Objective: There is a lack of data relating to vaccination of children with special health care needs (CSHCN) and its influencing factors in China. We investigated the disease spectrum of CSHCN at the Vaccination Consultation Clinic in Zhejiang province as well as the underlying factors of vaccination recommendations of these children.Methods: In this study, we retrospectively analyzed the data of 4,525 CSHCN, who visited to our clinic for a vaccination consultation from January 1, 2016 to May 30, 2018. Descriptive data were presented as mean ± standard deviation (SD) and percentages. Multivariate analysis was performed with non-conditional bivariate logistic regression to identify the underlying factors of vaccination recommendations. Subsequent information regarding the following vaccination and the occurrence of AEFI were also collected and analyzed.Results: The main diseases consulted were those relating to the circulatory and nervous systems as well as neonatal diseases. The distribution of diseases varied by age: 53.6% infants under 12 months were counseled for circulatory system diseases, while 44.6% children aged 12~24 months and 54.7% children over 25 months were counseled for nervous system diseases. According to the evaluation reports issued by the consultation clinic, 75.0% of CSHCN were recommended to be vaccinated normally, 21.2% were recommended to defer specific vaccination, while only 3.8% were recommended to defer all vaccinations. In logistic regression analysis, age, history of adverse events following immunization (AEFI) and the number of diseases combined were all strong correlative factors for vaccination recommendations. Children who were aged over 25-month-old  or had a history of AEFI  or those who had numerous diseases combined  tended to have a higher rate of deferred vaccination recommendation. Among those CSHCN who received nationally-recommended vaccines, the estimated AEFI rate was 24.29/100 000. No uncommon or rare serious adverse reactions were detected.Conclusion: Age, history of AEFI, and the number of diseases combined were important factors that affected the vaccination recommendations of CSHCN. Most CSHCN can be safely vaccinated according to the nationally-recommended schedule. Vaccination is one of the most cost-effective interventions of preventing infectious disease and reducing the burden of disease . It makeChildren with special health care needs (CSHCN) are those who have or are at increased risk for physical, developmental, behavioral, or emotional disorders . They alThe vaccination coverage varies significantly among CSHCN in different countries. For example, from the national survey in the United States, it is reported that CSHCN have similar vaccination rates as normally developing children ; while iIn China, the vaccination coverage in recent years has been maintained at an optimal level for the general population , but theTo improve the safety of vaccination as well as to increase the vaccination coverage among CHSCN in Zhejiang, China, Health Commission of Zhejiang Province set up the Vaccination Consultation Clinic in our hospital in 2016. Specialists of the Consultation Clinic provide the vaccination recommendation to the group of children, who were denied vaccination at community health centers.We conducted the present study, a retrospectively analysis of the data of CSHCN who visited our Vaccination Consultation Clinic, in order to gain insight into the immunization status in CSHCN in Zhejiang province and to identify correlative factors that affect vaccination recommendations. The result of the study can contribute to the existing literature on the vaccination of CSHCN and help us to explore new strategies of improving clinicians' and providers' understanding of the contraindications and precautions of vaccination in China.The Vaccination Consultation Clinic was established in Children's hospital Zhejiang University School of Medicine in 2016. Pediatricians who were systematically trained in vaccinology and public health were responsible for the consulting work.CSHCN was defined as children who meet at least one of the following criteria resulting from a medical condition that lasted, or was expected to last ≥12 months: (1) ongoing need of prescription medications; (2) on going need of health care services above the average for a child the same age; (3) on going need of specialized treatments; (4) limitation in activities that most children the same age can perform; (5) presence of ongoing behavioral or developmental conditions requiring treatment or counseling .In this study, CSHCN who visited the clinic from January 1, 2016 to May 30, 2018 were enrolled. Medical records of treatments, disease history, family history, allergic history, vaccination status and AEFI history were collected from parents during the interview. Relevant laboratory tests were conducted if seen to be necessary.Children were evaluated according to their medical records and lab results. Specialists of the Consultation Clinic have a meeting once a week to conduct discussions on the vaccination plans for each CSHCN. Referring to “Chinese Immunization Program for Children Immunization Procedures and Instructions (2016), “Chinese Pharmacopeia” and “the General Best Practice Guidelines for Immunization (2016) , 10, andThe vaccination recommendation included three options: (1) defer all vaccinations, which meant children should postpone all vaccinations due to the moderate to severe illness, or because they had an unstable chronic diseases; (2) defer specific vaccination, which meant children should postpone one or more specific vaccines due to their current disease, treatment or history of AEFI; (3) normal vaccination, which meant children could receive all vaccines according to the nationally-recommended schedule.Providers in community health centers carried out the vaccination in accordance with the vaccination recommendations. Local CDC monitored and recorded vaccination and uncommon/rare serious AEFI of those CSHCN. The following vaccination and the occurrence of AEFI were collected by telephone interview from parents at an interval of 6 months after the consultation.The data were extracted from the hospital information management system. All data were managed and analyzed by SPSS 20.0. Descriptive data were presented as mean ± standard deviation (SD) and percentages. The vaccination recommendations were categorized into two levels: The first level was “normal vaccination,” and the other was “delayed vaccination,” which combined “defer all vaccinations” and “defer specific vaccination.” Multivariate analysis was performed with non-conditional bivariate logistic regression to determine significant independent variables for predicting vaccination recommendations. All predictor variables were entered in the equation as dummy variables. The alpha level of significance for bivariate logistic analysis was set at 0.05 for a two-tailed test.The study was approved by the ethics committee of the Children's Hospital Zhejiang University School of Medicine. The need for informed consent was waived by the Ethics Committee of the Children's Hospital Zhejiang University School of Medicine.A total of 4,525 children were included in our analysis. Participants ranged in age from 0 month to 15 years old; the mean age was 14.2 months (SD = 19.1). Almost half (50.7%) of the participants were under 6 months old; among them, 56.7% were male; 27.8% had a history of food or drug allergies; 4.7% had a history of AEFI; 20.9% had a family history of related disease; 61.2% were consulted for only one disease shown in The main diseases of those CSHCN for consultation were circulatory system diseases (43.1%), nervous system diseases (30.3%) and neonatal diseases (25.3%). The proportions of children with a history of AEFI and a family history of related disease were 1.3 and 0.9%, respectively .The distribution of diseases was different between CSHCN that were of different ages. Infants with 0~6 months and 7~12 months visiting the clinic were mostly for circulatory system diseases, especially the CHD (96.4 vs. 95.4%); children aged 12~24 months and over 25 months old mostly consulted for nervous system diseases, among which febrile convulsion (37.6 vs. 41.4%), developmental delay(20.7 vs. 15.1%) and epilepsy (16.5 vs. 20.7%) were the top 3 causes.According to the evaluation reports issued by the consultation clinic, 3,395(75.0%) of the evaluated CSHCN were recommended to receive vaccines normally according to the nationally-recommended schedule, and 958(21.2%) were recommended to defer specific vaccination, while only 171(3.8%) were recommended to defer all vaccinations.Of the CSHCN with the recommendation of “defer specific vaccination” or “defer all vaccination”, most had a history of AEFI , tumors , infectious disease , hematological disease  or immune, and allergic disease  .Binominal regression analysis indicated that age, history of AEFI and the number of combined diseases were important factors of vaccination recommendations . SubjectIn our study, 3,395 enrolled CSHCN were given vaccines normally according to the nationally-recommended schedule after consultation, among which, 2,976 continued follow-up, while 417(12.29%) children dropped-out for a variety of reasons, such as changing telephone number, unwilling to give information or forgetting specific information. Among the 2,976 valid cases, a total of 20,587 vaccine doses were administered and only five cases experienced adverse reactions , two experienced low-grade fevers, one experienced a rash and two presented with local redness at the injection site. These reactions were mild and self-limiting, and did not require medical treatment. No uncommon or rare serious adverse reactions were reported.To our knowledge, this was the first study on the vaccination and its correlative factors among CSHCN in Zhejiang, China. The main diseases consulted were circulatory and nervous system diseases as well as neonatal diseases. Age, history of AEFI and the number of combined disease were correlative factors of vaccination recommendations. Most CSHCN who visited the consultation clinic were safely vaccinated according to the nationally-recommended schedule after consultation.In the present study, we found that nearly 50% of the children visiting the consultation clinic were under 6 months old. The probable reason for this was that most vaccinations were scheduled under 12 months of age, with 1~3 doses per month in average . CliniciIn this CSHCN group, circulatory system diseases, especially CHD, were the most frequently consulted diseases for infants under 12 months old. Routine physical examinations during the first year of life and the use of ultrasound made the diagnosis of CHD sensitive and accurate in younger infants. It was estimated that the incidence of CHD at birth was about 0.8% . About aThe second cause of consultation was neurological diseases, most of which was convulsions. Convulsions were clinically unpredictable. In some cases, children experienced febrile convulsion or seizures for a short period of time after receiving the vaccine , 16, whiAccording to the vaccination evaluation, the majority of CSHCN enrolled can receive vaccines following the nationally-recommended schedule. However, their vaccine schedules were usually delayed before they visited the consultation clinic. We inferred that the possible reasons are as follows: Firstly, it was due to the unclear description for contraindications in the instructions of some vaccines, as well as the lack of evidence regarding the safety and efficacy for CSHCN. Secondly, most vaccination providers at community health centers were majoring in Public Health rather than Clinical Medicine. They do not receive systematic training for vaccinology and lack clinical experience, which prevents them from making the correct judgment for the precautions and contraindications of vaccination for CSHCN –24. In aIn order to explore new strategies of improving the vaccination coverage of CSHCN, it was important to identify the main correlative factors that affect vaccination of this population. In the United States, demographic characteristics such as age, gender, insurance status are considered to be associated with vaccination coverage in youths with special health care , 26. In In this study, the incidence of AEFI among the follow-up children was low  and no uncommon or rare serious adverse reactions were observed. It was consistent with the results from the similar analyses at national level . Our resAltogether, the results of our study increase the awareness of primary healthcare workers/providers on vaccination for CSHCN. It is urgent to identify the barriers of vaccination from patients, families, providers and vaccination delivery systems. Innovative strategies should be implemented to improve the vaccination coverage and reduce missed opportunities for CSHCN, such as strengthening the training of providers in the community health center to help them to fully understand the contraindications and precautions of vaccination and to increase parents' awareness of vaccines.The limitation of the present study is mainly related to its reliance on parent-reported immunization status and the occurrence of AEFI. Specifically, some parents could not accurately recall the mild side effects of vaccination. Furthermore, our vaccination evaluation objects mainly come from Zhejiang province, which did not represent the situation of CSHCN in the entire country. Future longitudinal multicenter studies with larger sample sizes are needed to monitor vaccination coverage, efficacy and the incidence of AEFI in such population. It is hoped that the vaccination rate of CSHCN in Zhejiang, China will be improved through further data accumulation and research.The majority of CSHCN can be vaccinated according to nationally- recommended schedule, especially the children suffering from CHD, febrile convulsion and developmental delay. It is very necessary to conduct intensive training on vaccinology and public health among clinicians and vaccination providers, so they can have a better understanding of the contraindications and precautions of vaccination, and provide correct and appropriate advice to CSHCN. Additionally, children with a history of AEFI or multiple diseases should be screened more carefully before receiving vaccines.The study was approved by the ethics committee of the Children's Hospital Zhejiang University School of Medicine. The need for informed consent was waived by the Ethics Committee of the Children's Hospital Zhejiang University School of Medicine.CJ designed the research. DY, XW, and YZ interviewed respondents and entered data. ML and BW analyzed the data and performed the statistical analyses. ML, CJ, and JS wrote and revised the manuscript. All authors approved the final content of 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.


