Brucella abortus is a promising approach to developing small-molecule drugs against bovine brucellosis. Using the BLASTp algorithm, we identified ThrRS from Escherichia coli , which is 51% identical to ThrRS from Brucella abortus (BaThrRS) at the amino acid sequence level. EThrRS was used as the template to construct a BaThrRS homology model which was optimized using molecular dynamics simulations. To determine the residues important for substrate ATP binding, we identified the ATP-binding regions of BaThrRS, docked ATP to the protein, and identified the residues whose side chains surrounded bound ATP. We then used the binding site of ATP to virtually screen for BaThrRS inhibitors and got seven leads. We further characterized the BaThrRS-binding site of the compound with the highest predicted inhibitory activity. Our results should facilitate future experimental effects to find novel drugs for use against bovine brucellosis.Targeting threonyl-tRNA synthetase (ThrRS) of  Brucella spp. are important zoonotic pathogens worldwide [Brucella abortus, represents a major problem for livestock industry development worldwide and is also a threat to human health in many developing and underdeveloped countries [orldwide ,2,3. Bovountries . Gentamiountries . Howeverountries , novel aStaphylococcus aureus and Streptococcus pyogenes [Aminoacyl-tRNA synthetases (aaRSs), which play key roles in translation, catalyze the covalent attachment of amino acids to their cognate tRNAs, yielding aminoacyl tRNAs (aa-tRNA or charged tRNA) for protein synthesis ,8,9. Becpyogenes . In addipyogenes , includipyogenes , granatipyogenes , ochratopyogenes . Virtual screening, a complementary approach to high-throughput screening (HTS) ,17,18,19Brucella abortus (BaThrRS) and predicting interaction sites for substrates and inhibitors. To date, no experimentally determined 3D structures of Brucella abortus aaRSs have been published, and the rate at which aaRS structures are solved is insufficient to meet the need for development of drugs against brucellosis. Therefore, we used homology modeling to build a 3D structure of Brucella abortus aaRSs. Further refinement was achieved by subjecting the 3D model to molecular dynamics (MD) simulations. We also performed molecular docking studies to analyze the interactions among BaThrRS and its ligands, which should facilitate the design of novel drugs for the treatment of brucellosis. The 3D model of ThrRS obtained by comparative modeling analysis [This study was aimed at elucidating the 3D structural features of ThrRS from analysis ,38 proviEscherichia coli (EThrRS) (PDB code 1QF6) [From the BLASTp matches of BaThrRS, we selected the structure of ThrRS from de 1QF6)  as the mde 1QF6) ; these tde 1QF6)  for homoThe coordinates of the crystal structure of EThrRS were used as a template to build the BaThrRS structure. The 3D model of BaThrRS was constructed with Modeller 9.16 ,38. To dThe first validation was carried out using Ramachandran plot calculations, computed with the MolProbity 4.3 software, which checks the detailed residue-by-residue stereochemical quality of a protein structure . Then, oThe template, 1QF6, had a Ramachandran plot similar to that of the final model: 98.7% of residues in allowed regions vs. 1.25% in disallowed regions. The ERRAT score for the template was 90.047. Verify3D revealed that 99.53% of the residues in 1QF6 had average 3D–1D scores. Thus, the geometric properties of the backbone conformation, residue interactions, residue contacts, and energy profile of the final model were similar to those of 1QF6, suggesting that the homology model of BaThrRS was reasonable and suitable for examination of protein-substrate and protein-inhibitor interactions, as well as for virtual screening.ThrRS enzymes have a common substrate, ATP (ZINC18456332). The adenine moiety of ATP is sandwiched between Phe379 and Arg520 of EThrRS, and is specifically recognized by Ser517, which interacts with the N3 atom of ATP, and Glu365, which interacts with the N6 amino group. The ribose moiety is in the C3′-endo conformation, with its 2′ and 3′ hydroxyl groups essentially bound to Gln479 with two hydrogen bonds. The α-phosphate is stabilized by Arg363, which is also responsible for binding the carboxylate group of the cognate amino acid . Therefo3 and 12,968 Å3 for BaThrRS and 1QF6, respectively. Thus, the active site of BaThrRS is smaller than that of EThrRS, which may indicate that the affinities of ligands with BaThrRS may be different from those of same ligands with EThrRS.The cavity volume estimated by CASTp  is depenThe residues of BaThrRS and EThrRS that participate in their respective ATP-binding sites are listed in We docked the substrate ATP and the selective inhibitor borrelidin (ZINC30729522) to BaThrRS using AutoDock Vina . The griHydrogen bonds may play important roles in substrate binding. ATP and BaThrRS form eight hydrogen bonds, as seen in To identify new and potent inhibitors against BaThrRS, we performed docking-based virtual screening on the active site of BaThrRS. The ZINC Natural Products Database (NPD) , which i−1·nm−2. Finally, the