQ-5D* OR EQ5D* OR EQ 5D* OR EuroQol*). A second query added the search terms (cost OR cost*), to find all articles that used EQ-5D with respect to costs. This was used as a broad indicator of an \u2018economic context\u2019. Hence, the query became: (EQ-5D* OR EQ5D* OR EQ 5D* OR EuroQol*) AND (cost OR cost*). For the third query, we narrowed the economic context to QALYs: (EQ-5D* OR EQ5D* OR EQ 5D* OR EuroQol*) AND (cost OR cost*) AND (QALY OR QALY*). The search words above applied to title, abstract and contents of the papers.. After collecting yearly statistics until to the end of 2019, based on these three searches, data collection was terminated on May 15, 2020. This was necessary, as changes are continuously made to the PubMed database, through the addition of new journals, old archives being added, reclassification of publication years, etc. The titles and abstracts of the 2019 publications were imported into EndNote for the manual analysis and this information is available on request.These queries produced hits from 1990 onwards, as in that year the first article describing what was then called \u2018the EuroQol instrument\u2019 was published . The terMost papers identified related to the original version of EQ-5D. In 2007, the first two papers concerning the EQ-5D-5L version were published , 7, and All the titles and abstracts for the year 2019 were checked manually. If an article included both the search terms \u2018QALY\u2019 and \u2018cost\u2019, we checked whether the article did indeed report a cost per QALY ratio, to observe whether EQ-5D was used in a cost-effectiveness context, and not just as a way of reporting a QALY independently of costs. The paper searched was excluded if it did not refer to \u2018QALY\u2019 and \u2018cost\u2019. When checking the 2019 papers, we also categorized them in terms of the nature of the application.From 1990 to 2019, 10,817 papers were found using the EQ-5D search terms: (EQ-5D* OR EQ5D* OR EQ 5D* OR EuroQol*). Figure\u00a0Similar observations can be seen in Fig.\u00a0For 2019, there were 1409 papers, of which 407 (28.9%) contained \u2018cost\u2019 terms and 157 (11.1%) contained \u2018cost\u2019 AND \u2018QALY\u2019 terms see Fig.\u00a0. For EQ-The majority of the papers identified from 2019, 1289 (91.7%), focused on the use of EQ-5D in non-economic areas. When classifying the non-economic applications, it was difficult to construct mutually exclusive categories. For example, EQ-5D was often used in conjunction with other questionnaires, which could be either for comparison or for validation purposes. We arrived at eight categories that were considered the most informative.EQ-5D used as a HRQoL outcome measure in a clinical study. This included its use as a quality of life outcome in trials (randomized and non-randomized), burden of illness studies, and other epidemiological cohort studies such as patient reported outcome (PRO) or Routine Outcome Monitoring studies..Methodological research: EQ-5D presented as simply a tool, while something else was the real matter of interest, for example to validate another questionnaire.Methodological issues for EQ-5D itself: characteristics of EQ-5D investigated as the main objective of the research, with the intention of improving the instrument.EQ-5D in comparison with other quality of life instruments: tested against other instruments in patient cohorts to test comparative psychometric properties, such as validity and reliability.Mapping: EQ-5D utilities/values mapped onto a questionnaire not designed to calculate these.EQ-5D value sets.No cost per QALY ratio calculated: only cost and/or QALY estimated.Other .The eight categories were:The classification results are shown in Table (i)Clinical trial: EQ-5D was employed as a primary or secondary outcome in an investigation, where two groups of patients were compared, and one of the groups received an experimental treatment. This trial could be randomized \u201310, or n(ii)Burden of illness study: EQ-5D was used to express the severity of the disease and/or the relationship between the severity of the disease and the different medical and background variables that were examined , 14.(iii)Cohort study: a group or different groups of patients were followed through time and their trajectories compared or related to an event that could change their quality of life, such as a transplantation or a traumatic occurrence , 16. EQ-The most common use of EQ-5D (72.8%) was as an HRQoL outcome measure in trials (randomized and non-randomized), cohorts, burden of illness, and other epidemiological studies.As a tool for methodological research EQ-5D was mainly used to estimate the validity of another HRQoL questionnaire , 21.In cost per QALY studies, EQ-5D was indeed used to elicit utilities in the estimation of QALYs \u201324.In comparison studies, the acceptability, validity, responsiveness, and other psychometric properties of EQ-5D and other instruments , 26, notWith respect to those studies which investigated methodological issues concerning EQ-5D itself, these mainly explored the direct valuation of fewer health states in a value set study , 30, andWhen EQ-5D was employed alongside cost estimates but QALYs were not calculated, it was typically used to calculate the burden in HRQoL \u201339, alonFinally, a small number of studies mapped EQ-5D onto other instruments, and a few other papers derived value sets for EQ-5D , 43. In This is the first literature review of EQ-5D placed in the context of the instrument\u2019s use in economic appraisals of health and medical activities compared with its application in a wide variety of non-economic contexts.The most common use of EQ-5D was shown to be as a HRQoL outcome measure in trials (randomized and non-randomized), cohort studies, burden of illness studies, and other epidemiological studies.If a restrictive definition, namely, cost per QALY analysis, is used to characterize economic evaluation in health, only a very small proportion of EQ-5D papers fell into the economic evaluation category: in the year 2019 only 8.3% of the papers using EQ-5D were reports of cost per QALY analyses. Applying a less restrictive definition to papers deemed to have an economic context, this ratio increased somewhat. For example, there were articles that reported the \u2018utility\u2019 value of health states that could be used in future health economic modelling [http://www.euroqol.org) states that EQ-5D can be used in clinical trials, population health surveys, routine outcome measurement, and many other types of studies, where a generic measure of health status can be usefully applied. Indeed, such a broad base of applications has been displayed in our research.The EuroQol Group website ((i)The development of a short and simple generic health status instrument which aimed to minimise the burden of both the measurement and valuation of health status led first to its adoption by medical personnel, and then its application in economic evaluation in health care, drug appraisals, and HTA.(ii)Although for some years the EuroQol Group maintained that its instrument was to be used \u2018alongside\u2019 other instruments in evaluating medical programmes, and indeed this has often been the case, as shown above, EQ-5D increasingly came to be employed as a stand-alone instrument.(iii)The business model employed by the Group generated revenue from commercial users with which to fuel further research, while allowing free use of the instrument by academics.(iv)The \u2018open access\u2019 policy, including for Group publications, pursued from the outset.(v)EQ-5D is backed by 30\u00a0years of data, evidence, publications, and researcher and user experience. This enables new data to be compared to EQ-5D population norms, EQ-5D evidence from specific disease groups, and so on.This widespread use and application of EQ-5D can be attributed to a number of factors .(i)The dMoreover, EQ-5D has demonstrated satisfactory psychometric properties in the assessment of patients, has high utility for demonstrating changes in disease activity and disability , is avaiIt is worth recalling that the first clinical application of the EuroQol instrument was in 1993 . As the This paper has demonstrated a broad range of categories of EQ-5D usage, including a significant proportion of papers which do focus on economic aspects of health and medicine, in line with one of the original aims of the Group to develop a HRQoL instrument that could be used in valuing health and health interventions for the purposes of economic appraisal. We have also shown that applications of EQ-5D range well beyond the economic, and display an impressive richness and diversity, as exemplified by the category classification. It is especially evident that the instrument is a popular choice in the measurement of HRQoL, as emphasized in the proportion of papers retrieved in our search that used EQ-5D for this purpose.(i)The search was limited in that it was restricted to papers in data base, and the in-depth study of non-economics applications was limited to the year 2019. Hence some studies may have been missed. Specifically, (1) limiting the search to PubMed could lead to systematic omissions if some journals or some non-medical studies were not indexed on the PubMed portal; (2) some unpublished economic evaluation studies could be missed; (3) earlier studies not indexed in PubMed could be omitted. With respect to point (1), undertaking a systematic review following PRISMA guidelines would be expected to offer a more comprehensive picture of EQ-5D uses/applications.(ii)Although all practicable efforts were made to ensure that the classification of the 2019 papers was valid, it is still possible that some papers were misclassified.(iii)n\u2009=\u200987) and we classified these protocol papers based on their intended use of EQ-5D. Sometimes there were multiple intentions for the use of EQ-5D, e.g., as an outcome measure to examine the effect of interventions and used to calculate health utility for economic evaluation. In such case, we categorized the use as cost per QALY.The categories adopted were assumed to be mutually exclusive, but this may not have fully matched reality: many papers could be classified as falling into two or more of the categories used here. For example, we noticed many protocol papers (Our research also has some limitations.This study demonstrates that EQ-5D has been used extensively for a wide variety of purposes, both non-economic and economic. Detailed research with respect to applications in, e.g., specific disease areas, could provide further insight into how EQ-5D has been utilized."} +{"text": "Multiple sclerosis (MS) is an inflammatory, neurodegenerative disease of the central nervous system which results in disability over time and reduced quality of life. To increase the sensitivity of the EQ-5D-5L for psychosocial health, four bolt-on items from the AQoL-8D were used to create the nine-item EQ-5D-5L-Psychosocial. We aimed to externally validate the EQ-5D-5L-Psychosocial in a large cohort of people with MS (pwMS) and explore the discriminatory power of the new instrument with EQ-5D-5L/AQoL-8D.A large representative sample from the Australian MS Longitudinal Study completed the AQoL-8D and EQ-5D-5L (including EQ VAS) and both instruments health state utilities (HSUs) were scored using Australian tariffs. Sociodemographic/clinical data were also collected. External validity of EQ-5D-5L-Psychosocial scoring algorithm was assessed with mean absolute errors (MAE) and Spearman\u2019s correlation coefficient. Discriminatory sensitivity was assessed with an examination of ceiling/floor effects, and disability severity classifications.N\u2009=\u2009157 (10%) scored perfect health (i.e. HSU\u2009=\u20091.0) on the EQ-5D-5L, but reported a mean HSU of 0.90 on the alternative instruments. The Sleep bolt-on dimension was particularly important for pwMS.Among 1683 participants , over half (55%) had moderate or severe disability. MAE (0.063) and the distribution of the prediction error were similar to the original development study. Mean (\u00b1\u2009standard deviation) HSUs were EQ-5D-5L: 0.58\u2009\u00b1\u20090.32, EQ-5D-5L-Psychosocial 0.62\u2009\u00b1\u20090.29, and AQoL-8D: 0.63\u2009\u00b1\u20090.20. The EQ-5D-5L-Psychosocial is more sensitive than the EQ-5D-5L in pwMS whose HSUs approach those reflecting full health. When respondent burden is taken into account, the EQ-5D-5L-Psychosocial is preferential to the AQoL-8D. We suggest a larger confirmatory study comparing all prevalent multi-attribute utility instruments for pwMS.The online version contains supplementary material available at 10.1007/s11136-022-03214-y. Atlas of MS estimated that from 2013 to 2020, the global prevalence of MS increased by 500,000 to 2.8 million people are combined as POMS); relapse in the past 12\u00a0months (number of relapses and current relapse); and disability severity measured by the Patient Determined Disease Steps (PDDS) .Special surveys are also disseminated to AMSLS participants every year , 22. OthTo confirm the representativeness of our study sample, we compared the characteristics of respondents with non-respondents, and the broader AMSLS cohort.Supplementary Table 1 and Supplementary Fig.