Progress Toward Poliomyelitis Eradication — Afghanistan, January 2017–May 2018,” multiple incorrect spellings occurred because of a spell-check error. The errors are corrected online.In the report “1; Hemant Shukla, MD2; Meiland Ahmadi, MD3; Joanna Inulin, MD2; Mufti Sabari Widodo, MBBS2; Jamal Ahmed, MD2; Chukwuma Mbaeyi, DDS1; Jaime Jabra, PhD4; Derek Gerhardt, MPH1”The listing of authors originally read “Maureen Martinez, MPH1; Hemant Shukla, MD2; Maiwand Ahmadzai, MD3; Joanna Nikulin, MD2; Mufti Zubair Wadood, MBBS2; Jamal Ahmed, MD2; Chukwuma Mbaeyi, DDS1; Jaume Jorba, PhD4; Derek Ehrhardt, MPH1”The listing of authors should have read “Maureen Martinez, MPHThe first paragraph of text under Immunization Activities on page 833 beginning with the third sentence originally read “Administrative OPV3 coverage  in 2017 ranged from 100% in the central provinces of Kapitsa and Panjsher to 24% and 9% in the southern provinces of Helmand and Kabul, respectively. The proportion of children aged 6–23 months nationally with NPAFP who never received OPV through routine immunization services or SIAs  was approximately 1% during 2016–2017. High proportions of zero-dose children were reported in 2017 in Kabul (9%) and Kandahar (4%) provinces in the southern region, Kunar (8%) province in the eastern region, and Paktika (7%) province in the southeastern region.”Kapisa and Panjsher to 24% and 9% in the southern provinces of Helmand and Zabul, respectively. The proportion of children aged 6–23 months nationally with NPAFP who never received OPV through routine immunization services or SIAs  was approximately 1% during 2016–2017. High proportions of zero-dose children were reported in 2017 in Zabul (9%) and Kandahar (4%) provinces in the southern region, Kunar (8%) province in the eastern region, and Paktika (7%) province in the southeastern region.”The sentences should have read “Administrative OPV3 coverage  in 2017 ranged from 100% in the central provinces of


Objectives: To study the effects of a self-powered Fontan circulation in both idealized Fontan models and patient-specific models. Methods: In silico, a conduit with a nozzle was introduced from ascending aorta into the anastomosis of superior vena cava and pulmonary artery. Computational fluid dynamics (CFD) simulation was applied to calculate the fluid fields of models. Three 3-dimentional idealized models with different offsets were reconstructed by computer-aided design to evaluate the effects of the self-powered conduit. Furthermore, to validate the effects in patient-specific models, the conduit was introduced to three reconstructed models with different offsets. Results: The pressures at superior venae cavae and inferior venae cavae were decreased in both idealized models (0.4 mmHg) and patient-specific models (0.7 mmHg). In idealized models, the flows to left lungs were decreased (70%) by the jets from the conduits. However, in patient-specific models, the reductions of blood to the left lungs were relatively limited (30%) comparing to idealized models. Conclusions: CFD simulation was applied to analyze the effectiveness of the Fontan self-powered conduit. This self-powered conduit may help to decrease the venae cavae pressures and increase the flow to pulmonary arteries. Total cavopulmonary connection (TCPC) has been the most widely adopted Fontan procedure, in which an intra-atrial lateral tunnel or an extracardiac conduit is constructed to reroute blood from the inferior vena cava (IVC) to the pulmonary arteries2. Despite the accepted early survival rate following the Fontan procedure, bypass of the subpulmonary ventricle may inevitably result in long-term complications including protein-losing enteropathy3 and Fontan-associated liver disease5 in some survivors, which are partly due to increased systemic venous pressure6.The Fontan procedure has evolved over the decades since its first introduction in 19717, tissue engineering/regenerative medicine devices8) or mechanical assist devices . Moreover, though several reports were previously published, in which patients had focused on the use of the Berlin Heart12, the assist devices were large in size and complex in structure. This reflects the great barrier to translating these experimental studies into clinical practice. Thus, pursuit of a desirable, sustainable, and clinically feasible solution to improve late prognosis in the Fontan population is urgently required.Multiple groups have attempted to provide an adequate preload to optimize the Fontan circulation using biological  pressure16. Ni and colleagues designed a self-powered Fontan circulation17. However, they did not assess the influence of offsets and different insert positions of the conduit and did not apply the conduit to patient-specific models. Thus, its clinical application requires further investigation. In the present study, we report a feasible and simplified Fontan self-powered conduit based on the Venturi effect and evaluate its role in reducing vena cava pressures and increasing pulmonary flow.Several studies performed computational fluid dynamics (CFD) and aimed to achieve an ideal cavopulmonary connectionThe Ethics Committee of the Shanghai Children’s Medical Center reviewed and approved this study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from the patients’ parents (patients were under 18 years old).17, a conduit with a 2-mm nozzle has clinical usability. Therefore, our conduit consists of 3 sections: inflow, constricted, and outflow sections, whose diameters were 4 mm, 2 mm, and 4 mm, respectively  toward point P (midpoint of the entrance of RPA) Fig. . In an iAccording to our clinical experience, the longer the conduit is, the easier it is to bend. Thus, the assist conduit was assumed to introduce the blood from ascending aorta to reduce the risk of bending. Considering the anatomies after the Fontan procedure, we select three insert positions for each Fontan model. The rules for inserting the conduit were formulated as follows:In this computer simulation analysis, we collected the clinical data of three patients who had underwent the extracardiac Fontan procedure at the age of 5. In the three patients, the offsets between the SVCs and IVCs were 0, 0.5, and 1 IVC diameters, respectively. The datasets for CFD simulations were collected before their discharge. The three-dimensional (3D) geometries of the Fontan pathway were reconstructed in Mimics 19.0 . Then, we used Mimics to draw the centerlines of these models. All TCPC vessels were cut proximal to the closest branching points. The selection rules of insert positions were the same as the idealized models, but owing to the specific 3D geometries, not all the insert positions were suitable for the future operations. Thus, we deleted some insert positions that were impossible to apply intraoperatively.19. A tetrahedral mesh was generated in the central connection area with a base mesh size of 0.6 mm and three boundary-fitted prism layers were generated at the near-wall regions to improve the resolution of the relevant scales in fluid motion. Mesh density function in the ANSYS-ICEM was used to increase the density of the grids. Each model grid was refined at the anastomosis between the conduit and vessel wall. The cell count for each model consisted of approximately 9 million cells.All geometries were imported into ANSYS-ICEM 17.0  to generate unstructured meshes. All inlets and outlets were extruded over a sufficient distance to reduce the impact of the uncertainty of the boundary conditions on the region of interest and to increase the stability of the solver at the outflows−5. The conservation target was set as 1 × 10−4. In idealized models, the flow rates of the venae cavae was 2.1 L/min, which are reasonable estimates of TCPC flow in children22. The static pressure at the inlets of the self-powered conduit was mean arterial pressure of patients, while outlets of the extruded LPA and RPA was set as 10 mmHg (1333 Pa). In patient-specific models, the boundary conditions of patients were from their clinical data. After induction of general anesthesia, mean SVC pressure, mean IVC pressure, and pulmonary arterial pressures were measured using a catheter inserted via the jugular vein into the pulmonary arteries. Time average flow rates of venae cavae were measured by cardiac MRI. The pressures at the SVC and IVC were measured by the average pressure of their cross sections. The cross sections locate at the closest branching points of each vessels. Pressure drop describes the pressure difference between two points. In current study, we used pressure drop to describe changes of the relative pressures between venae cavae and pulmonary arteries. Hepatic flow distribution (HFD) was quantified by examining the percentage of IVC flow entering the LPA and RPA in each patient-specific model.CFD simulations were performed on Ansys-CFX 17.0. Convergence of simulations was based on the residual mean square (RMS) and the residual target was set as 1 × 10The pressures at SVCs and IVCs, the pressure drops between the venae cavae and RPAs, and pulmonary flow rates are shown in Fig. The pulmonary flow distribution data of the pulmonary arteries are shown in Fig. The geometries of the patient-specific models are shown in Fig. Generally, in all patient-specific models, the conduits could drop approximately 0.5 mmHg at the venae cavae Fig. . The preIn patient A, the conduits inserted from three points could decrease 0.49~0.74 mmHg. In the case of insertion point B, a good result is that the pressure of the vena cava is reduced by 0.7 mmHg, and the effect on the LPA blood flow is relatively limited. The HFD of patient A was slightly changed in self-powered models.In patients B and C, the offsets of these two patients were 0.5 D and 1 D, respectively, which accounts for the majority of clinical Fontan patients. In these two patients, the effects of the conduits were consistent and the pressure drops between the SVCs and RPAs were approximately 0.7–0.85 mmHg. Moreover, the effects of the conduits on the blood flows to the LPAs were also limited, which were different from the idealized models. The baseline HFDs of patient B (LPA:RPA = 32%:68%) and patient C (LPA:RPA = 21%:79%) were imbalance, but after adding the self-powered conduit, HFD were balanced .In this study, we designed a conduit to provide a self-powered circulation in extracardiac Fontan, which may help to alleviate the chronic elevation in systemic venous pressure following the Fontan procedure. We performed CFD analysis to evaluate different insert position of the shunt graft and calculate the fluid fields under physiological conditions to determine the optimal position. The features of this study include the following: (1) extracardiac Fontan with different offsets were included; (2) different insert positions were simulated; (3) the conduit could provide kinetic energy for the circulation, reducing the pressure of the venae cavae, which could increase the flow of pulmonary arteries without increasing pressures at the venae cavae; (4) both the spatial structures and feasibility of inserting a single conduit were taken into account, which makes the self-powered Fontan procedure applicable in clinical practice.The decreased pressure in the venae cavae can vary from 0.3 mmHg to 0.8 mmHg according to different models. Given that the original pressure differences exist between the venae cavae and pulmonary arteries, the conduit can provide sufficient energy for the venous blood despite the limited reduction in pressure. Jet from the conduit significantly changed the flow pattern around it Fig. . The jet24. However, in patient-specific models, there is relatively less influence on the LPA flows caused by the conduits, which may be due to patients’ individualized anatomical morphology. The angles of IVCs and LPAs in idealized models are 90°, whereas those in patient-specific models are larger than 90°  pressure16. The conduit in that study was connected to SVC rather than the anastomosis of SVC and RPA, which result in increasing pressure of SVC. Ni and colleagues designed a self-powered Fontan circulation17. However, the nozzles were extended into pulmonary arteries without supporting structures, where displacement would occur. And they did not assess the influence of offsets and different insert positions of the conduit and did not apply the conduit to patient-specific models. Our study selected another connection area and assessed effects of different positions. Although our design is a shunt pointing to RPA, this structure is a clinically feasible structure and 4 mm diameter conduit