production run was carried out for 20 ns, storing the coordinates of all atoms every 100 picoseconds for further analysis.The 658-amino acid sequence of the target protein, BaThrRS, was obtained from the National Center for Biotechnology Information (NCBI) Protein Database (NCBI Reference Sequence: WP_002969106.1) . The temTo obtain an accurate homology model, it is very important that appropriate quality assessment steps are built into the process. Therefore, in the modeling phase, model quality was assessed based on the geometric properties of the backbone conformation, residue interactions, residue contacts, and energy profile of the structure. Evaluation methods included ERRAT , Verify3The volume of the binding pocket was computed using the CASTp server  with defA docking study was conducted to evaluate the predictive ability of the BaThrRS homology model and its suitability for use in structure-based drug design studies. The structures of borrelidin (ZINC30729522) and ATP (ZINC18456332), a substrate of ThrRS enzymes, were obtained from the ZINC database . LigandsThe ZINC NPD,  which inThis paper describes how a reliable and reasonable 3D structure of BaThrRS was built using homology modeling techniques and MD methods. To determine the residues important for substrate (ATP) binding, we docked ATP to the protein. Arg372, Thr496, Glu374, Tyr476, Gln390, Phe388, and Arg534 were found to be important residues for ATP binding. Virtual screening of the ZINC NPD yielded 99 novel compounds. The scores of the seven top-scoring docking hits were lower than −12 kcal/mol (lower than that of ATP and borrelidin), indicating that they represent potential inhibitors of BaThrRS. We hope that our results will aid in future efforts to design inhibitors of BaThrRS for use in the treatment of brucellosis.


This retrospective study was aimed to identify risk factors of intravenous (IV) infiltration for hospitalized children. The participants were 1,174 children admitted to a general hospital, who received peripheral intravenous injection therapy at least once, and had complete records. Data were analyzed with frequency and percentage or mean and standard deviation were calculated, and odds ratio (OR) from univariate and multiple logistic regressions. The number and % of infiltrations were 92 and 7.8%, respectively. IV infiltration risk factors were lower limb (OR = 1.72), phenytoin (OR = 11.03), 10% dextrose (OR = 6.55), steroids (OR = 6.21), vancomycin (OR = 4.10), high-concentration electrolytes (OR = 3.49), and ampicillin/sulbactam combination (OR = 3.37). Nurses working at children’s hospitals should consider the risk of IV infiltration for children receiving IV infusion therapy and make a preventive effort to identify IV infiltration in high-risk children at an early stage. Peripheral intravenous (IV) catheter insertion is a common and universal medical practice to provide therapeutic IV medication ,2. IV inPrevious studies on the risk factors for IV infiltration simply described characteristics of children in which IV infiltration occurred, and they were based on small sized on samples, and they were uncontrolled trials or case reports ; therefoTherefore, the purpose of this study was to identify the physiological, device, and drug-related factors reported as to whether they have a relationship with IV infiltration occurrence using a large number of samples including children with and without IV infiltration.This investigation was a retrospective cohort study of children who did or did not experience IV infiltration after receiving an intravenous infusion during hospitalization at a children's hospital.The inclusion criteria were children hospitalized in three wards of a general hospital in Y city, South Korea, who received peripheral intravenous injection therapy at least once, and had complete records. Among a potential sample of 1758 children from August 1 to October 30, 2011, 1174 (66.8%) subjects met the inclusion criteria.One author developed a data collection tool which was named as 'Record on peripheral intravenous injection'. The tool consisted of four parts based on the existing literatures ,14,18–22The stage of IV infiltration was scored on a scale of 0 to 4 by using the criteria of Flemmer and Chan , where 0This study was conducted after receiving approval from The Pusan National University Yangsan Hospital Institutional Review Board (IRB no. 05-2013-029). The obtaining informed consent was waived because of a retrospective study without collecting personal identifiers. To identify the risk factors, one author collected data using study instrument. If a subject received peripheral intravenous injection therapy two or more times during the hospitalization period, data from the first round of therapy were collected. Device-related factors and drug-related factors were investigated from the start of intravenous catheter insertion to occurrence of IV infiltration for subjects with IV infiltration and to catheter removal for subjects without IV infiltration.The collected data were analyzed using SPSS/WIN software (ver. 18.0). A two-tailed test