\u00a01A/1B outline the descriptive systems of the EQ-5D-5L, EQ-5D-5L-Psychosocial, and AQoL-8D, including the conceptual mapping for the EQ-5D-5L-Psychosocial. Table The EQ\u20105D-5L asks participants to indicate whether they have problems on a five-level scale for each of the five dimensions of health: mobility, self\u2010care, usual activities, pain/discomfort, and anxiety/depression. The EQ-5D-5L describes 3125 health states and was developed to address the limited sensitivity (lack of descriptive richness and ceiling effects) of the EQ-5D-3L which deThe AQoL-8D was originally developed to achieve sensitivity not only in health states affected by physical disorders, but also in those affected by mental disorders . The AQoThe EQ-5D-5L-Psychosocial was developed to address the psychosocial gaps in the EQ-5D-5L by including the additional dimensions of vitality, relationships, sleep, and social isolation, which were adopted from four bolt-on questions from the AQoL-8D is the smallest difference in score in the outcome of interest that patients perceive as beneficial and would mandate a change in the patient\u2019s management , 27. A cThe primary outcome measure of this study was HRQoL, captured and assessed by the HSUs generated using Australian tariffs. A secondary measure was the EQ VAS score. In summary, we first investigated the external validity of the novel EQ-5D-5L-Psychosocial\u2019s HSUs for our AMSLS study population, compared with the study population and internal validation of the original development paper . Second,We investigated the external validity of EQ-5D-5L-Psychosocial\u2019s scoring algorithm using our AMSLS study population, as well as compared the goodness-of-fit statistics against the internal validation results reported in the original development paper . The meaSummary data describing the sociodemographic characteristics of the participants are presented as means with standard deviations (SD) for continuous variables and as percentages with frequency counts for categorical variables.Questionnaire completion was assessed with the individual responses to MAUI items (questions) using counts and proportions.Summary statistics were generated for HSUs and EQ VAS scores for the overall sample and then stratified by sociodemographic characteristics including age ; sex; Australian state or territory of usual residence, educational attainment , university (postgraduate)); MS phenotype (progressive and relapsing classifications) and disability severity , and years since diagnosis of MS to broadly reflect expected disability severity classifications since the time of diagnosis and to also provide some equivalency of groupings for further investigation of unadjusted HSUs.We also investigated the frequency distribution of the individual HSUs for each instrument including associated kurtosis.In regard to ceiling effects, we examined the counts and proportions for people who scored perfect health (HSU\u2009=\u20091.0) for the EQ-5D-5L and AQoL-8D and compared the individual HSUs and summary statistics of the HSUs generated for these participants on the EQ-5D-5L-Psychosocial, as described in our previously published work . We alsoWe adopted the same methodology for the examination of floor effects. In assuming the floor effect, we note that the algorithmic range for the Australian tariff of the EQ-5D-5L is substantially broader than the alternate instruments, being almost 0.5 utility points larger and scoring in the negative range namely \u22120.676 to 1.0 compared to EQ-5D-5L-Psychosocial (0.046\u20131.0) and AQoL-8D (0.09\u20131.0) Table . TherefoTo determine the interchangeability between the instruments, pairwise agreements between the HSUs for each instrument for each participant were assessed through the Bland\u2013Altman method of differences . In regaDisability was assessed with the PDDS, which was then mapped to the gold-standard Expanded Disability Status Scale (EDSS) for four classifications of MS-related disability severity classified as no disability (EDSS level: 0), mild disability (EDSS\u2009>\u20090\u20133.5), moderate disability (EDSS\u2009>\u20093.5\u20136), and severe disability (EDSS\u2009>\u20096\u20139.5) , 21. PDDWe assessed the sensitivity of the instruments in detecting the differences of different disability severities using a regression analysis in which a set of confounding factors (age and sex) were controlled for. To facilitate cross-instrument comparisons, the standardised coefficients are reported.All statistical analyses were conducted in STATA/SE 17.0 and R 4.0.2.n\u2009=\u20091651), EQ-5D-5L-Psychosocial (n\u2009=\u20091635), and AQoL-8D (n\u2009=\u20091630). Supplementary Table 2 provides counts for missing responses for the EQ-5D-5L and AQoL-8D .Figure\u00a0N\u2009=\u2009207 participants were reporting a current relapse event and 62.8% of the sample were people with RRMS (Table Table n\u2009=\u20091000 and n\u2009=\u20095000) with that our AMSLS study population which was 0.063. Figure\u00a0We first compared the MAE of the final mapping function from the development dataset of the new instrument of 0.058 and 0.059 generated a HSU of 1.0 . The distributions of the individual HSUs for these people with the EQ-5D-5L-Psychosocial are shown in Fig.\u00a0n\u2009=\u20097 (0.004%) reported an HSU of 1.0. This result was also mirrored for the AQoL-8D for the 1630 participants who generated a HSU: only n\u2009=\u20097 (0.004%) reported an HSU of 1.0 (Table Table n\u2009=\u2009157). This analysis revealed that for pwMS who are regarded as full health according to the EQ-5D-5L classification system for the five EQ-5D-5L items, when asked questions that directly relate to psychosocial health, the proportions of responses in levels 2 to 5 are substantial. Most importantly, these participants rated sleep as crucial to their psychosocial health , with almost 80% of these participants rating sleep quality as reduced from levels 2 to 5.Table n\u2009=\u20093 participants scored on the floor of the algorithmic range. However, 17 participants scored a HSU less than zero for the EQ-5D-5L; this is not possible for the EQ-5D-5L-Psychosocial and AQoL-8D with possible ranges of 0.046\u20131.00 and 0.09\u20131.00, respectively. Therefore, the summary HSUs for participants with severe disability were substantially lower for the severe disability category (mean 0.18) than the AQoL-8D (mean 0.50) and EQ-5D-5L-Psychosocial (mean 0.50) HSUs for this category.In regard to floor effects, for the EQ-5D-5L HSUs for health states less than \u22120.06, only Figure\u00a0In regard to the EQ-5D-5L-Psychosocial and AQoL-8D, the mean HSU difference for these instruments for the overall sample did not meet the MID for the AQoL-8D. Additionally, the Bland\u2013Altman analysis for the AQoL-8D and EQ-5D-5L-Psychosocial revealed no systematic variation and a relatively narrow limit of agreement suggesting that there was a pairwise agreement between the two instruments.Overall our results demonstrate that the EQ-5D-5L and EQ-5D-5L-Psychosocial are not interchangeable; however, the AQoL-8D and EQ-5D-5L-Psychosocial are interchangeable.Table\u00a0To our knowledge, this is the first study to validate the EQ-5-D-5L-Psychosocial in a large Australian cohort with a complex and chronic disease, namely MS. A comparison of the nine-item EQ-5D-5L-Psychosocial with its two source instruments, the five-item EQ-5D-5L and the 35-item AQoL-8D, revealed that the EQ-5D-5L-Psychosocial performed well with a reduced respondent burden compared to the AQoL-8D. We also found that the EQ-5D-5L-Psychosocial and EQ-5D-5L were not interchangeable, yet the AQoL-8D and EQ-5D-5L-Psychosocial were interchangeable. These findings suggest that the EQ-5D-5L-Psychosocial is preferential to the AQoL-8D for people living with MS when taking respondent burden into account. Finally, given its larger (and negative) algorithmic range, we also found that the EQ-5D-5L is preferentially sensitive for people with severe disability, whereas the EQ-5D-5L-Psychosocial is preferentially sensitive for pwMS with no to mild disability .Based on our results, we conclude that the original mapping algorithm developed by Chen and Olsen in 2020 is now eImportantly, our study showed that the new items for the nine-item bolt-on instrument are essential for capturing and assessing domains of health that are relevant for pwMS. Particularly, the sleep and vitality bolt-ons were important domains of health for pwMS, as over 30% of responses were at levels 4 and 5. Failure to assess sleep quality adequately may increase the risk of not fully capturing domains of HRQoL that are important when assessing pwMS. These findings align with the literature regarding fatigue for pwMS ; howeverRegarding sensitivity, we found that the EQ-5D-5L-Psychosocial revealed greater discriminatory sensitivity than the AQoL-8D or EQ-5D-5L for people with no disability to mild or moderate disability. However, comparing people with severe disability, the EQ-5D-5L-Psychosocial under-performs compared to the EQ-5D-5L. There are two potential reasons for this. First, as introduced in the methods section, in this study, the indicator used for classifying disability (both PDDS and EDSS) primarily assesses mobility and physical health, which are mainly captured by the EQ-5D-5L. Second, the greater utility range of the EQ-5D-5L is externally validated for a large MS cohort.The EQ-5D-5L-Psychosocial performed better than the EQ-5D-5L for the study population of pwMS with no disability to moderate disability. Additionally, when the respondent burden is taken into account, and given the interchangeability of the two instruments, the EQ-5D-5L-Psychosocial is preferential to the AQoL-8D for our study population of pwMS. This has implications regarding HTA guidelines that prescribe the EQ-5D-5L, particularly for disease-modifying therapies for pwMS. Future studies should consider further exploring the psychometric properties of other frequently used MAUIs such as the SF-6D for pwMS.Supplementary file1 (DOCX 412 KB)Below is the link to the electronic supplementary material."} +{"text": "The EuroQol Group has developed an extended version of the EQ-5D-Y-3L with five response levels for each of its five dimensions (EQ-5D-Y-5L). The psychometric performance has been reported in several studies for the EQ-5D-Y-3L but not for the EQ-5D-Y-5L. This study aimed to psychometrically evaluate the EQ-5D-Y-3L and EQ-5D-Y-5L Chichewa versions.The EQ-5D-Y-3L, EQ-5D-Y-5L and PedsQL\u2122 4.0 Chichewa versions were administered to children and adolescents aged 8\u201317\u00a0years in Blantyre, Malawi. Both of the EQ-5D-Y versions were evaluated for missing data, floor/ceiling effects, and validity .p\u2009>\u20090.05) with respect to gender and age, but not for school grade (p\u2009<\u20090.05). For empirical validity, the EQ-5D-Y-5L was 31\u201391% less efficient than the EQ-5D-Y-3L at detecting differences in health status using external measures.A total of 289 participants self-completed the questionnaires. There was little problem with missing data (<\u20095%) except in children aged 8\u201312\u00a0years particularly for the EQ-5D-Y-5L. Ceiling effects was generally reduced in moving from the EQ-5D-Y-3L to the EQ-5D-Y-5L. For both EQ-5D-Y-3L and EQ-5D-Y-5L, convergent validity tested with PedsQL\u2122 4.0 was found to be satisfactory (correlation\u2009\u2265\u20090.4) at scale level but mixed at dimension /sub-scale level. There was evidence of discriminant validity . The EQ-5D-Y-3L seems particularly suited for use in younger children (8\u201312\u00a0years) and the EQ-5D-Y-5L in adolescents (13\u201317\u00a0years). However, further psychometric testing is required for test re-test reliability and responsiveness that could not be carried out in this study due to COVID-19 restrictions.The online version contains supplementary material available at 10.1186/s41687-023-00560-4. The adult EQ-5D-3L, is one of the most widely used preference-based health-related quality of life (HRQoL) measures for health economic evaluations . DespiteThe youth friendly three response level, EQ-5D-Y-3L, and the experimental five response level EQ-5D-Y-5L have emerged from the adult EQ-5D versions , 7. The Neither the EQ-5D-Y-3L nor the EQ-5D-Y-5L have been psychometrically evaluated in Malawi where economic evaluation in health programs is becoming increasingly important . This stThe study recruited participants from a convenience sample of healthy and sick children (8\u201312\u00a0years) and adolescents 13\u201317\u00a0years) in urban Blantyre, Malawi. Children and adolescents attending schools and seeking any health care services through out-patient department at the Queen Elizabeth Central Hospital made up healthy and sick participants, respectively. Written assent and consent was obtained from children and their parents/guardians. For sick participants, the invitation came at the end of clinical care. For healthy participants, invitations were made through the school via a teacher. Participants took the study information leaflets and consent forms home for receipt of consent by their respective parents/guardians and these were brought back to the school the following day. For both sets of participants, once consent was obtained, the questionnaires were distributed by the research team at the end of clinical care or interviews were arranged on a school day. Once the participants completed the questionnaires , the forms were handed over and collected by the study staff. Only children who were literate (as evident from the written consenting process) and therefore able to self-complete the questionnaires were included, but the critically ill were excluded from recruitment. As previous research had revealed a tendency for respondents to avoid the middle responses when completing the adult EQ-5D-5L questionnaire if the EQ-5D-3L is administered first /ceiling/floorEQ-5D-Y-3L. It was hypothesized that the ceiling effect would be reduced both by age group and health condition when moving from the EQ-5D-Y-3L to the EQ-5D-Y-5L.The ceiling and floor effects of the EQ-5D-Y-3L and EQ-5D-Y-5L were defined as the proportion of children/adolescents scoring \u201cno problem\u201d (11111) or the \u201cmost severe problems\u201d (33333/55555) across all five