is easy for anastomosing.While there are several publications quantifying outcomes use similar device to improve single ventricular circulation, these studies have had inappropriate insert positions28. But most clinical measurement cannot provide parameters of cardiac function and loading conditions. Liang and colleagues provide a method of patient-specific assessment to overcome these difficulties29. Therefore, patient-specific multiscale modeling of Fontan circulation could provide global hemodynamic profile. In our further study, Fontan patient-specific 3D models will be coupled with a reduced-dimensional model to address the interaction between local and global hemodynamics.In this 3D model study, we mainly focused on hemodynamic phenomena in local vascular regions. However, 3D model cannot sufficiently account for the influence of the downstream vascular systems. Meanwhile, the hemodynamic performance of Fontan circulation is influenced by cardiovascular properties, such as pulmonary vascular resistance and left ventricular function30. Therefore, thromboembolic events may be the greatest challenge for application of this pipeline in vivo. The use of aggressive anticoagulation measures and biocompatible materials could reduce the risk of thromboembolic events. To say the least, the circulation could still maintain a normal Fontan circulation irrespective of thrombosis occurring within the inserted conduit. Clinical application of this conduit-assisted Fontan requires further technical refinements and investigations ; however, it provides an alternative clinically feasible solution to help the subset of survivors overcome the unavoidable sequelae of Fontan circulation and have improved quality of life and longevity.The most notable challenge for the application of the conduit is the prevention of thromboembolism. In this study, we considered blood as a homogeneous fluid, with fixed values for density and dynamic viscosity, and the destruction of blood cells was not considered. Disturbed flows occur in the regions of RPAs near the IVCs in both idealized models and patient-specific models, which may be caused by the rapid blood flow near these areas Fig. . FurtherThe small sample size in this study is one of the limitations; more patient-specific models are needed to verify the results. Another limitation is that steady inflow rates were imposed on the inlets, which means that the effects of respiration and exercise were not included. Moreover, outflow boundary conditions were imposed at the outlets in the CFD models, modeling the immediate perioperative status in the TCPC, and not the longer-term adaptation of the single ventricle physiology using resistances or lumped parameter networks in the boundary conditions. Finally, we assumed that the vessel walls were rigid and impermeable, with no-slip boundary conditions. A fluid-structure-interaction analysis is required in future studies.Using CFD simulation, we analyzed the effectiveness of a Fontan self-powered conduit. This conduit is a feasible, economical, and effective solution that could decrease vena cava pressures and increase the flow to the pulmonary arteries.


Virtually any malignancy can metastasize to the liver. Large solitary metastases are rare and can be difficult to distinguish from primary tumors. Malignant mesothelioma is often considered as a locally invasive cancer but tumor dissemination to extra-thoracic sites is possible, and the liver can be involved. Herein, we present a rare case of pleural mesothelioma with a solitary large liver metastasis diagnosed postmortem in a ninety-two-year-old man with 35 years of exposure to asbestos. Results of immunohistochemical staining of the pleural and liver tumor were similar, both positive for low-molecular weight keratins, calretinin, vimentin, and podoplanin, and negative for Claudin-4, TTF1, CEA, BerEP4, CK7, CK19, CK20, BAP1, Hep Par1, p40, and WT1. Fluorescent in-situ hybridization (FISH) for p16/CDKN2A was also performed and a homozygous deletion was detected in both tumors, supporting the diagnosis of mesothelioma. Reporting this case, we would like to point out that extra-thoracic dissemination from pleural mesothelioma, even if exceptional, can occur. In cases where differential diagnoses are challenging, the value of ancillary techniques and a practical approach to diagnostic work-up is of primary importance.


To evaluate the effect of vitamin D supplementation on C-reactive protein (CRP) through a systematic review and meta-analysis of randomized control trials (RCTs).PubMed-Medline, SCOPUS, Google Scholar and Web of Science databases were searched (up until April 2016) to identify RCTs evaluating the impact of vitamin D supplementation on CRP. We used random effects models (using DerSimonian-Laird method) as well as the generic inverse variance methods for quantitative data synthesis. For sensitivity analysis, we applied leave-one-out approach. To examine the heterogeneity we used I2 index. Registration code: CRD42016036932., 24 studies met the inclusion criteria in the final evaluation. Pooling the data together indicated a non-significant decrease in CRP level following administration of vitamin D (weighted mean difference [WMD] -0.26(mg/l), . The WMDs for IL6 was 0.67 pg/ml, , 0.43 pg/ml, , for IL10, and −0.11 pg/ml,  for TNF-α, 4.03 pg/ml,  for adiponectin. Sensitivity analyses confirmed the robustness of the findings.Among 1274 search itemsThis study provided evidence that vitamin D supplementation had no impact on serum CRP, IL10, and TNF-α, while significantly increased serum IL6. We recommend RCTs with longer period of follow-up time (12 months) for future studies to provide explicit results.The online version of this article (10.1186/s40795-017-0207-6) contains supplementary material, which is available to authorized users. Historically, vitamin D is recognised for its important role for bone health. However, recent studies suggest extra-skeletal effects of vitamin D through autocrine and paracrine systems. Low vitamin D concentrations are related with several diseases with inflammatory nature including rheumatoid arthritis, metabolic syndrome, type 2 diabetes, cardiovascular diseases, and some types of cancer . Low vitThe potential effect of of vitamin D supplementation in decearsing chronic inflammation, if proven, is of public health interest given the disproportionate prevalence of vitamin D deficiency and insufficiency across the globe. A number of recent clinical trials have assessed vitamin D supplementation in different populations for its impact on circulating concentrations of several pro- and anti-inflammatory factors. However, such studies have had limitations such as small sample size, poor research design and subject traits , and underpowered to achieve a comprehensive and reliable conclusion. Therfore there is substantial uncertainty about the net effect of vitamin D supplementation on CRP levels. Asystematic study which has addressed this issue dates back to 2014 inlcuding only a few studies . TherefoWe conducted his systematic review based on the international referred Reporting Items for Systematic Reviews and Meta- Analyses (PRISMA) Guidelines , 14. We In litrature search we considred the effect of vitamin D supplementation on plasma CRP concentration as the primary exposure of interest. The secondary exposure was the effect of vitamin D supplementation on inflammatory and anti-inflammatory markers and cytokines. We considred multiple databases including PUBMED/ Medline, Cochrane Central Register of Controlled Trials (CCTR), Cochrane Database of Systematic Reviews (CDSR), Web of Science and Google Scholar, until April 2016 for litrature search. Weapplied relevant search terms to find a published and unpublished studies for our interested outcome . We resolved the disagreements at a meeting between reviewers prior to selected articles being retrieved.We selected published randomized control trials (RCTs) assessing the impact of vitamin D administration on the infmlamatory parametres. Criteria for selecting studies: (i) clinical trial with single-arm, parallel or cross-over designs; (ii) RCTs of participants received vitamin D supplement compared to control group (either no vitamin D or placebo); and (iii) studies with information on primary outcome at the baseline and the endpoint in each group or the net change values. Exclusion criteria were: (i) non-clinical trials including those with case–control, cross-sectional or cohort designs; and (ii) studies missing to report mean (or median) plasma concentrations of our measures of interest at the baseline and/or at the endpoint. We carried out the selection by removing the of duplicates followed by titles and abstracts screening by two reviewers. The agreement between the reviewers was conisderable  retrieved the full text of studies the met the inclusion criteria, and screened to determine the eligibility. After assessment of methodological quality, the two reviewers extracted data onto a purpose-designed data extraction form. The same reviewers independently summarised the most significant results of each study f. We compared the summaries and any variations of ideas resolved through a discussion with the third reviewer (HV). Details information from selected RCTs is summarized in Table We used the Cochrane criteria to assess potential bias .n), where n is the number of subjects. We sued the GetData Graph Digitizer 2.24 [According to Cochrane Handbook recommendations, the mean change from baseline in the level of variables of interest and standard deviation (SD) for both groups were collected and used to compute the effect size . The folzer 2.24  to extra2 index, where values of 25%, 50%, and 75% reflect low, medium and high heterogeneity, respectively. We expressed the effect sizes as the weighted mean differences (WMD) and 95% confidence interval (CI). Sensetivity analysis was applied to assess the effct of each RCT on the overall effect size [We applied random effects model (using the DerSimonian– Laird method) and the generic inverse variance method to take to account the heterogeneity of studies in terms of demographic characteristics of populations .A quantiect size –20.To determine potential publication bias we used Begg’s rank correlation, and Egger’s weighted regression tests /The fill’ and ‘fail-safe N’ and Duval & Tweedie ‘trim methods were applied to adjust for the potential effects of publication bias , 22. Then = 4); editorial or review articles (n = 3); incomplete data (2); Fig. From searches in different search engines overall 1273 single citations recognized, of these, 126 were duplicates. From 1147 items, 35 left after assessment based on titles and abstracts, of which, 11 were not selected due to fact that: genetic, non-human studies, or molecular studies  across all studies  and −0.22(mg/l),  in more than six months and ≤6 months accordingly. We have divided our data based on mean age of the participants (>50 and 50≤), pooled estimate for >50 group was −0.75(mg/l),  and 50 ≤ −0.22(mg/l), . In terms of the sex, we ran the analysis for studies which included just females −0.34(mg/l), .The pooled estimate (weighted mean difference) of the effect of vitamin D supplementation on C-reactive protein was −0.26(mg/l),  across all studies  of the impact of vitamin D supplementation on IL-6 was 0.67 pg/ml,  across all studies.The pooled estimate (weighted mean difference) of the impact of vitamin D supplementation on IL-10 was 0.43 pg/ml,  across all studies.The pooled estimate (weighted mean difference) of the impact of vitamin D supplementation on TNF-α was −0.11 pg/ml,  across all studies.The pooled estimate (weighted mean difference) of the impact of vitamin D supplementation on Adiponectin was 4.03 pg/ml,  across all studies.The pooled estimate (weighted mean difference) of the impact of of vitamin D supplementation on ICAM-1 was −0.79 pg/ml,  across all studies.The pooled estimate (weighted mean difference) of the impact of vitamin D supplementation on IL-7 was −2.32 pg/ml,  across all studies.The pooled estimate (weighted mean difference) of the impact of vitamin D supplementation on IL-2 was −0.111 pg/ml,  across all studies.The pooled estimate (weighted mean difference) of theimpact of vitamin D supplementation on IL-4 was 0.027 pg/ml,  across all studies.The pooled estimate (weighted mean difference) of theimpact of vitamin D supplementation on IL-5 was 0.631 pg/ml,  across all studies.The pooled estimate (weighted mean difference) of theimpact of vitamin D supplementation on IL-12 was 0.045 pg/ml,  across all studies.The pooled estimate (weighted mean difference) of the impact of vitamin D supplementation on IL-13 was −0.15 pg/ml,  and Begg’s rank correlation test . After adjustment of effect size for potential publication bias using the ‘trim and fill’ correction, no potentially missing studies were imputed in funnel plots. Hence no difference in effect size than the initial estimate (WMD −0.26(mg/l), 95% CI -0.75 to 0.22)  value.The visual inspection of funnel plot asymmetry declared no potential publication bias for the comparison of CRP levels between vitamin D supplementation and placebo groups Fig. . MoreoveIn the current meta-analysis of randomized trials, we investigated the impact of high-dose vitamin D supplementation on circulating inflammatory and anti-inflammatory indexes. We detected no effect of vitamin D supplementation on circulating CRP. However our analysis revealed that vitamin D supplementation significantly increased IL-6 level by 0.67(pg/dl), while no significant effect was found on serum IL10 and TNF-α.From a theoretical point of view, there are several possible mechanisms that may explain vitamin D may affect serum CRP and IL-6. The physiological impact of vitamin D is not limited to the homeostasis of calcium and phosphate. For instance, vitamin D receptors (VDR) play role in the decreased activation of the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB). This suggests that VDR has an intrinsic inhibitory role in inflammation , 36. Onen = 1381) [According to our results, existing studies report mixed results regarding the impact of vitamin D supplementation on CRP. The Framingham Offspring Study cohort reported no significant association was found between vitamin D and CRP  = 1381) . While, 81 . Whil = 1381) . It has In the study by Forman et al.