was performed with a significance level (α) of 0.05. A univariate logistic regression analysis was performed to examine the physiological, device, and drug-related factors associated with IV infiltration. Prior to analysis, explanatory variables were generated or newly categorized. For instance, age was classified according to the pediatric classification system  as underFor the medical department category, cranial nerve, allergy, respiratory system, digestive system, kidney / internal secretion, heart, and hematologic tumors were grouped into the pediatric medicine department, and general surgery or otorhinolaryngology grouped into the surgery department.A general drug classification system was followed to evaluate drug factors. Saline solution, 5% dextrose, and 1:4 SD were classified as isotonic infusions while 10% dextrose, total parenteral nutrition, amino acids, and lipids were classified as hypertonic infusions. Among the injections administered, vesicant , nafciliFinally, a stepwise multiple logistic regression analysis was done to identify the risk factors of IV infiltration. Explanatory variables were significant factors by the univariate analysis.The majority (61%) of the study subjects were males with a mean age of 6.3 years and 66.6% of them had a normal body weight. Less than half (40%) of the subjects received treatment for allergies and respiratory system problems .Mean retention time of the intravenous injection was 59.96 hours. The size of 94% of the catheters used was 24-gauge. The insertion site was in the upper limb for 85% of the subjects .The largest portion of subjects (64%) received an infusion of 5% dextrose while 27% of the subjects were treated with cefotaxime .According to the univariate analysis, physiological factors related to the occurrence of IV infiltration were being underweight . PatientMultivariate analysis confirmed the findings on univariate analysis with the exception of being underweight, retention time, and use of cefotaxime. Results of the analysis identified insertion site, medical department admission, phenytoin, 10% dextrose, steroids, vancomycin, high-concentration electrolytes, and ampicillin/ sulbactam combinations as significant risk factors .p <0.001) and 6.55 , respectively. The OR for pediatric medicine admission was 3.97 , and the OR for the lower limb as insertion site was 1.72 .The ORs for use of phenytoin and 10% dextrose were 11.03 (95% CI = 2.92–41.68, Risk factors of IV infiltration were lower limb insertion site, pediatric medicine admission, and administration of phenytoin, 10% dextrose, steroids, vancomycin, high concentration electrolyte, ampicillin/sulbactam combinations in this study.Certain fluids and drugs can easily cause venous rupture when the venous endothelium and blood vessel walls are irritated, leading to IV infiltration or extravasation. Since strong acids or strong alkalinities can damage cellular proteins, reduce durability of the venous endothelium, and inflict venous rupture, pH ranging from 5 to 9 is recommended for intravenous injection . PhenytoLikewise, osmotic pressure within the range of 250–350 mOsm/L is permitted to be given peripherally ,27. HighSteroids are known to induce the formation of emboli when used over a long period of time . Emboli According to the results, nurses in clinical settings are recommended to select an IV site in upper arm rather than in lower limb, and need to make repeated observations to identify IV infiltration occurrence when they care for children in the pediatric medicine department, and administer high risk drug/fluid such as phenytoin, 10% dextrose, steroids, vancomycin, high-concentration electrolytes, and ampicillin/sulbactam.To our knowledge, this study is one of the few studies to identify the risk factors of IV infiltration considering different factors in a large group of subjects, divided into children with and without IV infiltration. However, careful interpretation of the results from the present investigation is required as this study has the following two limitations. First, there is limitation in generalizability because the study only included children hospitalized at a single children's hospital. Second, this study was a retrospective survey of medical records and thus failed to reflect characteristics used by the nurses to determine IV infiltration risk factors. This includes factors such as catheter insertion skill and appropriateness of catheter fixation after insertion.Risk factors related to the occurrence of IV infiltration in children were assessed in the present study. Based on the findings, nurses working at the children’ hospitals should consider the risk of IV infiltration for children receiving intravenous infusion therapy and make efforts to identify IV infiltration in high-risk children at an early stage to prevent damage. Furthermore, a replication study on children with different characteristics should be conducted to increase the possibility for generalization of the risk factors. A prospective study should also be performed to investigate the nurse-associated factors such as catheter insertion skill.