dimensions, respectively. A reduction (absolute or relative) in ceiling or floor effect would suggest enhanced classification efficiency. The absolute reduction was calculated as the difference in proportion scoring 11111 or 33333/55555 from the EQ-5D-Y-3L to the EQ-5D-Y-5L. The relative reduction was calculated as and the Shannon Evenness Index (J\u2032) informativity (absolute and relative) , 36. TheConvergent validity is the extent to which similar dimensions of two or more instruments are related. It is expected that similar dimensions will have a moderate to strong correlation. It was therefore hypothesized that the EQ-5D-Y-3L and EQ-5D-Y-5L sum and utility scores would be correlated (Pearson) with PedsQL\u2122 4.0 GCS total scale scores. It was further hypothesized that for both of the EQ-5D-Y versions, the dimensions of \u201cmobility\u201d, \u201cdoing usual activities\u201d, and \u201cfeeling worried, sad or unhappy\u201d would be correlated with PedsQL\u2122 4.0 GCS physical, school, and emotional functioning scores, respectively. It was hypothesized that the PedsQL\u2122 4.0 GCS correlation would be negative with the EQ-5D-Y levels sum score (better\u2009=\u2009lower score) but positive for the EQ-5D-Y utility score (better\u2009=\u2009higher score). A correlation\u2009\u2265\u20090.4 is considered moderate to strong .Discriminant validity is the extent to which unrelated dimensions between scales should not be similar. Further, it was anticipated that age, school grade and gender would not be factors in self-completion of the EQ-5D-Y-3L and EQ-5D-Y-5L. A Pearson correlation\u2009<\u20090.2 indicates lack of correlation. It was anticipated that there would be a lack of correlation between EQ-5D-Y-3L, EQ-5D-Y-5L sum and utility scores with age. It was also hypothesised that the correlation direction for sum score and age would be negative, and positive between age and utility scores. This is because a lower value is better for sum scores and vice versa for utility scores. No association at the 5% significance level was hypothesized between both the EQ-5D-Y-3L and EQ-5D-Y-5L sum and utility scores, with gender (t-test) and grade (one-way ANOVA). School grade was dichotomised based on general distribution and in line with the former scaling for primary school education in Malawi: grades 1\u20135 made group 1, grades 6\u20138 made group 2, and secondary/high school made group 3.Known-group validity is the extent to which scores differ for two or more groups that are known to be different in some other aspects e.g., health status. It was hypothesised that for the two EQ-5D-Y versions, sum and utility scores would be worse for the sick compared to the healthy children. A t-test evaluated the relationship and the effect size was interpreted according to Cohen\u2019s criterion:\u2009<\u20090.2 poor, 0.3\u20130.49 small, 0.5\u20130.8 moderate, and\u2009>\u20090.8 large , 38.The EQ-5D-Y-3L and EQ-5D-Y-5L are preference-based instruments used not only for measuring HRQoL but also in economic evaluation. As such, the EQ-5D measures the preference placed on specific health states . It is iThe relative ability to assess external indicators of health status was investigated by comparing the utility scores with self-reported general health and the PedsQL\u2122 4.0 GCS total scale scores using the relative efficiency (RE) statistic. RE was defined as \u2018the ratio of the square of the t-statistic of the comparator instrument over the square of the t-statistic of the reference instrument\u2019 . The EQ-Self-reported general health status was dichotomised using a frequency distribution into twoAll empirical validity analyses were based on participants who completed both the EQ-5D-Y-5L and EQ-5D-Y-3L, thus any respondents with missing responses for either measure were excluded from this analysis. However, for the PedsQL\u2122 4.0 GCS, a volume of missing values of\u2009<\u200950% are taken into account as per the scoring algorithm . There iA total of 289 participants completed the EQ-5D-Y, EQ-5D-Y-5L, and PedsQL\u2122 4.0 GCS, aged 8\u201317\u00a0years as presented in Table The EQ-5D-Y-3L had missing responses in all dimensions among children compared to none among adolescents Table . For theFor the analysis based on health condition , \u201clooking after myself\u201d (88%), and \u201cdoing usual activities\u201d (82%) had consistently higher proportions of \u201cno problems\u201d among adolescents, compared to children for the EQ-5D-Y-3L. Similarly, this was evident for the EQ-5D-Y-5L \u201cmobility\u201d (81%) and \u201clooking after myself\u201d (86%) dimensions.The ceiling effect (11111) for all dimensions was generally reduced (9%) from the EQ-5D-Y-3L to EQ-5D-Y-5L for all participants (8\u201317\u00a0years) and among adolescents for \u201chaving pain or discomfort\u201d in the acute and chronically ill. Additionally, there was a 6% ceiling effect reduction for \u201cmobility\u201d and \u201cdoing usual activities\u201d among the chronically ill. Among the healthy participants, the largest ceiling effect reduction was in \u201cfeeling worried, sad or unhappy\u201d. As with age, the floor effect, reporting most severe problems across all dimensions (33333/55555) ranged between 1 and 3% among the acutely and chronically ill. There was no floor effect reduction in any dimension for healthy participants.Inconsistent responses were similar across dimensions and age groups between gender and EQ-5D-Y-3L nor EQ-5D-Y-5L sum scores or utility scores with exception of the direction of the relationship (Table There was no significant difference p\u2009>\u20090.0 between There was a low Pearson correlation (0.1\u20130.2) and thus no association between age and both the sum and utility scores for the EQ-5D-Y-3L, and EQ-5D-Y-5L. The direction of correlation was as hypothesized in adolescents but not for children. However, this correlation between age and both the sum and utility scores improved (0.2\u20130.3) and was in the hypothesized direction in all respondents.p\u2009>\u20090.05), but this was statistically significant among adolescents (p\u2009<\u20090.05), and for all respondents (p\u2009<\u20090.001).There was no evidence of difference between either EQ-5D-Y version\u2019s sum (and utility) scores and school grade categories in children , the effect size was low (0.23) for the EQ-5D-Y-5L compared to high (\u2212\u20091.15) for the EQ-5D-Y-3L. In adolescents, effect sizes were generally higher (>\u20090.5) suggesting reasonably good known-group validity and the EQ-5D-Y-5L in adolescents (13\u201317\u00a0years). Other psychometric properties like test\u2013retest reliability and responsiveness also need to be evaluated in this context.Generally, the use of childhood preference-based HRQoL measures in sub-Saharan African settings is limited, as previously reported , and so The proportion reporting \u2018no problems\u2019 was similar between the EQ-5D-Y-3L and the EQ-5D-Y-5L, with the highest proportion for \u201clooking after myself\u201d and lowest in \u201chaving pain or discomfort\u201d for both versions. This is consistent with findings from other studies with general population samples , 20, 42.The greatest proportion of inconsistencies was in the \u201chaving pain or discomfort\u201d and \u201cfeeling worried, sad or unhappy\u201d dimensions across age groups. As observed elsewhere , 20, theThe discriminative power of the EQ-5D-Y-3L was marginally higher than that of the EQ-5D-Y-5L. This may imply that the informativity of dimensions does not improve on the EQ-5D-Y-5L in this setting. This has been observed in a previous study of idiopathic scoliosis , but is The evidence for convergent validity shows that pre-specified criteria were met at scale but not at dimension level. This might imply that the EQ-5D-Y-3L and EQ-5D-Y-5L are best suited to assess physical functioning as opposed to other aspects of HRQoL. While the adult EQ-5D-5L has been found to be highly correlated with other health measures compared to the EQ-5D-3L \u201350, thisThe discriminant ability of the EQ-5D-Y-3L and EQ-5D-Y-5L as regards gender and age is consistent with the adult EQ-5D-3L and EQ-5D-5L versions , 51. TheTests of empirical validity demonstrated that the EQ-5D-Y-3L was generally more efficient than the EQ-5D-Y-5L at detecting hypothesised differences in external health status. This was surprising as the adult EQ-5D-5L has demonstrated greater relative efficiency compared to the EQ-5D-3L \u201355. Our Finally, it should be noted that there were no major differences in the psychometric tests focussed on utility values and the sum scores. The only difference was in the direction of the correlation. While the higher values were associated with better health outcomes for the utilities and vice versa for lower values, the opposite was true for the sum scores.Limitations of this study include COVID-19 restrictions that led to collection of data in one wave and therefore test\u2013retest reliability and responsiveness could not be evaluated. Secondly, preference-based value sets are not available for the EQ-5D-Y-5L and these have only recently been developed in three countries (at the time of doing this research) for the EQ-5D-Y-3L \u201358. The The two EQ-5D-Y versions established convergent and known-group validity among children and adolescents. Both versions had issues with missing values in younger children and discriminant validity by school grade as well as utilization of response options suggesting that the instruments can be used with caveats in this setting. These issues are likely not to be specific to Malawi as shown by evidence from elsewhere. Although the EQ-5D-Y-3L could be used across the age groups studied, it seems particularly suited (due to less nuanced responses) for use in younger children (8\u201312\u00a0years) whilst the EQ-5D-Y-5L seems particularly suited for use in adolescents (13\u201317\u00a0years) in Malawian contexts. Further psychometric testing for test re-test reliability and responsiveness is required, which could not be carried out in this study.Additional file 1: Table S1. Proportion of reported problems in the EQ-5D-Y-3L and the EQ-5D-Y-5L by health conditionAdditional file 2: Table S2. Redistribution of the EQ-5D-Y-3L and EQ-5D-Y-5L dimension scoresAdditional file 3: Table S3. Convergent validity of the EQ-5D-Y and EQ-5D-Y-5L with PedsQL\u2122 4.0 self-report sub-scale.Additional file 4: Table S4. EQ-5D-Y-3L and EQ-5D-Y-5L sum score known group validityAdditional file 5: Table S5. Efficiency of the EQ-5D to detect differences in self-reported health status (utility set to between 0 and 1 only for both EQ-5D-Y and EQ-5D-Y-5L)"} +{"text": "Hepatocellular carcinoma (HCC) is one of the world\u2019s malignant tumors with high morbidity and mortality. Cuproptosis is a novel form of cell death. However, the prognostic evaluation and immune relevance of cuproptosis-related genes (CRGs) in HCC are largely unknown. In our study, we constructed a prognostic model of CRGs in HCC and performed immune infiltration, functional analysis, immune checkpoint and drug sensitivity analysis. Systematically elaborated the prognostic and immune correlation of CRGs in HCC. The results showed that 15 CRGs were up-regulated or down-regulated in HCC, and the mutation frequency of CRGs reached 10.33% in HCC, with CDKN2A having the highest mutation frequency. These 19 CRGs were mainly involved in the mitochondrion, immune response and metabolic pathways. Five selected genes were involved in constructing a prognostic CRGs model that enables the overall survival in HCC patients to be predicted with moderate to high accuracy. Prognostic CRGs, especially CDKN2A, the independent factor of HCC prognosis, may be closely associated with immune-cell infiltration, tumor mutation burden (TMB), microsatellite instability(MSI), and immune checkpoints. CD274, CTLA4, LAG3, PDCD1, PDCD1LG2 and SIGLEC15 may be identified as potential therapeutic targets and CD274 correlated highly with prognostic genes. Quantitative Real-Time PCR (qRT-PCR) and immunohistochemical were performed to validate the mRNA and protein expression levels of CDKN2A in adjacent normal tissues and HCC tissues, and the results were consistent with gene difference analysis. In conclusion, CRGs, especially CDKN2A, may serve as potential prognostic predictors in HCC patients and provide novel insights into cancer therapy. Hepatocellular carcinoma (HCC), one of the most common malignant tumors of the digestive system, has the second highest mortality rate after colorectal cancer. Among new cases and deaths of HCC worldwide in 2020, 905,677 (4.7%) were newly diagnosed, and 83,0180 (8.3%) were newly diagnosed . AccordiCopper is an indispensable trace element involved in various biological processes. Recent studies have shown that copper levels in serum and tumor tissue are significantly elevated in cancer patients compared to healthy patients . It has Therefore, our study will investigate the relationship between CRGs and the prognosis and immunity of HCC, evaluate the constructed model, elucidate the importance of CRGs for HCC, and lay a certain clinical foundation for future treatment.On 15 July 2022, We downloaded the RNA sequencing (RNA-seq) data and the corresponding clinical information from 371 HCC patients using the Cancer Genome Atlas (TCGA) database, and the clinical information of HCC patients are shown in Based on previous studies , a totalUsing the \u201cmaftools\u201d package generated Mutation frequencies and oncoplot waterfall plots for 19 CRGs in HCC patients. Using the \u201cRCircos\u201d package in R, the locations of CNV alterations in 19 CRGs on 23 chromosomes were mapped.http://vip.sangerbox.com/home.html, 19 CRGs were mapped in the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for