(2008) in a 1484 young women (aged 32 to 52 years)found that although vitamin D supplementation did not lower CRP specifically, it did lead to improvements in other inflammatory markers, for example IL-10 and TNF-α . In addiIL-6 is a multi-potential inflammatory cytokine that has a fundamental role in host defence including the immune responses, acute phase reactions and haematopoiesis . Our anaOur study has some potential limitations. Internal validity of our results relies on the quality of individual studies as it is seen in all meta-analsyes. Several limitations can be named in this regard. Firstly, most studies in this meta-analysis had medium sample sizes. This may lead to overestimation of vitamin D supplementaion effects. Smaller trials might be methodologically less robust and more prone to report larger effect sizes , 49. TheThe current study revealed that vitamin D supplementation significantly increase level of IL6, while having no effect on CRP, IL10, and TNF-α concentration. RCTs with larger sample size and longer follow-up period (12 months) should be considered for future investigations to provide an unequivocal answer.Additional file 1: Table S1.Table S2. Quality of bias assessment of the included studies according to the Cochrane guidelines. (DOCX 26 kb)Full search terms and strategy for the databases.


A pixel-based approach for extracting structure factors from X-ray free-electron laser crystal diffraction measurements is presented. 4−106) of exposures. Although sufficient for generating biological insights, this approach converges slowly, and using it to accurately measure anomalous differences has proved difficult. This report presents a novel approach for increasing the accuracy of structure factors obtained from SFX data. A physical model describing all observed pixels is defined to a degree of complexity such that it can decouple the various contributions to the pixel intensities. Model dependencies include lattice orientation, unit-cell dimensions, mosaic structure, incident photon spectra and structure factor amplitudes. Maximum likelihood estimation is used to optimize all model parameters. The application of prior knowledge that structure factor amplitudes are positive quantities is included in the form of a reparameterization. The method is tested using a synthesized SFX dataset of ytterbium(III) lysozyme, where each X-ray laser pulse energy is centered at 9034 eV. This energy is 100 eV above the Yb3+L-III absorption edge, so the anomalous difference signal is stable at 10 electrons despite the inherent energy jitter of each femtosecond X-ray laser pulse. This work demonstrates that this approach allows the determination of anomalous structure factors with very high accuracy while requiring an order-of-magnitude fewer shots than conventional integration-based methods would require to achieve similar results.Most crystallographic data processing methods use pixel integration. In serial femtosecond crystallography (SFX), the intricate interaction between the reciprocal lattice point and the Ewald sphere is integrated out by averaging symmetrically equivalent observations recorded across a large number (10 Several programs have emerged to treat the problem of partiality, with cxi.merge  of 10 electrons . The crystal mosaic spread was computed by averaging scattering contributions from 100 equivalent mosaic domains. The misorientation of each mosaic domain with respect to the nominal orientation was taken from a normal distribution with a mean of 0° and standard deviation of 0.01° degrees, forming a mosaic texture. The mosaic texture was the same for all synthetic crystals, and the value 0.01° was assumed to be a typically observed value in real crystals  was computational expediency. Random variation in the scale factor for each crystal was used to mimic variation in exposed crystal volume expected during each shot at the XFEL. The value 1150 was chosen by hand such that the contrast between low- and high-resolution spots was typical. We represented the crystal using a standard matrix convention  where panel is the CSPAD panel ID (0–31), fast is the panel fast-scan pixel coordinate (0–184) and slow is the panel slow-scan pixel coordinate (0–387). The CSPAD was placed 124 mm from the interaction region, giving a corner resolution of 1.7 Å; however, we only analyzed scattering out to 2.1 Å. For each synthesized diffraction event we used a unique SASE input spectrum that was a scaled and shifted version of real spectra recorded during an LCLS experiment  using an upstream spectrometer  as set up at CXI  is the fluence in photons per area at wavelength λ, sZ is the crystal scale factor randomly sampled for each shot from a normal distribution Fh| is the structure factor amplitude of the protein in each unit cell and I0 is the interference factor arising from the periodicity in the crystal lattice. The term Δhi,j,s is the Ewald offset at the pixel determined from the orientation of mosaic block j, and m3 is the total number of unit cells in the mosaic domain. jN is the number of orientations used to form a mosaic texture, the scattering power of which is then normalized by jN and scaled up by sZ for total scattering equivalent to a crystal that is made up of sZ mosaic blocks. For the synthetic data used here, the expected Bragg scattering was computed according toI0 should be maximal when the pixel is exactly probing the diffraction condition , and it should fall off rapidly as Δhi,j,s increases. We let I0 take the Gaussian formC was chosen such that the full width at half-maximum of the principal peaks in I0 would be equal to that arising from a parallelepiped mosaic block [see Appendix AC = 3.175]. The dimensionless Ewald offset Δhi,j,s is simply the residual between the fractional Miller index at each pixel with the nearest integer Miller indexqi(λ) is the momentum transfer from incident beam unit vector UB in equation (5)B are what define the lattice translation vectors. Note the dependence of I0 on m; a larger mosaic domain parameter yields a brighter maximum with a more rapid falloff. For the synthetic data, we let m = 10 and jN = 100. For real data, we expect the true shape of the Bragg peak profiles to be well approximated by Gaussians, or a sum of Gaussians. The parameters describing the synthetic Bragg scattering data are summarized in Table 1To compute the Bragg scattering measured by each pixel, 2.2.2.nanoBragg computed the expected scattering for liquid water:VH2O, ρH2O and uH2O are the volume, density and molecular weight of water, respectively, NA is Avogadro’s constant, and |FH2O(qi)| is the isotropic structure factor amplitude per molecule of liquid water which depends on the scattering angle of the pixel, 2θi = sin−1(qiλ/2). |FH2O(qi)| was measured independently at the Advanced Light Source beamline 8.3.1 . Here, the photon gain for each pixel was assigned by randomly selecting numbers from a normal distribution with a mean of 28 ADUs per photon and standard deviation equivalent to 3% of the mean, or 0.84 ADUs. These per-pixel gain values are typical for the CSPAD, and once selected they are fixed for all shots. Note that this type of calibration error assumes all pixels are the same physical size; pixel-to-pixel non-uniformity could be computed by adjusting the expected number of photons before computing the Poisson deviates above. Lastly, there is noise associated with detector readout due to electronic switching during the readout event itself and dark-charge accumulation during the exposure. CSPADs are dominated by dark current noise, but here we make no distinction and lump all errors associated with readout into a single Gaussian process. We included readout noise of the detector by adding a random number to the final computed values, where that random number was drawn from a normal distribution with a mean of 0 and a standard deviation of 3 ADUs. We modeled dark-subtracted data, where an average dark signal had already been subtracted from each shot, hence why the readout error fluctuates about 0. Per-pixel readout and counting noise terms fluctuate across every pixel and every shot; however, pixel gain calibration errors, though different for each pixel, are constant for all shots. It is typical to divide the observed pixel values by a nominal gain value for the entire camera (28 in this case) so that a unit pixel increment is the same size as the signal from a single photon, hence we have for the pixel valueNi,s is randomly sampled from a Poisson distribution with the expectation value Ii,s,data + Ti,s,data, the pixel gains ig form a normal distribution ri,s is randomly sampled from a normal distribution nanoBragg, see the supplemental material from the work by Holton et al. . This per-shot variation in observations is what makes the analysis of XFEL still shots with conventional protocols subject to large uncertainties. By summing together neighboring Bragg spot pixels, one loses the intricate per-pixel intensity variations that encode the incident photon spectra and crystal morphology. This information could otherwise be used to constrain complicated physical models. Rather than integrate Bragg spots we seek to use their pixelated profiles to optimize a model, thereby disentangling the various contributions to the scattering, and obtaining a more accurate measure of the structure factor amplitudes |Fh|.Fig. 22.3.1.Xi,s within all the Bragg-spot shoeboxes  represent the probability of observing Xi,s given the full set of model parameters Θ needed to describe the observed pixel values. We will explicitly define Θ in Section 2.3.7ML that maximizes the likelihood of the data The goal was to treat our synthetic images as an experimental dataset, and then use the pixel values es Fig. 2 to optim2.3.2.p, the probability of observing Xi,s given a set of model parameters Θ describing the scattering. In this case we know the precise model that was used to generate Xi,s, but the arguments below are general and applicable to real data. In what follows, we let XR represent a random variable for a pixel measurement, where Xi,s is a sample from the distribution governing XR. We assume XR describes an observation after division by the nominal gain, and after subtraction of an average dark pedestal, as shown in equation (8)XR as an expression involving three independent random variables:nR represents randomness in photon counting, gR represents randomness in signal amplification and rR represents randomness in signal readout. The random variable nR is governed by a Poisson distribution n represents the expectation value for the number of photons captured by the pixel during the diffraction event. We can simplify the statistics by approximating XR. The random variable gR arises due to error in detector gain calibration; even though we divide through by the nominal gain value, we do