Childhood cancer remains the leading cause of disease-related death among children 0 to 14 years and incidence varies by race, ethnicity, sex, geographic locale, and age at onset. However, data are unavailable in some regions, indicative of a need for such information for cancer awareness, education and prevention program. We utilized retrospective epidemiologic design to assess and characterize pediatric tumors in the Nemours Electronic Medical Records, between 2004 and 2014. Tumor frequency and children population size were used to determine the period prevalence as cumulative incidence (CI) proportion, as well as chi-square and Poisson Regression. The CI for overall childhood cancer in Delaware was 234 per 100,000 children, and varied by race, black , white . Similarly, sex variability was observed in CI, boys  and girls . The most commonly diagnosed malignancies were acute lymphoblastic leukemia (ALL), Central Nervous System (CNS)/brain and renal cancer. The geographic locales with relatively higher cancer CI in the state of DE were zip codes 19804 and 19960, but this does not imply cancer clustering. Differences in overall childhood cancer distribution occurred by race, sex, geography, and age. These findings are indicative of the need for cancer-specific health education, awareness and prevention programs in reducing the observed disparities in Delaware. Cancer is the leading cause of disease-related death among children 0 to 14 years old . DespiteRace, ethnicity, sex, age group, socioeconomic status, and geographic locale are common indicators of disparities in childhood cancer . Racial The estimation of cancer incidence sometimes present difficulties and complexities due to the unavailability of accurate population size data to serve as the denominator for cancer diagnosed in various settings. As a result of the limitation in population data, there are very few studies that have estimated incidence or cumulative incidence in childhood cancer. The current study was proposed due to the lack of data on cumulative incidence of childhood cancer in the state of Delaware, with incidence rate for cancer at all sites of 509 per 100,000 persons (2007–2011); which is higher than the U.S. incidence rate of 460 per 100,000 persons . The recThe study was approved by our Institutional Review Board to examine the distribution of cancer in children 0 to 17 years residing in the state of Delaware at the time of their diagnosis, 2004 to 2014. We estimated the overall pediatric cancer cumulative incidence between 2004 and 2014 and stratified childhood malignancies by health disparities indicators, zip code, and county to examine trends and patterns.We collected data on 592 patients from the Nemours Electronic Medical Records (NEMR) diagnosed with pediatric cancer. Of these 592 study subjects, five were excluded because their zip codes did not have 2010 U.S. Census population size data. There were 106 subjects from the remaining 587 who were diagnosed with pediatric cancer in the years 2001 to 2003 and were therefore excluded from the study sample. This exclusion was due to the lack of information on some of the main health disparities indicators examined in this study. Therefore our study population consisted of 481 children, 0 to 17 years old diagnosed with cancer from 2004 to 2014, and followed for the disease. The children population size was obtained from the U.S. Census data, 2010.We used the NEMR which includes data on all types of childhood malignancies from A.I. duPont Children’s Hospital, Wolfson Children’s Hospital in Jacksonville FL, Nemours Children’s Hospital in Orlando, FL, and The Children’s Hospital at Sacred Heart in Pensacola, FL. The Nemours database gathers information on age at diagnosis, height and weight, tumor histology, radiation therapy, chemotherapy, stem cell and bone marrow transplants, exposure to tobacco and smoke, surgery received, family history of cancer, and insurance type. Since cancer exposure data collection in our institution began in 2010, we were unable to assess the effect of tobacco and smoke as well as family history of cancer on healthography. Demographic information including, zip code, sex, race, and ethnicity were available. Population of children under the age of 18 and the median income were extracted for each zip code using the United States Census Bureau American FactFinder based on the 2010 United States Census . For theThis study