enrichment analysis in HCC.By p-values, hazard ratios (HRs), and 95% confidence intervals. Based on these prognostic CRGs, a prognostic model was constructed using LASSO Cox regression analysis. The resulting model risk score was formulated as follows: risk score = exp-gene1 * coef-gene1 + exp-gene2 * coef-gene2+ \u2026+ exp-genei * coef-genei. According to the median risk score, TCGA-HCC patients were divided into low-risk and high-risk subgroups. The Kaplan-Meier analysis compared the two subgroups\u2019 overall survival (OS) time. Predictive accuracy and risk scores were assessed for each gene by performing a temporal receiver operating characteristic (ROC) analysis. Predictive nomograms were developed to predict overall survival at 1, 3, and 5\u00a0years.A prognostic overall survival (OS) analysis was performed to identify potential prognostic characteristics. Log-rank tests were performed to calculate https://cistrome.shinyapps.io/timer/), a web portal that comprehensively analyzes tumor-infiltrating immune cells to analyze the association between prognostic CRGs and immune infiltration. TIMER\u2019s \u201cGenes\u201d module allows visualizing the correlation of gene expression with immune infiltrate levels in HCC. In the analysis of TMB and MSI, Spearman correlation analysis was used to calculate the correlation between gene expression and TMB and MSI scores, and p < 0.05 was considered statistically significant.Then, we used the Tumor Immunity Estimation Resource was added to the sample tissue according to the actual manufacturer\u2019s instructions and incubated on a shaker for 15\u00a0min at room temperature. Total RNA was subsequently extracted from target samples. One microgram of RNA was reverse transcribed into cDNA using Revert Aid First Strand cDNA Synthesis Kit . Quantitative RT-PCR was then performed with Pro Taq HS Premix Probe qPCR Kit . The GAPDH gene was used as an endogenous control gene for normalizing the expression of target genes. Primers used in this study included CDKN2A, GAPDH .The tissues were sectioned and embedded in paraffin. Sections were incubated overnight at 4\u00b0C with anti-CDKN2A antibody . Slides were washed with phosphate-buffered saline (PBS) and incubated with a goat anti-rabbit IgG secondary antibody conjugated with fluorescein isothiocyanate for 30\u00a0min with washed slides. After washing with PBS, they were incubated with an antifade reagent . Staining was visualized to determine protein expression levels using an Olympus CX41 fluorescence microscope . The analysis results were performed using ImageJ software.We first explored the different expressions of 19 CRGs in HCC and the adjacent normal tissues by the UCSC Xena. In HCC, 15 CRGs were up-regulated or down-regulated . More spThen we summarize the incidence of copy number variants and somatic mutations in 19 CRGs in HCC. Using GO and KEGG databases, pathways were analyzed to reveal the function of CRGs. We found that GO was divided into three categories: biological pathway (BP), cytological component (CC), and molecular function (MF). In the BP, these 19 CRGs are mainly involved tricarboxylic acid cycle, acetyl COA metabolic process, aerobic respiration and immune response . In the p-values for the 19 CRGs (p = 0.002), DLAT (p = 0.002), DLST (p = 0.014) and GLS (p = 0.022) and PDHA1 (p = 0.023) had better survival than the high expression group. A five-gene model was constructed according to the optimum \u03bb value obtained by LASSO Cox regression analysis (p = 0.00155), and 1, 3, and 5 years ROC curves were showed AUCs of 0.735, 0.643, and 0.631, respectively , CD8+ T cells (p = 9.56e-07), CD4+ T cells (p = 5.26e-04), neutrophils (p = 1.33e-06), macrophages (p = 6.01e-06), and medullary dendritic cells (p = 1.40e-08) (p = 6.19\u201304), CD8+ T cells (p = 1.64e-02), CD4+ T cells (p = 1.66e\u201405), neutrophils (p = 2.00e\u201413), macrophages (p = 1.03e-08), and medullary dendritic cells (p = 2.13e-07) (p = 1.91e-01), CD8+ T cells (p = 6.64e-01), CD4+ T cells (p = 1.90e-03), neutrophils (p = 5.24e-05), macrophages (p = 8.62e-02), and medullary dendritic cells (p = 1.91e-02) (p = 8.66e-05), CD8+ T cells (p = 2.90e-04), CD4+ T cells (p = 6.60e-22), neutrophils (p = 9.07e-20), macrophages (p = 6.77e-23), and medullary dendritic cells (p = 1.21e-07) (p = 9.29e-04), CD8+ T cells (p = 1.28e-03), CD4+ T cells (p = 5.01e-03), neutrophils (p = 8.72e-13), macrophages (p = 2.81e-10), and medullary dendritic cells (p = 8.07e-04) and HCC immune infiltration using the TIMER database. The results showed that CDKN2A expression was positively associated with the expression of B cells (.40e-08) . DLAT ex.13e-07) . DLST ex.91e-02) . Moreove.21e-07) . PDHA1 e.07e-04) . This stp = 2.9e-05), DLTA (p = 0.027), GLS (p = 0.0056) was significantly different at the pTNM stage in HCC. In addition, CDKN2A (p = 2e-24), DLAT (p = 8.8e-09), DLST(p = 0.00047), GLS(p = 2.5e-13) and PDHA1 (p = 8.3e-14) were significantly different at the in HCC patients with high tumor grades, low tumor grades and adjacent normal tissues.We analyzed the association of prognostic genes with tumor grade and pTNM stage. The expression of CDKN2A (p = 0.044) but no significant correlation between MSI and CDKN2A, DLAT, DLST and GLS (p = 1.64e-4) and negatively correlated with GLS (p = 7.74e-05) but not significantly associated with DLAT, DLST, PDHA1 .Drug therapy is an important means of cancer treatment. In this study, drug sensitivity analysis and immune checkpoint analysis provide a certain basis for the mechanism of drug therapy. In drug sensitivity analysis, the expression of CDKN2A, DLAT, PDHA1, and GLS was negatively associated with some or most drugs in the Cancer Therapy Response Portal database, and the expression of DLST positively correlated . HCC samqRT-PCR was performed to validate CDKN2A expression levels in HCC and adjacent normal tissues. The results showed the expression level of CDKN2A in HCC was significantly higher than that in adjacent normal tissues , which wHCC accounts for the majority of primary liver cancers. Globally, liver cancer is the fourth most common cause of cancer-related death and ranks sixth among incident cases . There hBy gene expression profiling, the results showed that ATP7A, LIAS, LIPT1, LIPT2, DLD, DLAT, PDHA1, PDHB, MTF1, GLS, CDKN2A, and DLST were up-regulated, while NLRP3, SLC31A1, and DBT were down-regulated in HCC compared to adjacent normal tissues, which is consistent with previous studies. In previous studies, CDKN2A was highly mutated and expressed in advanced HCC, which may be related to the pathogenesis of HCC , but no In GO enrichment, acetyl coenzyme A (acetyl COA) is the main raw material of the tricarboxylic acid (TCA) cycle, and CRGs are involved in the acetyl COA metabolic process, which has been demonstrated that copper directly binds to the fatty acylation component of the TCA cycle, resulting in toxic protein stress and ultimately cell death . TCA fluFurther prognostic studies showed that DLAT, PDHA1, GLS, CDKN2A, and DLST were potential prognostic biomarkers in HCC. Another study also showed that CDKN2A promoter methylation is associated with enhanced HCC risk and plays a key role in the progression of HCC ; GLS can+ T cells and CD4+ T cells. Recently, similar results have emerged in immune infiltrates concerning CRGs in other types of cancer. Previous studies have demonstrated that CDKN2A expression is positively associated with the level of infiltration of immune cells in HCC were involved in constructing a prognostic CRGs model that enables the overall survival in HCC patients to be predicted with moderate to high accuracy. Prognostic CRGs, especially CDKN2A, the independent factor of HCC prognosis, may be closely correlated with immune-cell infiltration, TMB, MSI, and immune checkpoints. The qRT-PCR and immunohistochemical staining results verified the mRNA and protein expression levels of CDKN2A in adjacent normal tissues and HCC tissues. CD274, CTLA4, LAG3, PDCD1, PDCD1LG2 and SIGLEC15 may be identified as potential therapeutic targets and CD274 correlated highly with prognostic genes. However, due to the limitations of this study, further studies should be performed to validate this finding."} +{"text": "During all the assembly stages of a polymer electrolyte membrane fuel cell (PEMFC) stack, gas diffusion layers (GDLs) endure clamping loads in the through-plane direction several times. Under such complicated assembly conditions, GDLs have to deform with the changes in structure, surface roughness, pore size, etc. A comprehensive understanding of the compressive performance of GDLs at different clamping phases is crucial to the assembly process improvement of PEMFCs. Two typical clamping compression was designed and performed to get close to the actual assembly conditions of PEMFCs. The results indicate that the initial clamping compression and the magnitude of the maximum clamping load have great impacts on the segmented compressive properties of GDLs. The nonlinear compressive performance of the GDL is mainly attributed to the unique microstructural information. The rough surface morphology contributes to the initial compressive characteristics where the big strain along with the small stress occurs, and the irreversible failures such as carbon fiber breakages and adhesive failures between fibers and binders account for the hysteresis between different compression stages. Importantly, it is found that the clamping compression hardly influences the small pore distribution below 175 \u03bcm but affects the large pore distribution over 200 \u03bcm. Polymer electrolyte membrane fuel cells (PEMFCs), as the most promising candidate of fuel cells , has beeCarbon paper-type GDLs, generally with a global thickness of 100\u2013400 \u03bcm , are widAlthough the compressive properties of GDLs have been investigated, the majority of the research focused on how the mechanical performance behaves under cyclic compression with constant loads and simplified clamping conditions. In practice, the clamping stress during all the assembly processes of a PEMFC stack is extremely complicated even with variable compression load magnitudes. Studying the mechanical properties of GDLs under the clamping conditions closed to the actual assembly procedures of a PEMFC stack is significant for the reliable stress simulation of fuel cells, which can build an accurate link between mechanical properties, and the other performance such as porosity, electrical and thermal conductivity, contact resistance, etc. The present study experimentally investigated the mechanical response of a commercial carbon paper GDL to a series of clamping compression. Particularly, two representative clamping conditions with constant maximum loads and variable maximum loads were designed and performed to stimulate the assembly processes of a PEMFC stack. Furthermore, the pristine and compressed GDLs were characterized by thickness, surface morphology, roughness, and pore size distribution. All the achieved structural information was employed to interpret the failure mechanism and reveal the effects of clamping compression on the mechanical performance of GDLs.Carbon paper-based GDLs are commonly composed of a substrate with randomly arranged carbon fibers and an MPL coated with PTFE. In general, the thickness of a substrate almost accounts for over 70% of the total thickness of GDLs. Carbon fibers are anisotropic with a longitudinal modulus of 225 GPa and a transverse modulus of 15 GPa , and theNormally, conventional assembly processes of a PEMFC stack can be summarized in three key steps, as presented in The mechanical performance changes of GDLs caused by clamping loads play an important role in influencing the overall performance of PEMFCs . With reDue to the uncertainty of clamping loads in each assembly step, this research designed two typical compression conditions to stimulate the clamping processes. In the first case, GDLs were performed cyclic compression for five cycles. In the cyclic compression, the maximum pressure was kept constant, and two maximum loads were employed. In the second case, GDLs were conducted with compression with variable loads twice. Details of the clamping compression are illustrated in In addition to clamping compression experiments, the surface morphology of GDLs was observed by a scanning electron microscope . A representative SEM image of a pristine GDL is shown in Although the SEM apparatus is good at the microstructure observation in two dimensions (or in-plane directions) in the micro scale size, it has a very limited ability to exhibit the structural characteristics of GDLs in a large scale size, particularly in the through-plane direction. With a confocal laser microscope , more morphology information of GDLs can be captured in a larger scale, especially surface roughness. The surface profile and roughness of a pristine GDL are presented in With SEM and surface roughness images, it can be intuitively observed the appearance profile and architecture of GDLs. To quantitatively characterize the effective structural characteristics of GDLs, an automatic mercury porosimeter was employed to measure the pore size distribution. The original pore size distribution of a pristine GDL is described in Compared to pristine GDLs, compressed GDLs after a series of clamping compression were also observed with SEM images and surface roughness and measured with the pore size distribution. With these elaborate structural descriptions of GDLs, it could help us further understand the structural changes caused by clamping pressure and guide us to a reasonable assembly procedure of a PEMFC stack with excellent performance.From individual elements to an overall PEMFC stack, GDLs suffer clamping