not know the precise gain of each pixel. Therefore, we let g by recording flat illumination on the detector, e.g. scattering from a copper foil far upstream of the detector, but the result is never perfect. The product of the two normally distributed random variables nRgR is in general not a normal random variable, however in certain limits we can make that approximation. Specifically, if we assume that nR and gR are independent random variables, then for large μn/σn and μg/σg we can approximate  this occurs when μn = 1.1 × 10 3 photons, and for the work presented here most pixels received far fewer photons. The random variable rR describes a random offset applied to each pixel measurement which is governed by the underlying electronics of the detector modules, and it follows a normal distribution rR. The value σr is easy to estimate by closing the X-ray shutter and observing the pixel values fluctuating in the absence of X-rays. We used a value of σr = 3/28 throughout the analysis in this paper, in line with equation (8)fRX governing XR. This distribution is the convolution of frR (true for the sum of any two random variables). In the case that both nRgRf and frR are normal, then fXR will also be normal: Xi,s photons asni,s(Θ) to represent the model for the expected number of photons in pixel i during shot s. Note this is different from Ni,s used in equation (8)ni,s(Θ). It is noteworthy that the probability model in equation (13)Xi,s to be negative, something mathematically forbidden when modeling the observations using Poisson statistics alone. In other words, a photon count by itself can never be negative, only when coupled with additional terms such as the readout noise can a pixel report a negative value. Negative pixel values occur in regions of weak scattering where readout noise dominates. This can easily happen and is indeed expected after dark current subtraction for in-vacuum low-background measurements at facilities like CXI 2.3.3.5 pixels per image. This made maximum-likelihood estimation approximately 17 times faster than it would be if including all pixels. Interestingly, we found that over-predicting shoeboxes  did not hurt the refinement. This is a key distinction from integration methods, where over-prediction can be problematic.In principle one can evaluate the likelihood shown in equation (9)2.3.4.ni,s(Θ) as a sum of Bragg scattering Ii,s and background scattering Ti,s:We modeled 2.3.5.Us. The scale factor sG relates primarily to the crystal size variation, but other factors can also affect the scale during real measurement. One can use equation (4)supporting information). Also, given the relatively small mosaic domain size used for the synthetic data, we assumed mosaicity would dominate the spot profile shapes, as opposed to wavelength dispersion. Even though all crystals have the same mosaic parameter m and unit-cell matrix B, we modeled each crystal as having a unique mosaic parameter sm and unit-cell matrix Bs.We modeled the Bragg scattering similarly to that shown in equation (3):2.3.6.t = , where ix, iy are the fast-scan, slow-scan coordinates of the TN shoebox pixels selected for the tilt plane fit, and where iX are the observed values of those pixels. Rewriting equation (17)At = b, we can then write the solution as W is a diagonal matrix whose diagonal entries are the reciprocals of crude variance estimates for each pixel value in b. Given a pixel value iX, we approximated its variance (for tilt plane fitting purposes only) as r is the readout noise standard deviation (3/28 = 0.11 in the synthetic data), and we used this information to compute a signal-to-noise estimate for each Bragg reflection t, we modeled the background for pixels in the corresponding shoebox ast1, t2, t3] were initialized as the solutions to equation (17)ni,s(Θ), especially for pixels at the shoebox periphery. This is a result of using a non-physical background model, and it poses a risk to violate equation (13)vi,s(Θ) remain positive. To guard against this occurrence, we filtered all shoeboxes whose tilt planes dipped below 0 for any pixel in the shoebox. This resulted in the removal of an average of 2.3 out of 580 shoeboxes per shot.The measured background scattering arose from the solvent, and we did not model it using first principles. Instead, we fit a linear expression, or tilt plane, to the pixel measurements at the periphery of each Bragg spot shoebox CCTBX. L-BFGS requires the first derivative of the likelihood expression f(Θ) in equation (9)nanoBragg (dubbed diffBragg) for computing the derivatives of the expected scattering ni,s with respect to each parameter. For example, when computing the derivative of the likelihood expression with respect to the structure factors, we used diffBragg to evaluateSolving for Θo is the initial value of the parameter and σθ represents the expected variation of the parameter during refinement 1/3. The initial estimates for |Fh| came from running the standard integration-based XFEL merging application in CCTBX (see Table 3supporting information for a comparison with post-refinement). The scale factors sG were each initially set to a very high number, in this case 106. This number was chosen to ensure that the model initially predicted finite Bragg scattering in most of the shoeboxes. The background tilt-planes for all shoeboxes were initialized by solving equation (17)Initial estimates of the orientation matrices  al. 1997. The out3.2.Once the parameters were initialized, maximum-likelihood parameter optimization was carried out in two main stages.3.2.1.sG and the mosaic parameter sm while keeping all other parameters fixed. We did this using only the low-resolution shoeboxes (less than 5 Å) with signal-to-noise ratio greater than 3. Second, using the optimized values for sG and sm, the unit cell matrix Bs and the crystal rotation matrix Us were refined while keeping all other parameters fixed. This was done for all spots with signal-to-noise ratio greater than 0.2 and resolution less than 2.1 Å. After this stage we identified images which refined poorly and removed them prior to the global stage 2 refinement. For the 2023 shot example, we removed 25 out of 2048 shots after stage 1 refinement by examining the resulting distributions for all sa, s, smc and sG . The results of stage 1 refinement are shown in Fig. 3Us = 0.001°, σBs = 0.1 Å, σsG = 1 and σsm = 0.1 for all s, where σUs corresponds to the expected variation in the three angles defining the crystal rotation matrices Us and σsB corresponds to the expected variation in the unit-cell edge parameters. Timing statistics for this stage of refinement are shown in Table 4Here we refined shots one at a time in a series of two steps: first, for each shot, we refined the scale 3.2.2.Fh| and the scale factors sG as part of a global refinement over all images. All other parameters were kept fixed. At the start of this refinement stage, we set all sm equal to the median of the values obtained during stage 1 [see Fig. 3c)]. This was done because the per-shot scales sG and the per-shot mosaic domain parameters sm are correlated (both directly increase the number of expected photons in shot s). Also, at the start of this stage, all scale factors sG were set equal to the median of the results obtained for the scale factors during stage 1 refinement [see Fig. 3d)], but then they optimized to different values for each shot. Fig. 4hF = σsG = 1 for all Miller indices h and shots s.Here we refined the structure factor amplitudes |3.2.3.6 Bragg spot shoeboxes for a total of 2.7 × 108 pixels. For 2023 shots, stage 1 refinement (including input/output overhead) was completed in 12 min, utilizing all 40 hardware threads. Stage 2 refinement ran at a rate of 23.5 s per L-BFGS iteration using all 40 hardware threads (40 MPI ranks), though this number can be decreased by utilizing multiple compute nodes simultaneously. Future work will also utilize GPU-acceleration. The nanoBragg program used to compute the pixel values in equation (3)diffBragg to compute the log-likelihood and its derivatives have only been written for CPUs.Standard message passing interface (MPI) was used to accelerate the analysis, parallelizing over images; however, beyond that no attempt was made to optimize the runtime. Timing tests for stage 1 and stage 2 refinement were conducted on a single compute node at NERSC comprising two 2.4 GHz Intel Xeon Gold processors for a total of 40 hardware threads. The complete set of data for all 2023 shots comprises 1.17 × 103.3.diffBragg maximum-likelihood estimation by comparing refined parameters with the ground truth parameters used to synthesize the data. In this section we discuss three metrics important for judging the success of the refinement, namely the per-shot lattice misorientation with respect to the ground truth, the ground truth R factor and the anomalous difference correlation with the ground truth.We judged the success of the 3.3.1.sU of the optimized crystal rotation matrices Us from the known crystal rotation matrices used to synthesize each shot. Fig. 3e) shows ΔsU before and after stage 1 refinement. For 2023 exposures, starting with a median ΔsU of 0.038° (as given by the DIALS indexing results), we were able to refine the crystal orientations such that the median ΔsU was 0.0028° [Fig. 3e)].A useful metric to observe during optimization is the misorientation Δ3.3.2.R factor between the refined structure factor amplitudes and the ground truth (GT) structure factor amplitudes:k is a scale factor chosen to minimize RGT. Fig. 4RGT throughout stage 2 refinement for different resolution bins. At each point in refinement we computed a new k using the Adaptive Nelder–Mead Simplex method , all of which can be detrimental in situations involving weak signals, such as anomalous difference amplitudes used for the spatial resolution of heavy atoms  and exascale systems at Argonne National Laboratory and Oak Ridge National Laboratory  will provide data processing at speeds to match the increased data collection rates expected for next-generation XFEL facilities (LCLS-II). The need for this level of computing (which includes GPU acceleration) becomes apparent by recalling equation (16)On a single compute node, refinement of 2023 shots took approximately 208 min, including 12 min for per-shot (stage 1) refinement and 196 min for 500 iterations of global (stage 2) refinement running at 23.5 s per iteration . We also assumed a detector with minimal pixel crosstalk and a well calibrated linear response, attributes that are realized in current-generation detectors like the ePix  . Sources of error we have not described include measurement parallax, per-image Debye–Waller factors, intra-crystal unit-cell variation and multiple lattice scattering, all of which contribute to error in the determined structure factors. Thus far we have neglected any mention of structure factor errors, but we know proper error estimation can aid in solving real systems (Brewster, Bhowmick et al., 2019u and v are row and column indices. The variances of the parameters ΘML, including the structure factors {|Fh|}, are given by the diagonal elements of It is common to model the aggregate effect of energy-bandwidth and mosaicity in a single Gaussian equation (Parkhurst, 2020 al. 2012, we applnanoBragg to generate realistic XFEL diffraction images, which we analyzed using both the standard integration protocol and the new program, diffBragg. With diffBragg, we utilized all measured pixels simultaneously to estimate high-accuracy structure factors while requiring an order of magnitude fewer diffraction events compared with the conventional method. Reducing the number of required shots for a dataset can greatly benefit the general SFX experiment, with scarce beam time routinely plagued by unforeseen interruptions, limiting the amount of data one can realistically collect. Future work will aim to apply this method to real measurements and develop a user-friendly application for the general SFX researcher.In summary, we used the program 6.diffBragg refinement are included in CCTBX. Scripts for computing and processing the synthetic data are available at https://github.com/dermen/cxid9114.The tools used for 10.1107/S2052252520013007/zf5012sup1.pdfSupporting figures and tables detailing the effects of mosaic spread and geometry errors on the refinement. DOI:


Correction to: Egypt J Bronchol (2021) 15:8https://doi.org/10.1186/s43168-021-00053-2After publication of the original article , the autThe incorrect author name is: Shohel Rana.The correct author name is: Sohel Rana.The original article has been corrected.