examined the cumulative incidence of childhood cancer in the NEMR and differences in cancer frequency by race, sex, age at diagnosis, and geographic locale. Race, which was self- reported, was classified as Black, White, or Other. We used this category since granular race and ethnicity data collection in our institution began in 2010. Age at diagnosis was grouped into (a) 0–4 years; (b) 5–9 years; (c) 10–14 years; (d) 15–17 years. Geographic locale was sub-classified by zip codes and counties in Delaware.proportionate morbidity estimate, implying for example, a given race count as numerator divided by the total childhood cancer count as the denominator. Descriptive statistics were used to examine the percentages of childhood cancer per zip code with the total tumor frequency in the state of Delaware as the denominator. Tumor histology was examined to determine the eight most commonly diagnosed pediatric tumors in our institution. Age-category was used to represent the distribution and percentage of tumors in each age group. Temporal trends were examined by assessing summary statistics on childhood cancer count and percentages for 2004 to 2008 vs. 2009 to 2014.We performed summary statistics on variables of interest including zip codes, counties, sex, race, age group, ethnicity, major malignancies, and survival status. To examine the tumor and specific tumor proportion within zip code or race category for instance, we utilized the n = 481) divided by the total population of children 0 to 17 years old in Delaware . Cumulative incidence for the eight major childhood malignancies was estimated by the frequency of a specific major tumor divided by the total children population in Delaware. Similarly, cumulative incidence by sex was determined by the cancer count for boys or girls for a specific tumor divided the specific sex population size of children 0 to 17 years old. All estimates for cumulative incidence utilized 100,000 children as the multiplier and age adjusted to the 2010 U.S. standard population.The overall cumulative incidence was estimated by the total number of cancer cases  as well as some zip code population sizes, was too small for sufficient power to detect a statistically significant difference at 0.05 type I error tolerance level. Therefore in this study, the probability value The geo-mapping and racial mapping of the overall pediatric malignancies was performed by distributing the tumor counts by zip code and by race. In effect given a zip code, tumor counts were populated in that geographic locale and the same applied to race  by geography.All test were two-tailed and the significance level (type I error tolerance) was set at 0.05 (Poisson regression model). STATA 13.0  was used for the entire analyses .These data represent cumulative incidence of childhood cancer in the state of Delaware between 2004 and 2014, implying a decade experience with the leading cause of disease- related death among children 0 to 14 years. A total of 481 childhood cancers were diagnosed during this period. The population of children ages 0 to 17 in Delaware was 205,765 children. The cumulative incidence (CI) of childhood cancer was 234 per 100,000 children, while the annual cumulative incidence was 23 per 100,000 children.n = 26, 5.4%), 19702 , 19709 , 19720 , 19805 , 19808 , 19901 , and 19904 . Within the state of Delaware, the proportionate cancer morbidity (tumor counts per zip code) indicated the distribution by zip codes: 19701 (n = 296 (65%), Kent County n = 91 (18.9%), and Sussex County n = 94 (19.5%). These data reflect the proportionate morbidity of cancer within these geographic areas. The cumulative incidence was comparable among the three counties during this time period, New Castle County , Kent County , and Sussex County .p = 0.04. There were a total of 15 cancers diagnosed with seven malignancies between 2004 and 2008 and eight malignancies between 2009 and 2014 in zip code 19804. Additionally, an estimated nine malignancies were diagnosed among boys and six among girls. Acute lymphocytic leukemia accounted for three cases, CNS/Brain two cases, and lymphoma two cases. Other malignancies diagnosed included Acute myeloid leukemia (AML), renal, Hodgkin’s, endocrine gland, salivary gland, skin, placenta, and cancer of no specific site. Similarly, relative to the state of Delaware children in zip code 19960 were two times as likely to be diagnosed with childhood