compression several times due to the specific assembly procedures. Even in a running PEMFC stack, GDLs still service under a certain compressive load. This research mainly focused on investigating the mechanical performance of a carbon paper-type GDL under different clamping conditions. Two typical clamping compression with homogeneous stress was designed and performed. The key findings can be found as follows.In the current, the mechanical performance of GDLs under cyclic compression has been widely investigated ,49,50 wiBesides, the surface profile of compressed GDLs was observed and their surface roughness was also measured, as exhibited in During all the assembly processes of a PEMFC stack, the clamping loads are flexible, and they cannot keep constant in each assembly step. Besides constant maximum clamping loads, it is also very significant to investigate how the mechanical performance of GDLs behaves under variable maximum clamping loads. In the previous section, it can be found that the initial compression plays a crucial role in the determination of the mechanical performance of GDLs, and the continuous compression after two cycles makes no big difference. This section concentrated on the impacts of variable maximum clamping loads on GDLs. The dashed black line in By SEM images as shown in Besides SEM images, the surface profile of GDLs after repetitive compression with variable maximum clamping loads was observed, and their surface roughness was measured, as exhibited in It is not convincing to use the appearance architecture of GDLs to represent the overall structural characteristics of GDLs without any descriptions of their interior structure. In the current, it is almost impossible to observe the internal structure of compressed GDLs without any damage treatment due to the lack of reliable experimental methods. In this study, the pore size distribution of GDLs measured by an automatic mercury porosimeter was employed to quantitatively state the effective structural characteristics, as presented in In general, the pore size of pristine and compressed GDLs mostly concentrates on a range from 25 \u03bcm to 75 \u03bcm. Even though the clamping compression barely influences the small pore size distribution where the pore diameter is below 175 \u03bcm, it affects the large pore size distribution where the pore size diameter is over 200 \u03bcm. The pristine GDL has the most pore size diameter beyond 300 \u03bcm, compared to compressed GDLs. The fact that broken fibers and binders fall into pores and fill voids might account for this phenomenon. Notably, even though the magnitude of maximum clamping loads shows great impacts on the compressive performance and mechanical failure degrees of GDLs, it exhibits minor effects on the pore size distribution.This research focused on the impacts of clamping loads on the mechanical performance of a commercial carbon paper-type GDL. Owing to the complicated assembly procedures of a PEMFC stack, two representative clamping compression with constant and variable maximum clamping loads were designed and performed to get close to real clamping conditions that GDLs endure in fuel cells. By SEM images, surface profile, interfacial roughness, and pore size distribution of GDLs, their structures were characterized to interpret the compressive mechanism.In conclusion, the mechanical characteristics of the carbon paper GDL without an MPL behave in a nonlinear manner, and the initial clamping compression has a huge influence on its mechanical performance. Significantly, the compressive performance of GDLs is very sensitive to the magnitude of the maximum clamping load. With SEM images, it can be seen that larger clamping loads result in more serious mechanical failures such as breakages of carbon fibers and adhesive failures between binders and fibers. By surface profile, it can be concluded that the surface roughness contributes a lot to the mechanical characteristics of GDLs at the very beginning stage of applying pressure. In addition, the pore size distribution of GDLs was measured to quantitatively describe their effective structural changes. Although the clamping compression plays a decisive role in the determination of the mechanical performance of GDLs, it hardly influences the distribution of small pores with a diameter under 175 \u03bcm but affects the distribution of large pores with a diameter over 200 \u03bcm. The findings in this research could guide the assembly procedures and reliable stress simulation of PEMFCs with better performance."} +{"text": "Myxococcus xanthus is the best-studied member of the phylum Myxococcota, but the bacteriophages infecting it and their characterization remain limited. Here, we present complete genomes of Mx1, the first Myxococcus phage isolated, and of an Mx4 derivative widely used for generalized transduction, both unclassified Caudoviricetes with long, contractile tails. Myxococcus xanthus is a model organism to study bacterial multicellularity, motility, social behavior, light response, predation, and biosynthesis of lipids, steroids, and secondary metabolites (\u2013AF396866.1) and Mx9 (GenBank accession no. OQ709411.1) (OK085710.1) and high-frequency transduction mutant , and Mx4 hrm-1, a host range mutant (obtained spontaneously) that can infect wild-type M. xanthus strains and the 85710.1) . Here, w85710.1) , 15, andsduction . Mx4 ts2 strains , 18.M. xanthus strain DK1050 was determined by mapping the input reads to the final assemblies using Bowtie2 (https://www.ebi.ac.uk/interpro), HHPred , 86 located on the positive strand and 164 on the negative strand, but most CDSs (187) exhibited no homology to characterized proteins or domains, and 22 tRNA genes were annotated. The genome start point was assigned to the first of several CDSs oriented in the same direction in a region containing the putative large terminase gene. Ninety CDSs were annotated in the 56,975 bp Mx4 ts27htf-1hrm-1 genome, which is 99.96% identical to that of Mx4, but differs in 21 substitutions, 15 of which are non-synonymous; the sequenced lysate population was also heterozygous at position 39,140 . Eight variants distributed among four predicted structural genes may account for the phenotypical differences between Mx4 and its derivative. In conclusion, the Mx1 genome is larger than Mx4, has a considerably lower GC content than Mx4 or the host, and encodes 22 tRNA genes (Mx4 encodes none), suggesting a longer period of Mx4 coevolution with its bacterial host.The 169,621-bp Mx1 genome has 55.5% GC content, compared to 70.1% for the 56,975-bp Mx4"} +{"text": "Robot measurement systems with a binocular planar structured light camera (3D camera) installed on a robot end-effector are often used to measure workpieces\u2019 shapes and positions. However, the measurement accuracy is jointly influenced by the robot kinematics, camera-to-robot installation, and 3D camera measurement errors. Incomplete calibration of these errors can result in inaccurate measurements. This paper proposes a joint calibration method considering these three error types to achieve overall calibration. In this method, error models of the robot kinematics and camera-to-robot installation are formulated using Lie algebra. Then, a pillow error model is proposed for the 3D camera based on its error distribution and measurement principle. These error models are combined to construct a joint model based on homogeneous transformation. Finally, the calibration problem is transformed into a stepwise optimization problem that minimizes the sum of the relative position error between the calibrator and robot, and analytical solutions for the calibration parameters are derived. Simulation and experiment results demonstrate that the joint calibration method effectively improves the measurement accuracy, reducing the mean positioning error from over 2.5228 mm to 0.2629 mm and the mean distance error from over 0.1488 mm to 0.1232 mm. Industrial robots are preferred for their high flexibility, low cost, and wide working range, and they have started to gradually replace manual labor in many scenarios, such as welding, shot peening, and palletizing ,2,3. A 3The working process of a robot measurement system is illustrated in Some achievements have been made in the separate calibration of each error. The D-H method, screw method, and Lie algebra method are often used to model robot error ,11,12. TIt is crucial to highlight that separate error calibration is inadequate in ensuring the accuracy of the measurement system. Instead, a joint calibration method that considers robot, hand\u2013eye matrix, and camera errors is necessary. Few researchers have paid attention to this aspect. Some studies established joint error models based on the D-H method and used geometric constraints to calibrate robot and hand\u2013eye matrix errors simultaneously ,27. HoweWe thoroughly consider the advantages and limitations of previous studies and propose a joint calibration approach that takes into account three types of error. A standard sphere is used as the calibrator considering the features of the 3D camera, and the sphere center is captured as the raw data for calibration. Separate models are established for each type of error. The error models for the robot and hand\u2013eye matrix are established using Lie algebra, whereas a pillow model is created for the camera based on its error distribution and measurement principles. These error models are then integrated to construct the joint error model, which is used to establish a stepwise optimization problem for joint error calibration. Our contribution can be concluded as follows.(1) The camera error, which is proved to influence the calibration accuracy in the simulation, is taken into account during joint calibration for the first time.(2) A camera error model is established based on the measurement principle and error distribution, whereas a joint error model is established for joint calibration.(3) The calibration problem is transformed into an optimization problem that minimizes the sum of the relative position error between the calibrator and robot, avoiding reliance on external equipment.The typical robot measurement system is shown in This paper focuses on a joint calibration method for the robot, hand\u2013eye matrix, and camera errors. Separate error models and the joint error model are first proposed. Then, the calibration problem is transformed into a stepwise optimization problem, and analytical solutions for each calibration parameter are derived.The principle of the joint calibration method is depicted in ith joint coordinate system. The transformation matrix Firstly, the robot error is modeled. ) method , where iEquation .(1)v\u2227=vThe hand\u2013eye matrix Equation .(3)CET\u2032B, tilt angles Finally, the camera error is modeled. The measurement principle of the 3D camera is illustrated in studies ,22,29,30The Surface HD50 camera is used in this study, with a repeatability accuracy of \u00b10.15\u00a0mm, an optimal working distance of 500\u00a0\u00b1\u00a0250 mm, and an FOV of H55\u00b0\u00a0\u00d7\u00a0V36\u00b0. To observe the camera\u2019s error distribution, a ceramic standard sphere with a diameter of 38.1\u00a0mm and a diameter deviation of no more than 1\u00a0\u00b5m is placed within the camera\u2019s 250\u2013400\u00a0mm working distance. The fitted diameter is compared with the theoretical diameter to observe the camera\u2019s error. This process is illustrated in i, j, l, and m are positive integers. The coordinate of point S for the used camera is theoretically In Equation , \u0394x,\u0394y,\u0394The calibration value Equation , can be m sampling poses, and Equation minEDefine Equation is obtaiEquation .(10)minDefine Equation by solviR and t represent the rotation matrix and translation vector of quations\u00a0 and 13)\u2202e\u03bek\u2227Ckj\u2212Then, the hand\u2013eye matrix calibration parameter quations\u00a0\u201313)..\u03beEC is dFinally, Equation can be tEquation .