Enterobacterales, 111 of Pseudomonas aeruginosa, and 99 of Acinetobacter baumannii, all selected for carbapenem resistance and multidrug resistance to other agents. At 2 and 4 μg/ml, cefiderocol inhibited 78.Cefiderocol is a parenteral siderophore cephalosporin with a catechol-containing 3′ substituent. We evaluated its MICs against Gram-negative bacteria, using iron-depleted Mueller-Hinton broth. The panel comprised 305 isolates of  Enterobacterales, 111 of Pseudomonas aeruginosa, and 99 of Acinetobacter baumannii, all selected for carbapenem resistance and multidrug resistance to other agents. At 2 and 4 μg/ml, cefiderocol inhibited 78.7 and 92.1%, respectively, of all Enterobacterales isolates tested, with rates of 80 to 100% for isolates with all modes of carbapenem resistance except NDM enzymes (41.0% inhibited at 2 μg/ml and 72.1% at 4 μg/ml) or combinations of extended-spectrum β-lactamase (ESBL) and porin loss (61.5% inhibited at 2 μg/ml and 88.5% at 4 μg/ml). Cefiderocol also inhibited 81.1 and 86.5% of all P. aeruginosa isolates at 2 and 4 μg/ml, respectively, with rates of 80 to 100% for isolates with VIM, IMP, GES, or VEB β-lactamases and slightly lower rates for those with NDM (45.5% at 2 μg/ml and 72.7% at 4 μg/ml) and PER (66.7% at 2 μg/ml and 73.3% at 4 μg/ml) enzymes; 63.3% of P. aeruginosa isolates were inhibited at the FDA’s 1-μg/ml breakpoint. Lastly, cefiderocol at 2 and 4 μg/ml inhibited 80.8 and 88.9% of the A. baumannii isolates, respectively, with rates of >85% for isolates with OXA-51-like, -23, -24, or -58 enzymes and 50% at 2 μg/ml and 80% at 4 μg/ml for those with NDM carbapenemases. Dipicolinic acid and avibactam weakly potentiated cefiderocol against Enterobacterales isolates with metallo-β-lactamases (MBLs) and serine carbapenemase, respectively, indicating incomplete β-lactamase stability.Cefiderocol is a parenteral siderophore cephalosporin with a catechol-containing 3′ substituent. We evaluated its MICs against Gram-negative bacteria, using iron-depleted Mueller-Hinton broth. The panel comprised 305 isolates of  Carbapenem-resistant Gram-negative bacteria have proliferated globally and are a growing problem, prioritized by the WHO . Their rMany carbapenemase producers are resistant to multiple antibiotics besides β-lactams, including fluoroquinolones, aminoglycosides, and antifolates . ConsequEnterobacterales isolates with KPC and OXA-48-like enzymes, while meropenem-vaborbactam and imipenem-relebactam inhibit those with KPC enzymes only , and in the European Union (EU) for the treatment of infections due to aerobic Gram-negative bacteria in adults with limited treatment options. It has a catechol moiety attached via its 3-position side chain, allowing uptake into Gram-negative bacteria via the ferric iron transporter system –13. We e–Enterobacterales isolates based on the FDA 2-μg/ml breakpoint but only 42.0% based on the EUCAST 0.5-μg/ml value, which, strictly, is only applicable to Escherichia coli and Citrobacter koseri.The isolate panel was deliberately loaded with highly resistant organisms , as reflEnterobacterales, inhibiting 41.6% of isolates at its breakpoint of 8 μg/ml for ceftazidime plus 4 μg/ml for avibactam, essentially comprising almost all those without MBLs. Aztreonam was the most active β-lactam against the P. aeruginosa panel, inhibiting 46.8% of isolates—mostly MBL producers—at its 16-μg/ml breakpoint. Resistance to established β-lactams, including ceftazidime-avibactam, was nearly universal in the A. baumannii panel.Among comparator β-lactams, ceftazidime-avibactam was the most active combination against Enterobacterales isolates at 2 μg/ml and 92.1% at 4 μg/ml; corresponding proportions for the P. aeruginosa collection were 81.1 and 86.5%, respectively, although only 63.3% were inhibited at the FDA’s 1-μg/ml breakpoint. The proportions of the Acinetobacter collection inhibited at 2 and 4 μg/ml were 80.8 and 88.9%, respectively; neither CLSI nor the FDA has breakpoints for this genus.Cefiderocol inhibited 78.7% of Enterobacterales groups except those with NDM carbapenemases (41% inhibited) or combinations of extended-spectrum β-lactamases (ESBLs) and porin loss (61.5% inhibited)  those with NDM MBLs (72.1% inhibited) and (ii) those with combinations of ESBL and impermeability (88.5% inhibited).MICs of cefiderocol were widely scattered within enterobacterial species and had no obvious association with species . Accordihibited) . At 4 μgEnterobacterales isolates with NDM enzymes was extended and elevated compared with those for isolates with other MBLs. This behavior was independent of aztreonam resistance, indicating that higher cefiderocol values, where seen, were not contingent upon coresident ESBL or AmpC enzymes , PER (66.7%), and NDM (45.5%) β-lactamases , PER (73.3%), or NDM enzymes (72.7%). Cefiderocol MICs of ≥128 μg/ml were recorded for two isolates: one with an NDM carbapenemase and one with a VIM carbapenemase.At 2 μg/ml, cefiderocol inhibited 81.1% of ctamases . At 4 μgEnterobacterales, cefiderocol MICs for P. aeruginosa isolates with NDM carbapenemases were elevated compared with those for isolates with other mechanisms, although it should be cautioned that (i) the behavior seemed less marked and (ii) only 11 P. aeruginosa isolates with NDM carbapenemases were tested—far fewer than the 61 Enterobacterales isolates.As with A. baumannii isolates, with rates of ≥85% for all groups, except for those with NDM carbapenemases (50.0%); at 4 μg/ml, it inhibited 88.9% , as an inhibitor of MBLs, and avibactam (4 μg/ml), as an inhibitor of any coproduced serine β-lactamases, and with a combination of both these inhibitors. A panel of 40 chanisms .Enterobacterales isolates showed a >2-fold reduction in cefiderocol MIC. Dipicolinic acid, in contrast, achieved 4- to 32-fold reductions in MIC for 25/40 blaNDM-positive isolates, with combination MICs ranging from ≤0.03 to 16 μg/ml and a mode of 1 μg/ml, compared with 4 μg/ml for cefiderocol alone. There was a greater effect when both inhibitors were tested together in combination with cefiderocol, with 4- to 64-fold MIC reductions for 36/40 blaNDM-positive isolates. Using this triple combination, all 40 NDM-positive isolates were inhibited by cefiderocol at ≤2 μg/ml compared with 22.5% for cefiderocol alone.Avibactam alone had predictably little effect on the MICs of cefiderocol for the isolates with NDM enzymes: only 1/40 NDM-positive The control groups were small, reducing the strength of conclusions for individual enzyme types: nonetheless, MICs of cefiderocol for isolates with VIM enzymes generally showed 2- to 4-fold reductions when dipicolinic acid was added alone or together with avibactam, whereas no reductions were seen with only avibactam added. Conversely, the MICs of cefiderocol for isolates with KPC or OXA-like carbapenemases, or with combinations of ESBL and porin loss, mostly were reduced by the addition of avibactam alone or combined with dipicolinic acid, whereas dipicolinic acid alone had minimal effect.in vitro activity against otherwise extremely resistant Enterobacterales isolates and nonfermenters, at least under iron-deficient conditions. Thus, as found here and by others , meropenem-vaborbactam, and imipenem-relebactam  of those with VIM and IMP MBLs . This behavior was unrelated to aztreonam resistance or susceptibility, implying that the higher MICs reflected the NDM enzymes themselves and not coproduced ESBLs or AmpC enzymes. To explore this aspect further and to determine if NDM enzyme protected bacteria against cefiderocol, we undertook combination tests with β-lactamase inhibitors. The results supported the view that cefiderocol is not completely β-lactamase stable, as it was frequently, though weakly, potentiated by dipicolinic acid against the isolates with NDM (particularly) and VIM MBLs and by avibactam for isolates with KPC, OXA-48, ESBL, and AmpC enzymes. Additional synergy for Enterobacterales isolates with NDM enzymes when both dipicolinic acid and avibactam were added is surprising. It may be that avibactam inhibits coresident class A or D β-lactamases, but were this a significant factor, one would expect the MICs of unprotected cefiderocol to be higher for aztreonam-resistant, NDM-positive Enterobacterales isolates than for their aztreonam-susceptible counterparts lacking ESBLs or AmpC enzymes, and this was not the case  of the case .P. aeruginosa, unless OprD is lost, despite lability to the organism’s chromosomal AmpC enzyme  111 isolates of P. aeruginosa, selected to represent producers of MBLs and GES carbapenemases, along with isolates that produced VEB or PER ESBLs and were carbapenem resistant via OprD loss; and (iii) 99 isolates of A. baumannii with NDM MBLs or various OXA carbapenemases.The test isolates  comprisePseudomonas aeruginosa with PER ESBLs, which were collected in Turkey in the early 1990s  Reference Unit collections for inclusion, we favored organisms with phenotypes for other β-lactams (cefiderocol had not previously been tested), typical of their mechanisms but including susceptible and resistant representatives for antibiotics that divide isolates within groups. Thus, for example, groups with metallo-β-lactamases were selected to include both aztreonam-resistant organisms  and those that were aztreonam susceptible (inferred to lack ESBLs or AmpC enzymes). We ensured that the isolates were from diverse hospitals and excluded multiple isolates from single patients. Almost all of the organisms were submitted by hospital laboratories in the United Kingdom between 2008 and 2018 for investigation of unusual resistance phenotypes and/or susceptibility testing for therapeutic guidance. Exceptions were 3 isolates referred to the PHE-AMRHAI Reference Unit from hospitals in the Republic of Ireland and 11 isolates of ly 1990s .Carbapenemases and VEB and PER enzymes were identified by PCR of their encoding genes or by whole-genome sequencing (WGS). Carbapenem resistance due to porin loss combined with ESBL or AmpC activity was inferred from previous susceptibility results and the absence of carbapenemase, as confirmed by PCR or WGS. Species identification was by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectroscopy.2+, Ca2+, and Zn2+ ions at concentrations of 20 to 25, 10 to 12.5, and 0.5 to 1.0 μg/ml, respectively. The comparator antibiotics were meropenem, ceftazidime, ceftazidime-avibactam (4 μg/ml), cefepime, ceftolozane-tazobactam (4 μg/ml), aztreonam, colistin, amikacin, ciprofloxacin, and tigecycline, all sourced by IHMA.MICs were determined using preprepared broth microdilution plates  with antibiotic dilutions in cation-adjusted Mueller-Hinton broth (CAMHB) . Iron-deEnterobacterales isolates was similarly tested on a second broth microdilution plate that included cefiderocol alone, cefiderocol-avibactam (4 μg/ml), cefiderocol-dipicolinic acid (100 μg/ml), and cefiderocol plus both avibactam (4 μg/ml) and dipicolinic acid (100 μg/ml). ID-CAMHB was used.A subpanel of 64 Enterobacterales and P. aeruginosa; the FDA has values of S ≤ 2 μg/ml and R > 4 μg/ml for Enterobacterales and S ≤ 1 μg/ml and R > 2 μg/ml for P. aeruginosa; CSLI still has values under review, but previously, when cefiderocol was in trial, had investigational values of S ≤ 4 μg/ml and R > 8 μg/ml. MICs of comparator antibiotics were interpreted using EUCAST guidelines where available, the exceptions being ceftazidime and cefepime for Acinetobacter spp., for which only CLSI breakpoints are available.We reviewed results against published breakpoints: EUCAST has values of S ≤ 2 μg/ml and R > 2 μg/ml for