malignancy . Although imprecise, there were increased risk of childhood malignancies in 19701 (IRR 1.12), 19707 (IRR = 1.27), 19709 (IRR = 1.13), 19801 (IRR = 1.26), 19808 (IRR = 1.34), 19934 (IRR = 1.21), 19938 (IRR = 1.32), 19958 (IRR = 1.19) relative to the state of Delaware. In contrast, childhood malignancies were lower in zip codes 19713 (IRR = 0.75), 19810 (IRR = 0.67), 19966 (IRR = 0.67), 19973 (IRR = 0.58) compared to the state of Delaware. Poisson Regression Model) of pediatric malignancy by county using the state of Delaware as the reference group and the 2010 Census data as the population size for the children age (0–17 years). The cumulative cancer incidence risk was comparable for the three counties albeit a 1% imprecise increased risk in New Castle County during the study period .vs. 38.6 per 100,000 children. Similarly, lymphoma  was more common among boys, as well as AML . In contrast, girls were more likely to be diagnosed with renal  as well as endocrine gland malignancies (18.8 vs. 11.4).n = 153, 32%), followed by age group 10–14 .n = 268 (56%), blacks, n = 120 (25.3%), and other, n = 87 (18.3%). White children  had a lower cumulative incidence than black children . Racial variance was observed in ALL, with whites (20.5%) more commonly diagnosed relative to blacks (10.0%), χ2 (2) = 8.3, p = 0.02. Although imprecise or statistically marginally significant, CNS/Brain tumors were more commonly diagnosed among blacks (20.8%) relative to whites (16.8%), χ2 (2) = 1.0, p = 0.62. Similarly although imprecise, renal carcinoma was more commonly diagnosed among blacks (10.0%) compared to whites (5.6%) χ2 (2) = 2.7, p = 0.36. Likewise, although imprecise, blacks (8.3%) were slightly more likely to be diagnosed with endocrine gland tumors relative to their white (6.3%) counterparts, χ2 (2) = 1.19, p = 0.55. Among whites endocrine gland malignancy was more common among girls (70.6% vs. 29.4%) but was equally distributed among blacks (50.0% vs. 50.0%). The basis for analytic cancer epidemiology is the descriptive finding, which allows for hypotheses generation in order to examine cancer determinants, risk markers, progression, and survival. With the limitations in this area (descriptive childhood cancer epidemiologic data) in the state of Delaware, we proposed to characterize childhood malignancies by geographic locale (zip code), sex, race, as well as temporal trends, and age at onset. There are a few relevant findings from our study. First, the cumulative incidence  was 234 per 100,000 children, implying an annual cumulative incidence of 23 per 100,000 children. Secondly, childhood cancer varies by geographic locale namely zip code. Thirdly, ALL significantly varied by race and brain/CNS was statistically marginally significant, while renal, endocrine gland, AML, lymphoma, and osteosarcoma although imprecise varied by sex and age at onset.Cumulative incidence of cancer remains an important finding in understanding the burden of malignancies across specified populations. We have demonstrated that the cumulative incidence  of childhood cancer in the state of Delaware is 234 per 100,000 children, implying an annual cumulative incidence of 23 per 100,000 children. Our findings indicate an increase relative to the National Cancer Institute (NCI) data on the annual incidence for childhood cancer in the nation . The obsWe observed significant geographic variation in our data using geo-coding mapping based on the zip codes in the state of Delaware. The zip codes with highest cumulative incidence were 19804 (Newport) and 19960 (Lincoln). The observed cumulative incidence in these zip codes is indicative of some environmental factors or other known or unmeasured potential carcinogens as well as cancer predisposing factors including, though not limited to, lifestyle variables, paternal occupation, pre and postnatal exposure, maternal hormone, host factor , family history or gene-environment interaction . Risk anWhile cancer has a very long induction period and insulting pathogens may never be known , the preLincoln, zip code 19960, is a largely agriculturally-based community, indicating the possibility for potential carcinogens around the environment, given the nature of pesticides used on the land that may affect the respiratory tract as well as other organ systems involving inhalation or ingestion . PaternaAdditionally, because of the genetic predisposition to cancer, it is not