(14)minEquation by solvix is calculated, and the objective function value x into Equation (L has been reached. Algorithm 1 presents the pseudo-code for the iterative process. The initial Algorithm 1: The pseudo-code of calibration processIn each iteration, the calibration parameter Equation . The iteeference . \u00a0\u00a0Algox, and calibrated values x. Finally, the performance of the joint calibration method is evaluated by comparing the calibrated values with ideal values The simulation is designed to verify the joint calibration method. Firstly, the simulation data are generated. The robot is modeled using the MDH method with parameter errors to generate Equation , is intrOne hundred training data sets and one hundred testing data sets are generated. The initial x, and the results are presented in Next, It has been observed that To illustrate the importance of joint calibration, the methods are classified into methods 1\u20134 based on whether robot, hand\u2013eye matrix, or camera errors are taken into account, as shown in The results are presented in Notably, the proposed method\u2019s optimization objective is to minimize the relative error in Equation . It doesTo investigate the impact of random noise on each method, we gradually increase the random noise by multiplying a coefficient \u201cnoise level\u201d to To investigate the impact of camera error on each method, we gradually increase the preset value of The calibration system, as shown in The initial x, and the results are presented in Next, To evaluate the performance of methods 1\u20134, distance accuracy is used instead of positioning accuracy, since Method 3 exhibits the largest The individual compensation values for the robot, hand\u2013eye matrix, and camera errors are shown in The The robot measurement system is mainly calibrated to measure workpieces precisely. To evaluate each method in practical measurements, the calibrated robot measurement system is used to measure the standard sphere, which is placed at another position, different from that during the calibration process. Multiple point clouds of the standard sphere are collected from different poses and stitched together, and the performance of each method is assessed based on the stitching quality. For better visualization, only three point clouds are stitched first, and the stitching process and results are presented in the upper parts of After that, point clouds from more sampled poses are stitched together and fitted to spheres, as shown in the bottom part of This paper proposes a joint calibration method for a robot measurement system, which considers robot kinematic, camera-to-robot installation, and 3D camera measurement errors. This method establishes separate error models for each type of error and constructs a joint error model based on homogeneous transformation. Based on the joint error model, the calibration problem is formulated as a stepwise optimization problem, and analytical solutions are derived. The superiority of the proposed method is validated through simulation and experiment. In the simulation, the proposed method can reduce the mean positioning error from over 2.5228 mm to 0.2629 mm and the mean repeatability error from over 0.1831 mm to 0.0112 mm, compared to methods without considering all error types. In addition, the anti-noise simulation results demonstrate that the proposed method can achieve reliable high-precision calibration, even in increasing random noise. In the experiment, the proposed method can reduce the mean distance error from over 0.1488 mm to 0.1232 mm and the mean repeatability error from over 0.1045 mm to 0.0957 mm compared to other methods. When applied to actual measurements, the proposed method outperforms other methods in stitching and fitting point clouds, reducing the fitted diameter error from over 0.57 mm to 0.23 mm. However, based on the experimental results, the proposed method can only partially calibrate the 3D camera measurement error. Other forms of 3D camera measurement error, except for that proposed in"} +{"text": "All cocrystals were found to exhibit an improvement in thedissolution rate of QUE. Further, the QUE plasma levels in Sprague\u2013Dawleyrats showed a 64-, 27-, 10- and 7-fold increase in oral bioavailabilityfor QUEBET\u00b7MeOH, QUEPTF, QUEPRO, and QUETHP, respectively, comparedto QUE anhydrate. We rationalize our in vivo and in vitro findingsas the result of dissolution\u2013supersaturation\u2013precipitationbehavior.Quercetin (QUE) is a widely studied nutraceutical witha numberof potential therapeutic properties. Although QUE is abundant inthe plant kingdom, its poor solubility (\u226420 \u03bcg/mL) andpoor oral bioavailability have impeded its potential utility andclinical development. In this context, cocrystallization has emergedas a useful method for improving the physicochemical properties ofbiologically active molecules. We herein report a novel cocrystalof the nutraceutical quercetin (QUE) with the coformer pentoxifylline(PTF) and a solvate of a previously reported structure between QUEand betaine (BET). We also report the outcomes of in vitro and invivo studies of QUE release and absorption from a panel of QUE cocrystals:betaine (BET), theophylline (THP), Crystal engineering can combinewestern and traditionalChinese medicine to unlock the therapeutic potential of nutraceuticals. Artemisia annua plant.2 Thisdiscovery led to subsequent changes to the standard of care for malaria.This has renewed interest in nutraceuticals as a means to uncovera new generation of active pharmaceutical ingredients (APIs) for usein drug products. Nutraceuticals have been defined as a \u201cfood(or part of food) that provides medical or health benefits, includingprevention and/or treatment of disease.\u201d3 Despite the prevalence of nutraceuticals in many plant-basedfoods,4 poor bioavailability may preventtherapeutic in vivo concentrations from being attained, limiting theirpharmacological response.In 2015, Professor Tu Youyou won the NobelPrize for her discoveryof the antimalarial drug artemisinin, traditionally extracted fromthe 5 andother potentially beneficial therapeutic effects such as anti-inflammatory,antiviral, anticarcinogenic, metal chelation, and cardioprotectiveeffects.6 The extensive work on flavonoidanalogues such as rutin (quercetin-3-O-rutinoside)8 attributes two reasons for this poor bioavailability, the rapidfirst-pass metabolism of QUE in the body and low aqueous solubility.One study reported that <1% of unchanged QUE was absorbed in thegastrointestinal (GI) tract,9 while otherstudies reported a significant decline in the pharmacological activityof the metabolites compared to its aglycon form (which confers increasedwater solubility).10 The BiopharmaceuticalClassification System (BCS)11 is a usefulguide for categorizing drug molecules based on their solubility andpermeability properties. APIs classified in this way undergo differentpreformulation screening routes in order to improve their bioavailability.With an aqueous solubility of approximately \u226420 \u03bcg/mL,QUE is practically insoluble, equilibrating in water as quercetindihydrate (QUE\u00b72H2O).12 Despite its low bioavailability, QUE is known to possess a Papp of 10\u20135 cm/s.13 Consequently, QUE has been categorized as a BCS class II14 and IV15 molecule.Quercetin (QUE), 3,3\u2032,4\u2032,5,7-pentahydroxyflavone,is a polyphenolic flavonoid nutraceutical found in various foods includingcitrus fruit, onions, and asparagus. It is widely recognized for itspotential antioxidative properties16 solvate and polymorphscreening,17 amorphous dispersions,17 and cocrystallization.18 Cocrystals are \u201csolids that are crystalline single phasematerials made up of two or more different molecular and/or ioniccompounds generally in a stoichiometric ratio that are neither solvatesnor simple salts19\u201d and have provento be a promising approach to increase the solubility and bioavailabilityof QUE.9 Some of us9 previously reported four new cocrystals of QUE, two containing caffeine(one anhydrous and one methanolate), which exhibited a significantincrease in solubility and bioavailability(>10-fold) in rats compared to QUE dihydrate. Liu et al. reporteda cocrystal of QUE with the highly soluble API, isoniazid.20 They observed that the dissolution rate of QUEincreased by 52-fold and bioavailability by 29-fold. Other notableapproaches taken to improve the solubility and bioavailability ofQUE include microemulsion formulation,21 inclusion complexes,22 and solid dispersions.23 Despite the wealth of literature on QUE formulationapproaches, there is a need to better understand the relationshipbetween in vitro and in vivo behavior for cocrystal systems.Various routine solid-form optimization strategies are employedto improve the solubility of APIs. These include salt formation (forionizable compounds),Herein, we report the synthesis and pharmacokinetic characterizationof cocrystal forms of QUE with four coformers, betaine (BET), proline(PRO), theophylline (THP), and pentoxifylline PTF, 1. The phl-proline(PRO), and all solvents and reagents were purchased from Sigma-Aldrichat the highest purity available and used as received. QUE anhydrous was purchased for dissolution and pharmacokineticstudies.QUE dihydrate was supplied by Bio-TechnologyCo., Ltd. and used for synthesis without further purification. Betaine(BET), theophylline (THP), pentoxifylline (PTF), The CambridgeStructural Database wasused to search for archived quercetin cocrystal structures. The calculatedpowder X-ray diffraction (PXRD) patterns from the database were usedto verify success in the synthesis of known cocrystal structures.2O and PTF were dissolved in 3 mL of iso-propylalcohol (IPA) by mild heating at 40 \u00b0C. Thesolution was left to slowly evaporate at room temperature. Yellowneedles of QUEPTF were harvested after 3 days. m.p.: 180 \u00b0C.These single crystals enabled the structure of QUEPTF to be determinedby SCXRD.QUE\u00b72H2O and PTF were slurried in 6 mL of IPA for24 h. The cocrystal product was filtered and air-dried. The bulk purityof the product was verified by PXRD.QUE\u00b72H2O and THP were addedto a pestle and mortar with 500 \u03bcL of IPA and ground for 10min, forming a homogenous powder. The yellow powder was analyzed byPXRD, TGA, and DSC. m.p.: 225 \u00b0C. The experimental PXRD datamatched that calculated from the reported cocrystal (REFCODE: JATPIH).QUE\u00b72H2O and THP were slurried in 6 mL of IPA for24 h. The cocrystal product was filtered and air-dried. The bulk purityof the product was tested by PXRD.QUE\u00b72HQUE\u00b7MeOH and BET were slurried in 6 mL of 1:1 MeOH/water for24 h. The cocrystal product was filtered and air-dried. The bulk purityof the product was tested by PXRD, TGA, and DSC. Single crystals ofsufficient quality could not be obtained to elucidate the structure.m.p.: 125 \u00b0C.2O and PRO were slurried in 5 mL of ethanol/waterfor 24 h. The cocrystal product was filtered and air-dried. The bulkpurity of the product was tested by PXRD and matched the calculatedPXRD of the reported cocrystal (REFCODE/EJERES). m.p. 220 \u00b0C.QUE\u00b72HK\u03b11,2 (\u03bb = 1.5418 \u00c5)). A scan speed of 0.5 s/step (6\u00b0/min)with a step size of 0.05\u00b0 in 2\u03b8 was used at room temperature.PXRD studies of microcrystallinesamples were performed in Bragg\u2013Brentano geometry on a PANalyticalEmpyrean diffractometer ,a Photon II CPAD detector, and an Oxford Cryosystem 800. Data wereintegrated with the APEX program suite and empirically corrected forabsorption correction. The structure solution was conducted throughdirect methods in OLEX2. All heavy atoms were found on the electrondensity map and refined anisotropically against all \u20131 of N2 with a heating rateof 10 \u00b0C min\u20131 for all cocrystals.DSC analyseswere carried out on a TA Instrument DSC Q20 under a sample purge of50 mL min2 with a heatingrate of 10 \u00b0C min\u20131. The balance purge was40 mL min\u20131 and the sample purge was 60 mL min\u20131 of N2.TGA curves were performedon a TA Instrument Q50 TG under the flow of NAccelerated stabilitytesting was conducted by taking 100 mg of QUEPTF, QUETHP, QUEBET,and QUEPRO and placing them in a humidity chamber at 75% relativehumidity achieved using a NaCl-saturated solution at 40 \u00b0C for14 days. The samples were analyzed before and after testing by PXRDand TGA.3) column.The wavelength was set to 370 nm with an injection volume of 20 \u03bcLand a flow rate of 1 mL/min with an oven at 40 \u00b0C. The isocraticmobile phase comprised 0.1% orthophosphoric acid/methanol (65:35 v/v).QUE concentrations in the range of 25\u2013250 \u03bcg/mL showeda good linear relationship (R2 > 0.99).Powder dissolution studieswere carried out on QUE anhydrous, QUEBET\u00b7MeOH, QUEPRO, QUETHP,and QUEPTF. 20 mg equivalent of QUE anhydrous with a particle sizebetween 75 and 100 \u03bcm (achieved by using a standard-mesh sieve)was added to 200 mL of phosphate buffer solution (PBS) 6.8 at 37 \u00b10.5 \u00b0C with stirring at 100 rpm. 1 mL of aliquots were collectedat the following time points: 1 3, 5, 10, 15, 30, 60, 120, and 240min. Each aliquot was filtered through a 0.45 \u03bcm Whatman syringefilter. 500 \u03bcL of the filtrate was diluted with 500 \u03bcLof methanol and injected into an HPLC instrument. All dissolutionexperiments for QUE anhydrous and cocrystals were carried out in triplicate.The aliquots were analyzed using a Shimadzu (LC-20A) HPLC instrumentwith a Gemini C18 and housed in a temperature- and humidity-controlled room witha 12 h light/dark cycle and free access to food and water. Rats werefasted with free access to water for 18 h before drug administration.A 0.5% sodium carboxymethyl cellulose (CMC-Na) solution was selectedas the gavage vehicle. The QUE formulations were delivered via oralgavage at a dosage of 100 mg of QUE/kg body weight. Approximately200 \u03bcL of blood was collected at the following time points:0, 5, 10, 15, 30, 45, 60, 120, 240, and 480 min. Blood samples werecentrifuged immediately and a preservative solutionat 10% (v/v) concentration was added to each separated plasma to ensurethe integrity of QUE during storage. This preservative was composedof 20% ascorbic acid and 0.1% EDTA. The samples were stored at \u221280\u00b0C until they were analyzed for their QUE content.R2 > 0.99).Xinbo PharmaceuticalResearch Co., Ltd. analyzed the plasma samples fortheir QUE content using LC with tandem mass spectrometry. The standardswere prepared as follows: A 1.00 mg/mL stock solution of QUE was accuratelyprepared in MeCN. Standards were prepared using the appropriate blankrat plasma with ascorbic acid and EDTA preservative. 