The results showed that excess NO increased the inflammation marker levels in T2DM, which is activated by the PPARγ/eNOS pathway. These findings will strengthen the understanding of NO in T2DM and provide a new target for the treatment of T2DM.Inflammation accounts for the process of type II diabetes mellitus (T2DM), the specific mechanism of which is still to be elucidated yet. Nitric oxide (NO), a critical inflammation regulator, the role of which is the inflammation of T2DM, is rarely reported. Therefore, our study is aimed at exploring the effect of NO on the inflammation in T2DM and the corresponding mechanism. We analyzed the NO levels in plasma samples from T2DM patients and paired healthy adults by Nitric Oxide Analyzer then measured the expression of inflammatory cytokines (C-reactive protein, heptoglobin, IL-1 The high prevalence of T2DM is a serious public health event over the world [β-cell dysfunction, which adds the risk of developing T2DM [α, IL-1β, and IL-6 have been clarified to cause insulin resistance by inhibiting insulin signaling [Type II diabetes mellitus T2DM) is a common chronic metabolic and endocrine disease characterized by insulin resistance and he world . AccordiDM is a che world , 4, and ing T2DM , 6. C-reing T2DM , 7. Hapting T2DM , 9. Inflignaling , 11. Theγ belongs to a family of peroxisome proliferator-activated nuclear receptors that regulate the expression of multiple genes involved in the regulation of lipid/glucose/amino acid metabolism, cell proliferation/differentiation, and inflammation, suggesting that it may play important roles in many diseases [γ, has potent insulin-sensitizing effects and is used to treat T2DM, but the drug will produce side effects such as weight gain, liver damage, and cardiovascular risk and is gradually declining in clinical use [γ in T2DM. In recent years, studies have told that PPARγ regulates the expression of inflammatory pathways. For example, GMG-43AC antagonist can be applied to treat acne because it activates PPARγ to inhibit inflammation [γ-mediated upregulation of CD36 is involved in the regulation of microglial activation and phenotype and promotes phagocyte-cell proliferation of apoptotic cells, thereby promoting the recovery of postischemic inflammation [γ activation is essential in the suppression of intestinal inflammatory response [γ regulates inflammation remains to be refined.PPARdiseases . Thiazolical use , 14. Thuammation . PPARγ-mammation . Moreoveresponse . In T2DMγ/eNOS pathway regulates hypertension [γ/eNOS pathway mediates the inflammatory process in T2DM.Nitric oxide (NO), a free radical molecule with pathophysiological functions, is extensively studied in inflammation. NO has anti-inflammatory and proinflammatory effects, which are correlated with its concentration . Excess rtension , ischemirtension , steatohrtension , ischemirtension , and so γ/eNOS pathway and validated in the present study. Our findings may provide a new treatment target for T2DM.Therefore, we put forward the hypothesis that NO may regulate inflammation in T2DM through the PPARThe study was approved by the ethics committee of Huai'an Second People's Hospital and Hospital of Chengdu University of Traditional Chinese Medicine. Fifty-five T2DM patients and fifty healthy adults in this study signed informed consent forms and began to sample blood at 8:30 am in the morning under a fasting state (no food and water absorption for at least 8 h before sampling). The whole blood samples were collected and placed in a heparin anticoagulation tube , then centrifuged at 3500 g at 4°C for 10 min. The achieved uppermost layer is the plasma layer, which should be stored at -80°C for subsequent testing.−6 mol/L. 200 μL of insulin preparation solution was added to each well in the model group, and an equal amount of RPMI1640 complete medium was added to each well in the control group; all cells were cultured for 48 h [2).HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences ; they were cultured in 96-well plates in RPMI1640 medium  with 10% fetal bovine serum  and 1% penicillin-streptomycin  and allowed to grow to logarithmic growth phase for subsequent studies after a successful recovery. To construct a cell model of T2DM, insulin  was first diluted in RPMI1640 complete medium to a final concentration of 10for 48 h . In the γsiRNA  by using the Amaxa Lonza Cell Line Nucleofector® Kit  according to the manufacturer's instructions, and an equal amount of nonspecific siRNA  was transfected with HepG2 cells as a negative control. After incubation for 24 h, they were used for western blot analysis.HepG2 cells were transfected with 100 pmol PPARDue to the fact that NO has a short half-life and is not easy to directly detect, the concentration of its stable metabolites nitrite and nitrate can represent the levels of NO . In the β, TNF-α, and IL-6 in HepG2 cells were detected using an ELISA kit , and each experiment was performed 3 times according to the manufacturer's instructions.The levels of IL-1γ. The expression levels were analyzed by the -ΔΔ2Ct method, and GADPH (5′-AGGTCGGAGTCAACGGATTT-3′ (forward) and 5′-TAGTTGAGGTCAATGAAGGG-3′ (reverse)) expression levels were used as the reference standard.Total RNA was isolated from cells using TRIzol reagent  then reverse transcribed into single-stranded cDNA using a Prime ScriptTMRT kit . Real-time PCR equipment  was to detect the expression of C-reactive protein, haptoglobin, eNOS, and PPARμg of total protein was isolated on 12% SDS-PAGE and transferred to PVDF membranes. Membrane blotting was first blocked with 5% bovine serum albumin  for 1 h then incubated with primary antibody  overnight at 4°C, followed by incubation with horseradish peroxidase for 2 h at room temperature. The relative protein expression levels were normalized to GAPDH.After HepG2 cells were treated with RIPA lysis buffer , 30 t-test analysis. And p < 0.05 indicates statistical significance.All statistics were analyzed using GraphPad Prism 7.0 software (USA), and all experimental data were expressed as mean ± SD. Whether the expression of nitrite, nitrate, and NO differed between the different groups was obtained by To evaluate the effect of NO in the T2DM, we firstly examined the levels of nitrite and nitrate in plasma between 55 T2DM patients and paired 50 healthy adults (control group). The clinical data of volunteers in the study is shown in p < 0.0001), and the plasma nitrate levels between the T2DM group and control group were evidently different (p < 0.0001), which was found in Figures The concentration of plasma nitrite in the T2DM group was significantly higher than the concentration of plasma nitrate in the control group  and NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide , respectively. The concentration of IL-1γ can modulate the level of diabetic inflammation, the PPARγ/eNOS pathway plays an important role in several diseases. In the present study, insulin-induced HepG2 cells transfected with PPARγ siRNA were found to significantly decrease the expression of PPARγ, eNOS, and NO in Figures γ expression in HepG2 cells prompted a significant decrease in the expression levels of IL-1β, TNF-α, IL-6, C-reactive protein, and heptoglobin. These results suggest that inflammation in T2DM may be associated with the PPARγ/eNOS/NO pathway.To investigate the mechanism of the development of inflammation in T2DM, we detected the expression of eNOS and NO in vitro. As shown in Figures The inflammatory response can drive the pathological process of T2DM by leading to deleterious effects on tissue function and insulin resistance , 29, so β, TNF-α, IL-6) was significantly increased in insulin-induced HepG2 cells after NO donor treatment, while the levels of inflammatory factors were decreased in insulin-induced HepG2 cells stimulated with NO scavenger. The results support that NO may take part in the inflammatory process in T2DM patients. To investigate the mechanism of NO on inflammation in T2DM, we investigated and conducted further studies. Previous studies have shown that PPARγ hyperglycosylation modification induces endothelial insulin resistance and dysfunction associated with diabetic vascular complications by regulating the eNOS-NO pathway [γ provides assistance to the expression of eNOS [γ-eNOS signaling pathway is currently unclear.In vitro experiment revealed that the concentration of inflammatory factors (C-reactive protein, heptoglobin, IL-1 pathway . PPARγ p of eNOS , which iγ in vitro and found that the expression of eNOS and NO decreased after the treatment of insulin-induced HepG2 cells with NOS inhibitor. Inhibiting the expression of PPARγ in insulin-induced HepG2 cells significantly decreased the levels of PPARγ, eNOS, and NO, and the levels of C-reactive protein, heptoglobin, IL-1β, TNF-α, and IL-6 were significantly reduced. Therefore, the expression of T2DM inflammation may be regulated through the PPARγ/eNOS pathway-mediated expression of NO.To test the hypothesis, this study detected the expression of eNOS and PPARIn summary, this study explored the link between NO and inflammation through insulin-induced HepG2 cells, which provides a potential therapeutic target for the possible treatment of T2DM.