50 \u03bcL ofplasma was used as the aliquot volume and then 10 \u03bcL of standard/blankwas added for all samples. Except for the double blanks, 200 \u03bcLof the internal standard (IS) spiking solution (50 ng/mL flurbiprofenin MeCN) was added to all samples. Tubes were then capped, vortexedfor 2 min, and centrifuged at 12,000 rpm for 5 min. Approximately200 \u03bcL of the supernatant was then transferred for analysis.The concentration range of the standard curve was 5\u2013500 ng/mLof QUE. The results indicated that the standard curve performancewas within the acceptable range for bioanalytical method acceptance(m/z 301.1 (parent ion) to m/z 151.1(product ion) for QUE and m/z 243.0(parent ion) to m/z 199.0 (production)for the IS flurbiprofen.Plasma analysiswasconducted using a Thermo Scientific Ultimate 3000 liquid chromatographand an Applied Biosystems Sciex Triple Quad 5500 mass spectrometerwith an electrospray ionization (ESI) source. The chromatographicseparation was performed on a Waters Symmetry Shield RP8 column and the mobile phaseconsisted of acetonitrile/water with 0.2% acetic acid .The flow rate was 0.5 mL/min and the injection volume was 1 \u03bcL.The mass spectroscopy was performed in a negative mode. The sprayvoltage was \u22124500 V, and the capillary temperature was 550\u00b0C. Multiple reaction monitoring (MRM) of MS/MS was used forspecific detection of QUE and the internal standard (IS) by measuringthe characteristic ion transitions of Cmax) andarea under the curve (AUC) were obtained graph set.24 This is highlighted in The 1:1 cocrystal QUEPTF was crystallized inthe triclinic space group b-axis bridged by these PTF H-bonds on either side . See Table S2 for the crystallographic table.This same \u2212OH group from the chromone ringsforms a supramolecularheterosynthon by simultaneously H-bonding with the CO moiety of theamide ring of PTF .The second \u2212OH of the catechol group forms a different heterosynthonwith the ketone of PTF . The QUE dimers are slipped-stacked along the her side 1b. The NFigure S1\u20134). PXRD diffractograms were compared to the calculated patterns forknown phases and to the calculated pattern from SCXRD for QUEPTF.Crystal forms were synthesizedby the slurry; successful synthesis and cocrystal purity was confirmedby PXRD, TGA, and DSC respectively occurring at 92 \u00b0C and 65 \u00b0C, respectively; all forms decomposedbeyond 250 \u00b0C. DSC confirmed that all solids employed in thisstudy contained a single crystalline phase. QUEBET\u00b7MeOH appearsto recrystallize immediately after the first melt and subsequentlymelt again at ca. 140 \u00b0C, which may indicate the recrystallizationof an anhydrous phase. QUETHP melts at 160 \u00b0C with a recrystallizationpeak at 225 \u00b0C and a melt at 230 \u00b0C, indicating the meltof QUETHP hydrate and possible recrystallization subsequent meltingof the anhydrous form.TGA and DSC were used to analyze thethermal behavior of all four cocrystals. DSC thermograms illustratethe melting points of the four cocrystals, which are presented in 2O during stability testing, which makes thermal analysis challengingfor the post-accelerated stability testing sample. Pure QUETHP appearsto lose some mass at <150 \u00b0C, which maycorrespond to adsorbed water. A second degradation peak occurs at180 \u00b0C , possibly indicating dehydration. Further,two degradation steps can be identified, but it is unclear what elementof the cocrystal is degrading, as 30% remains at > 350 \u00b0C.Thestructurally similar cocrystal, QUEPTF, has two broad, overlappingdegradation peaks with a first apparent weight loss of 40%, 2 mg from250 \u00b0C, and a second from 325 \u00b0C.Beyond an approximate numerical description,it is difficult to precisely determine what is degrading and whenbecause of the broad degradation peaks that are incomplete even upto 400 \u00b0C. QUETHP appears to convert partially to QUE\u00b72HQUEBET appears tolose some weight at temperatures around 100 \u00b0C, suggesting desolvation, and appears to degrade furtherat 280 \u00b0C, which may correspond to the loss of betaine . QUEPRO again has two degradation steps initially from 250 \u00b0C and another from 300 \u00b0C.2O, andno apparent phase change was observed for the remaining three samplestested . TGA confirmedmoisture uptake with QUETHP and no uptake with the remaining cocrystals.QUEPTF, QUETHP, QUEBET\u00b7MeOH,and QUEPRO were stored in a 75% relative humidity chamber at 40 \u00b0Cfor 14 days. PXRD and TGA data were collected on samples before andafter testing. After 14 days, the PXRD diffractograms indicated apossible phase change for QUETHP to QUE\u00b72H25 In this contribution,dissolution experiments were conducted on QUE anhydrous, QUEPTF, QUEBET\u00b7MeOH,QUEPRO, and QUETHP in triplicate. PBS pH 6.8 at 37 \u00b1 0.5 \u00b0Cwas used as the dissolution medium, and no change in medium pH wasobserved during dissolution testing. Some studies have reportedthat QUE falls below the limit of detection when performing dissolutiontests in water or PBS buffer media.26 QUE anhydroushad a maximum solubility of 40.7 \u00b1 1.7 \u03bcg/mL after 5 minbefore plateauing to 25.8 \u00b1 1.6 \u03bcg/mL, corresponding toconversion to QUE\u00b72H2O. All four cocrystals exhibitedan increase in maximum solubility in comparison to QUE anhydrous within15 min; QUETHP improved QUE solubility by 1.5-fold to 63.7 \u00b13.9 \u03bcg/mL, while QUEPRO and QUEBET\u00b7MeOH increased by over2-fold to 85.3 \u00b1 1.7 and 85.5 \u00b1 3.4 \u03bcg/mL, respectively.QUEPTF exhibited the highest maximum solubility of 192.9 \u00b1 11.9\u03bcg/mL, close to 5 times more than that of QUE dihydrate. Notably,QUEBET\u00b7MeOH exhibited a prolonged \u201cparachute\u201d effect.The residue at the end of the experiments was analyzed by PXRD toconfirm that all four cocrystals had converted to QUE dihydrate asexpected . These datasuggest that all forms generated a supersaturated solution with respectto QUE\u00b72H2O and that some cocrystal forms can sustain that supersaturation longer than others. Forexample, QUE from QUEPTF desaturated within 60 min, while QUE fromQUEBET\u00b7MeOH appears to sustain supersaturation for up to 4 h.All four cocrystals display a \u201cspring\u201deffect intheir dissolution profiles.Cmax, and Cmin togain insights into how these compounds might be expected to behavein vivo and AUC (QUEPTF> QUEBET\u00b7MeOH > QUEPRO > QUETHP > QUE).In terms of total exposure to QUE, QUEPTF enabledthe highest absorptionover the course of the experiment, with a 64-fold improvement in bioavailabilityversus the parent drug (AUC = 1705.3 \u00b1 224.0 ng.h/mL). QUEBET\u00b7MeOHrevealed the second highest AUC with 743.0 \u00b1 182.0 ng.h/mL, closeto a 28-fold increase in comparison to QUE. QUEPRO exhibited a 10-foldincrease with 262.8 \u00b1 67.6 ng.h/mL, whereas QUETHP exhibiteda 7-fold increase compared to QUE with 184.4 \u00b1 54.1 ng.h/mL.As predicted by the in vitro analysis, two distinct pharmacokineticcharacteristics are conferred by the cocrystal forms. Supersaturationleads to a rapid increase in Capp of 10\u20135 cm/s for QUE (BCS II).13 While current guidance suggests that drug bioavailability can beused as a substitute for (and is sometimes preferred to) permeabilitydue to the large inter-lab variation in traditional CaCo-2 models,27 the use of bioavailability in this context could have ruled outthe rationale for improving QUE bioavailability by enhancing aqueoussolubility. A further semantic point is that it is difficult to placenutraceutical solubility in the context of the BCS classes, as wedo not know what the therapeutically effective dose is. This suggeststhe need for a modified developability framework for nutraceuticalsor nomenclature consensus.28QUEis a poorly soluble polyphenol nutraceutical with potentialbeneficial therapeutic effects ranging from anti-inflammatory andantioxidant to anticancer properties. Access to these properties ispotentially limited by its poor solubility and bioavailability. AlthoughQUE can be classified as either a BCS class II or a BCS class IV,BCS nomenclature may be inappropriate for nutraceuticals like QUE.The literature (and our findings) suggest that QUE does not meet thethreshold of high permeability, as it has a bioavailability of \u226a85%. However, one study finds a P29 QUE is a particularly suitable candidate for crystal engineeringdue to its high propensity toward forming strong intermolecular interactionsvia H-bonds. Consequently, it is perhaps unsurprising that there are40 QUE cocrystal structures deposited on the CSD, 11 are pharmaceuticallyrelevant, as their coformers are either another APIs or a GRAS-listedcoformer. The other 29 structures were prepared to develop crystalengineering principles through hierarchical and systematic studies(Table S1).Cocrystalshave proven generally effective at modulating the physicochemicalproperties of APIs, including nutraceuticals.30 They describea \u201chover\u201d phenomenon, where their cocrystal equilibratedat a higher concentration after 72 h, instead of conventionally returningto the QUE dihydrate concentration. They attribute this to the complexationof BET and QUE in solution, which may rationalize the slower transformationof the hydrated QUEBET\u00b7MeOH cocrystal to QUE2\u00b7H2O. Further, solubility studies have been performed for this cocrystal,which found evidence of complexation. However, in their study,31 they employ just 40 mL of PBS buffer with 100mg equivalent of QUE. These equilibrium solubility measurements employlarge amounts of the excess drug but do not detail the phase, whichremains at completion. In contrast, our results do not suggest thatthe conditions enable complexation, as the QUE concentration after240 min was equivalent to that of QUEBET, suggesting complete conversionof QUEBET to QUE\u00b72H2O. Evidence of complexation wouldinclude the increased concentration of QUE compared to that of QUE\u00b72H2O (converted from QUE anhydrate) for the QUEBET dissolutiontest. QUEPRO was evaluated previously in 0.5% Tween, finding thatQUE possessed apparently superior dissolution behavior to QUEPRO.32 The contrast between those results and our ownfor this cocrystal system emphasizes the need for biorelevant conditions,as our methodology enabled us to identify that QUEPRO has advantagesover QUE , and these apparent in vitro advantagesare also found to translate to the in vivo system. QUETHP and QUETHP.0.5H2O have been evaluated by other authors in a 0.5% Tween 80medium.33 Although these authors foundthat QUETHP enhanced the dissolution rate and extent of solvationof QUE, they performed their experiments in media that contained surfactantand placed 500 mg of cocrystal into a 20 mL vessel. The conditionsemployed in these studies depart considerably from typical in vitrodissolution testing experiments, and our study represents conditionsmore closely reflecting the in vivo environment.Interestingly, QUEPTFappeared to convert back into QUE dihydratewithin 1h, while QUEBET\u00b7MeOH exhibited a \u201cparachute\u201deffect, transforming to QUE dihydrate at a slower rate. Zhang et al.reported the anhydrous form of QUEBET and conducted dissolution testsin PBS 6.8 where they also experienced a \u201cspring effect\u201din their dissolution profile.2O within around 30 min.There are two reasonable hypotheses to explain this pharmacokineticprofile in QUE cocrystals. (1) The cocrystals comprise substratesand inhibitors for a particular metabolic enzyme, which can competitivelyinhibit first-pass metabolism of each other during absorption andsubsequent metabolic passes . (2) An intuitive possibility is that the shift in the circulationlifetime for these compounds is due to the presence of undissolvedcocrystals that, upon eventual dissolution, enabled absorption; thisis supported by our in vitro solubility data. This dissolution\u2013supersaturation\u2013precipitationbehavior36 has also been observed previouslyfor meloxicam cocrystals.37 However, bothprocesses may be occurring concurrently.Of our newforms, QUEPTF can produce unpreceded in vivo exposureto quercetin in cocrystal forms to date. This pharmacokinetic behaviorcannot readily be explained by the in vitro experiments. This raisesthe following question: why are levels raised well beyond the spring-and-parachuteeffect seen in the in vitro model? Indeed, in vitro results illustratethat QUE in solution released from the cocrystal forms returns tolevels comparable to QUE2\u00b7HREL) was used to evaluatethe QUE cocrystals in this contribution to those reported in the literature.FREL relates AUC[cocrystal]/AUC[QUE]. Some of this team previously published four novel cocrystals ofQUE with isonicotinamide (INM), theobromine (QUETBR) as a dihydratecocrystal, caffeine (QUECAF), and a methanolate solvate with caffeine(QUECAF.MeOH). Pharmacokinetic data are tabulated in FREL compared to theother QUE cocrystals in the literature with FREL of 63.6and 27.7, respectively. The relative Cmax and melting points of these cocrystals were also compared againstthose in the literature. These data indicate that QUEPTF and QUEBET\u00b7MeOHalso exhibit the largest relative maximum concentration of QUE inthe literature. This is stated precariously, however, as the QUE controlin this study is lower than that in other studies.Relative bioavailability(FQUE is a polyphenol nutraceutical thatexhibits beneficial biologicalactivity but its utility as an API is hindered by low solubility andfirst-pass metabolism. Herein, we report the synthesis and characterizationof two novel QUE cocrystals, one methanolate with BET and one anhydratewith PTF. Dissolution and pharmacokinetic studies were conducted onthese novel solid forms as well as QUETHP and QUEPRO, two cocrystalspreviously reported. All four cocrystals were found to improve thesolubility and bioavailability of QUE with QUEPTF exhibiting the largestimprovement in bioavailability out of all QUE cocrystals reportedto date. Both QUEBET\u00b7MeOH and QUEPTF might be regarded as candidatesfor further development based on their in vivo and in vitro performance.This study further validates that cocrystals can modulate the physicochemicalproperties of biologically active molecules in order to achieve improvedbioavailability."} +{"text": "Information-theoretic quantities reveal dependencies among variables in the structure of joint, marginal, and conditional entropies while leaving certain fundamentally different systems indistinguishable. Furthermore, there is no consensus on the correct higher-order generalisation of mutual information (MI). In this manuscript, we show that a recently proposed model-free definition of higher-order interactions among binary variables (MFIs), such as mutual information, is a M\u00f6bius inversion on a Boolean algebra, except of surprisal instead of entropy. This provides an information-theoretic interpretation to the MFIs, and by extension to Ising interactions. We study the objects dual to mutual information and the MFIs on the order-reversed lattices. We find that dual MI is related to the previously studied differential mutual information, while dual interactions are interactions with respect to a different background state. Unlike mutual information, interactions and their duals uniquely identify all six 2-input logic gates, the dy- and triadic distributions, and different causal dynamics that are identical in terms of their Shannon information content. A\u00a0dependency, or\u00a0interaction, is called higher-order if it is inherently a property of more than two variables and\u00a0if it cannot be decomposed into pairwise quantities. The term has been used more generally to refer simply to complex interactions, as for example in nth orderIn Y asY is isolated by conditioning on all other variables being zero. This expression is well-defined, as the restriction of a derivative is the derivative of the restriction. A\u00a0pair of variables Y:n-point interactions.We start by re-defining the interactions introduced in . We defiDefinition\u00a01 (n-point interaction with respect to outcome Y).Let p be a probability distribution over a set X of random variables where additive, which they distinguish from multiplicative interactions. However, when the outcome is chosen to be the log of the joint distribution X, then the additive and multiplicative interactions are equivalent and simply related through a logarithm [This definition of interaction makes explicit the fact that interactions are defined with respect to some outcome. The authors of refer toogarithm . SettingDefinition\u00a02 (model-free n-point interaction between binary variables).A model-free n-point interaction (MFI) is an n-point interaction between binary random variables with respect to the logarithm of their joint probabilitywhere If the variables Definition\u00a03 (derivative of a function with respect to a binary variable).Let The linearity of the derivative operator then immediately and uniquely defines the higher-order\u00a0derivatives.S and\u00a0any permutation It is symmetric in terms of the variables, as Conditionally independent variables do not interact: If n, e.g., ,34.The interactions are model-free; no knowledge of the functional form of The MFIs are exactly the Ising interactions in the maximum entropy model after observing moments of the data. This can be readily verified by settingUsing this definition, the n-point interactions become model-free in the sense that they are ratios of probabilities that do not involve the functional form of the joint probability distribution. For\u00a0example, writing n-point interaction can only be non-zero if all n variables are in each other\u2019s minimal Markov blanket.An If Furthermore, in n-point interaction as a derivative of a derivative is reminiscent of Gregory Bateson\u2019s view of information as a difference which makes a difference [The definition of an fference ; howeverS, its powerset Note that all MI-based quantities can be written thusly as sums of marginal entropies of subsets of the set of variables. Given a finite set of variables n-variable Boolean algebra forms an n-cube. Furthermore, for any finite n, the n-variable Boolean algebra forms a bounded lattice, which means that it has a greatest element, denoted as least element, denoted as This poset P, we define the M\u00f6bius function On a poset incidence algebra of P. In fact, P is the Boolean algebra with This function type makes )\u2223x\u2223\u2212\u2223y\u2223 ,37. This follows ,39:(15)MDefinition\u00a04.Let P be a poset ).Let P Equation , and letThe M\u00f6bius inversion is a generalisation of the derivative to posets. If\u00a0Equation is just Equation additionX:Y, we can write this explicitly asP: SummaryMutual information is the M\u00f6bius inversion of marginal entropy.Pointwise mutual information is the M\u00f6bius inversion of marginal surprisal.Instead of starting with entropy, we could start with a quantity known as surprisal, or\u00a0self-information, defined as the negative log probability of a certain state or realisation:With mutual information defined in terms of M\u00f6bius inversions, the same can be done for the model-free interactions. Again, we start with\u00a0(negative) surprisal. However, on\u00a0Boolean variables a\u00a0state is just a partition of the variables into two sets: one in which the variables are set to 1, and\u00a0another in which they are set to 0. That means that the surprisal of observing a particular state is completely specified by which variables Definition\u00a05 (interactions as M\u00f6bius inversions).Let p be a probability distribution over a set T of random variables and let For example, when Theorem\u00a01 .The interaction Proof.\u00a0We have to show thats is some binary string; thus, we have to show that the same strings appear with the same\u00a0sign.Both sides of this equation are sums of \u00b1a and b, n Boolean variables to S of m variables becomes function composition:First, note that the Boolean algebra of sets ordered by inclusion as in is equivs contains an odd number of 0s receive a minus sign. This can be summarised asFrom this, it is clear that a term quations\u00a0 and 45)f(s) appeNote that the structure of the lattice reveals structure in the interactions, as previously noted in . On\u00a0the X and Y tend to be off . CalculS and a morphism opposite category Lattices have the property that a set with the reverse order remains a lattice; that is, if\u00a0P with X isX [X. Note that a similar construction was already anticipated in [X can be written out as follows:Let us start with mutual information. We can calculate the dual mutual information, denoted ng out X , i.e., wpated in and thatpated in . On\u00a0the X isX isX describes the effect on the mutual information from marginalising over X, whereas the dual interaction of X describes the effect on an interaction when fixingaveraged quantity and the latter a pointwise quantity.To find the dual to the interactions, we start from Equation and consZ is always 1 in the data under\u00a0consideration.Dual interactions should probably be called co-interactions; however, to avoid confusion with the term co-information, we instead refer to them simply as dual interactions. Dual interactions are interactions that are conditioned on certain variables being 1 instead of 0. This makes them no longer equal to the Ising interactions between Boolean variables; however, there are situations in which an interaction is more interesting in the context of SummaryMutual information is the M\u00f6bius inversion of marginal entropy on the lattice of subsets ordered by inclusion.Differential mutual information is the M\u00f6bius inversion of marginal entropy on the dual lattice.Model-free interactions are the M\u00f6bius inversion of surprisal on the lattice of subsets ordered by inclusion.Model-free dual interactions are the M\u00f6bius inversion of surprisal on the dual lattice.Dual interactions of a variable X are interactions between the other variables where X is set to 1 instead of 0.T and let i as a and b are independent do we have linear dependencies in P is an endomorphism of the set P, defined asTo summarise these relationships diagrammatically, note that surprisals form a vector space as follows. Let For the case where While mutual information and model-free interactions are related, there are several important differences in terms of how they capture dependencies. Note, for\u00a0example, that higher-order information quantities are not independent of the lower-order quantities. The mutual information of three variables is bounded by the pairwise quantities as follows:ximators ,43,44 anximators ,45. Ther\u00a0A\u00a0\u00a0B\u00a0\u00a0C\u00a0\u00a00\u00a0\u00a0\u00a00\u00a0\u00a0\u00a01\u00a0\u00a00\u00a0\u00a0\u00a01\u00a0\u00a0\u00a00\u00a0\u00a01\u00a0\u00a0\u00a00\u00a0\u00a0\u00a00\u00a0\u00a01\u00a0\u00a0\u00a01\u00a0\u00a0\u00a01\u00a0which describes an XNOR gate. Let I, its logical negation will have a 3-point interaction \u00a0A\u00a0\u00a0B\u00a0\u00a0C\u00a0\u00a0D\u00a0\u00a00\u00a0\u00a0\u00a00\u00a0\u00a0\u00a00\u00a0\u00a0\u00a00\u00a0\u00a00\u00a0\u00a0\u00a00\u00a0\u00a0\u00a01\u00a0\u00a0\u00a01\u00a0\u00a00\u00a0\u00a0\u00a01\u00a0\u00a0\u00a00\u00a0\u00a0\u00a01\u00a0\u00a01\u00a0\u00a0\u00a00\u00a0\u00a0\u00a00\u00a0\u00a0\u00a01\u00a0\u00a00\u00a0\u00a0\u00a01\u00a0\u00a0\u00a01\u00a0\u00a0\u00a00\u00a0\u00a01\u00a0\u00a0\u00a00\u00a0\u00a0\u00a01\u00a0\u00a0\u00a00\u00a0\u00a01\u00a0\u00a0\u00a01\u00a0\u00a0\u00a00\u00a0\u00a0\u00a00\u00a0\u00a01\u00a0\u00a0\u00a01\u00a0\u00a0\u00a01\u00a0\u00a0\u00a01\u00a0This is a three-input XOR gate, i.e., I, the three-input OR and AND gates receive a 4-point interaction Under the assumption of a causal collider structure The 3-point interactions are able to separate most two-input logic gates by sign or value, leaving only AND\u223cNOR and OR\u223cNAND. Mutual information has less resolving power. Assuming a uniform distribution over all four allowed states from a gate\u2019s truth table, a\u00a0brief calculation yieldsThe logic gate interactions and their duals are summarised in J-interaction. When the MFIs are interpreted in the context of an energy-based model, such as an Ising model or a restricted Boltzmann machine, then the interactions have dimensions of energy, meaning that the J-interactions correspond to the difference in the energy contribution between a triplet and a pair. These J-interactions of the input nodes A and B assign a different value to each logic gate J-interaction However, note that because of a difference in sign convention dual mutual information is a difference between two mutual information quantities, while dual interactions are a sum of two interactions. Based on this, we can consider the difference of two interactions and define a new quantity tas from , inheritNote that while J-interactions of the input nodes uniquely assign a value to each gate proportional to the synergy of its logic. The hierarchy is Thus, the multiplicative dynamics) or\u00a0the mean of its parent nodes (for additive dynamics), plus some zero-mean Gaussian noise with variance To illustrate how different association metrics reflect the underlying causal dynamics, consider data generated from a selection of three-node causal DAGs as follows. On a given DAG C, as on colliders A and B vanishes by\u00a0definition. data in . The mutThat the interactions have such resolving power over distributions of binary variables is perhaps not very surprising in light of the universality of RBMs with respect to this class of distributions. More surprisingly, their resolving power extends to the case of categorical variables. In , the autThe two distributions are defined on three variables, each taking a value in a four-letter alphabet The authors of found thOf particular interest here are the two quantities That is, the additively symmetrised 3-point interaction is zero for the dyadic distribution and\u00a0strongly negative for the triadic distribution. These two distributions, which are indistinguishable in terms of their information structure, are distinguishable by their model-free interactions, which accurately reflect the higher-order nature of the triadic\u00a0distribution.In this paper, we have related the model-free interactions introduced in to inforOne might wonder why the interactions enjoy this advantage over entropy-based quantities. The most obvious difference is that the interactions are defined in a pointwise way, i.e., in terms of the surprisal of particular states, whereas entropy is the expected surprisal across an ensemble of states. Furthermore, the MFIs can be interpreted as interactions in an Ising model and as effective couplings in a restricted Boltzmann machine. As both these models are known to be universal approximators with respect to positive discrete probability distributions, the MFIs should be able to characterise all such distributions. What is not immediately obvious is that the kinds of interactions that characterise a distribution should reflect properties of that distribution, such as the difference between direct and indirect effects and\u00a0the presence of higher-order structure. However, in the various examples covered in this manuscript the interactions turn out to intuitively align with properties of the process used to generate the data. While the stringent conditioning on variables not considered in the interaction might make it tempting to interpret an MFI as a causal or interventional quantity, it is important to be very careful when doing this. Assigning a causal interpretation to statistical inferences, whether in Pearl\u2019s graphical do-calculus or in RuN are rare in most datasets. Moreover, the stringency in the conditioning might worry the attentive reader. Estimating One major limitation of MFIs is that they are only defined on binary or categorical variables, whereas many other association metrics are defined for ordinal and continuous variables as well. As states of continuous variables no longer form a lattice, it is hard to see how the definition of MFIs could be extended to include these\u00a0cases.Finally, it is worth noting that the structure of different lattices has guided much of this research. That Boolean algebras are important in defining higher-order structure is not surprising, as they are the stage on which the inclusion\u2013exclusion principle can be generalised . However" \ No newline at end of file