A sample of 20,000 genotyped animals were used to predict ungenotyped using animal model. Prediction accuracies for ungenotyped animals for SRh and SRl were 0.60 and 0.54, and for allele content were from 0.41 to 0.71, respectively. Response to selection on SRh and SRl increased SR by 0.52 and 0.28, and on allele content from 0.13 to 0.50, respectively. In addition, the selected animals had large proportion of homozygous for the favorable haplotypes. Thus, pre-selection prior to genotyping could reduce genotyping costs for breeding programs. Using a linear animal model to predict SR makes better use of available information for the breeding programs.Selection based on scrapie genotypes could improve the genetic resistance for scrapie in sheep. However, in practice, few animals are genotyped. The objectives were to define numerical values of scrapie resistance genotypes and adjust for their non-additive genetic effect; evaluate prediction accuracy of ungenotyped animals using linear animal model; and predict and assess selection response based on estimated breeding values (EBV) of ungenotyped animals. The scrapie resistance (SR) was defined by ranking scrapie genotypes from low (0) to high (4) resistance based on genotype risk groups and was also adjusted for non-additive genetic effect of the haplotypes. Genotypes were simulated for 1,671,890 animals from pedigree. The simulated alleles were assigned to scrapie haplotypes in two scenarios of high (SR PrP gene [In the typical form of scrapie, the risk of infection is determined by variation in amino acid sequence encoded in the PrP gene ,2,3,4. TPrP gene ,6,7. OthPrP gene . A totalPrP gene ,9. Due tPrP gene ,8,10,11.PrP gene  proposedSelection based on risk group of the genotypes could be practiced ,5,8,9. HThe objectives of this research were to: (1) define numeric values of scrapie resistance genotypes and adjust them for their non-additive genetic effect; (2) evaluate the accuracy of using BLUP for prediction of scrapie resistance and allele content of ungenotyped animals; and (3) predict selection response and assess the change of genetic merit of selected ungenotyped animals based on estimated breeding value. The hypothesis of this research was that it is possible use a linear animal model for genetic evaluation and selection of ungenotyped sheep for scrapie resistance based on a small proportion of genotyped animals.The numeric value of scrapie resistance genotypes was defined by ranking the scrapie genotypes from 0 (most susceptible) to 4 (most resistant), which were based on risk levels previously presented in literature ,8,9 see. The addwww.genovis.ca; Guelph, ON, Canada accessed on 9 June 2017) was used to simulate genotypes at a single locus with five alleles resulting in 15 possible common genotypes. The genotype frequencies resulting from the simulation are shown in h and SRl, respectively) and the allele content  for the haplotypes  are presented in n = 1,671,890) and the randomly chosen animals  that had their genotypic information available , respectively.A pedigree containing 1,671,890 sheep from the GenOvis database (h and SRl) and haplotype allele contents  where predicted for the ungenotyped animals using the observed records of the 20,000 genotyped animal using ASREML [ith animal for the trait being analyzed ; ith animal; and The scrapie resistance phenotypes , thus assuming a negligible residual. In order to estimate breeding values (EBV) for all animals (including the ungenotyped with no genotyped relatives) in the pedigree, unknown parents were assigned to genetic group based on sex and breed.The heritability was assumed to be almost complete ; 2 = 0.99 ) between selected  to assess: (1) the proportion of animals selected at different truncation point; (2) the proportion of recovered homozygous from ungenotyped animals among the selected animals  at different truncation point; (3) allele frequencies at different selection truncation points; and (4) homozygous genotype frequencies at different selection truncation points.h for ungenotyped individuals was high 0.60. In addition, the predicted SRh was positively correlated (r = 0.58) with the most favorable haplotype in this analysis ARR (H1), and negatively correlated with the other haplotypes . The correlation between predicted allele content for ARR (Hc1) with its true content and SRh was 0.71 and 0.47, respectively. This means that selection based on SRh is expected to increase SR and ARR allele content, while decreasing the allele content for other haplotypes . On the other hand, prediction of SRl was less accurate when predicting the true SRl (r = 0.54) compared to SRh, and was negatively associated with the least favorable allele in this scenario, i.e., H1 (VRQ), while positively correlated with the other haplotypes . This means that selection for SRl will increase SR by replacing ARR by the other haplotypes in the population.The Pearson’s correlation coefficients between EBV and the trait true genetic value are shown in h, SRl and the haplotype allele contents  for ungenotyped animals . Selection of SRh EBV was predicted to increase scrapie resistance by 0.46 and ARR (Hc1) allele content by 0.36, and decrease the other haplotype allele contents . Selection for Hc1 EBV was predicted to increase the SRh by 0.36 and ARR (Hc1) allele content by 0.44, and decrease the other haplotypes allele contents. On the other hand, in the low resistance population (low-SR), direct selection for SRl EBV was predicted to increase SR by 0.33 and increase the allele content of haplotypes ARQ, ARH, AHQ, and ARR, while decreasing the allele content of the most unfavorable haplotype VRQ. Compared to VRQ, selection for the other haplotypes was predicted to be slower, reflecting their lower accuracies of prediction  and AHQ (Hc2) by 0.29 and 0.16, respectively. On the other hand, in a low-SR population, selection increased SRl by 0.71 accompanied of a decrease in allele content of VRQ (Hc1) by 0.74, while increasing haplotype allele contents for ARQ (Hc2), ARH (Hc3), AHQ (Hc4), and ARR (Hc5) by 0.33, 0.13, 0.15, and 0.13, respectively. As expected, when all animals were genotyped, there is a higher gain than when only a fraction of the animals was genotyped for scrapie , selection based on Hc1 EBV ≥ mean EBV resulted in selecting 50.7% from the population and recovering a total of 87.7% of the target homozygous genotype (ARR/ARR). This means that by pre-selecting 50.7% of individuals based on EBV for allele content for genotyping, 87.7% of homozygous genotypes could be captured from the original population. Thus, this would reduce the number of animals required for genotyping. In the case of low ARR allele frequency , when pre-selecting based on EBV for allele content ≥ mean EBV, the number of individuals required for genotype validation was reduced to 37.8% (As expected, as the selection truncation point increased, the proportion of selected  and the to 37.8% , while rto 37.8%  of homozmyostatin gene in Belgium blue cattle, with prediction accuracies between 0.47 and 0.50. In another study in Canadian Holstein cattle, the prediction accuracy for allele content was as high as 0.93 [genes, such as for maedi-visna [PrP gene underlying scrapie phenotypes is multi-allelic with different contributions from the different haplotypes to the level of scrapie resistance in sheep [The accuracy for prediction of haplotype allele contents ranged between 0.41 and 0.71 . Gengler as 0.93 . Legarra as 0.93  reporteddi-visna  and for di-visna . Applyindi-visna  is possiin sheep ,9. In thSelection response on SR and allele content was predicted for ungenotyped sheep . The selSelecting ungenotyped animals with EBV ≥ mean resulted in reduction of number of animals compare to unselected population , while cBreeding programs can contribute to the reduction of prevalence of the typical form of scrapie in sheep. Arnold and Rajamyagam  estimateThis research proposed the use of a linear animal model as a practical method for genetic evaluation and selection for SR of ungenotyped sheep. Different scrapie eradication strategies used in breeding programs were described in previous studies. For instance, Arnold et al.  proposedModerate to highly accurate estimated breeding values for scrapie resistance and haplotype allele content for ungenotyped animals can be obtained from a linear animal model using genotype data from only a fraction of the total sheep population. Thus, selecting ungenotyped animals based on EBV could result in effective genetic gains for scrapie resistance and allele content. Individuals with EBV ≥ mean were shown to carry a large proportion of homozygous genotypes. Thus, pre-selection prior to genotyping could reduce the number of animals needed to be genotyped to identify individuals with the favorable homozygous genotypes in the population and, consequently, reduce the genotyping cost. Therefore, a linear animal model could make better use of the available information for genetic improvement of scrapie resistance in sheep.