unlikely to expect the populations in these zip codes with the highest cumulative incidence of pediatric cancer to have a family history of cancer relatively higher than other zip codes, indicative of epi-genetic predisposition. Despite this possibility, it is highly unlikely that the excessive cumulative incidence of childhood cancer in these zip codes (19804 and 19960) is due to genetic, family history of cancer or host factors only, but gene-environment interaction remains a possible explanation. Overall, our findings support previously published data on cancer incidence in both adult and childhood population by geographic locale in Delaware .vs. 210 per 100,000). Epidemiologic data continue to associate higher incidence of childhood cancer, ALL, and thyroid with white children [et al. on leukemia using SEER data from the 13 registries, 1973–2006 [Black children had a higher cumulative incidence of cancer of all sites than whites  if the population projection is comparable across the zip codes. Secondly, our inability to compare these findings with that of the state of Delaware Health and Social Services as a result of the state’s cancer registry recent findings that lump pediatric cancer with adult malignancy by using the age at diagnosis category of 0 to 39 years. Thirdly, Nemours Data Warehouse does not communicate in terms of record linkage with the Delaware Cancer Registry, which may result in the possibility of tumor diagnosis misrepresentation. Fourthly, due to the lack of individual level income data, we were unable to characterize tumor distribution by income level, restricting our ability to examine the effect of SES on childhood cancer incidence in our sample. Lastly, our descriptive cancer estimates may indicate underestimation due to children from the state of Delaware receiving cancer treatment from other hospitals, namely, the Children’s Hospital of Philadelphia (CHOP). Despite this flux to other states for care, our cancer patients represent an estimated 80+% of children with cancer in the state of Delaware, implying a representative sample for a reliable estimate.In summary, the cumulative incidence of childhood cancer as well as the annual cumulative incidence is higher in the state of Delaware relative to the nation. There are geographic variation in all-sites cancer, sex variations in renal carcinoma and endocrine tumor, while racial variability in leukemia remains.


Here we use a genetically amenable, widely used model system, Drosophila melanogaster, to develop an in vivo system, and investigate the role of non-thermal plasma in blood cell differentiation. Although the blood system in Drosophila is primitive, it is an efficient system with three types of hemocytes, functioning during different developmental stages and environmental stimuli. Blood cell differentiation in Drosophila plays an essential role in tissue modeling during embryogenesis, morphogenesis and also in innate immunity. In this study, we optimized distance and frequency for a direct non-thermal plasma application, and standardized doses to treat larvae and adult flies so that there is no effect on the viability, fertility or locomotion of the organism. We discovered that at optimal distance, time and frequency, application of plasma induced blood cell differentiation in the Drosophila larval lymph gland. We articulate that the augmented differentiation could be due to an increase in the levels of reactive oxygen species (ROS) upon non-thermal plasma application. Our studies open avenues to use Drosophila as a model system in plasma medicine to study various genetic disorders and biological processes where non-thermal plasma has a possible therapeutic application.Non-thermal plasma is increasingly being recognized for a wide range of medical and biological applications. However, the effect of non-thermal plasma on physiological functions is not well characterized in  How. Howin vin vitro and ex vivo systems, and to establish the safety of non-thermal plasma as a feasible therapeutic option in an in vivo system. Hence, Drosophila will make an excellent candidate for investigating effects of non-thermal plasma application in an in vivo model.Drosophila provides a genetically amenable and well-established system that has been used over the past 100 years . It has In this study, we standardized application distance, treatment time, and an appropriate dose of floating electrode microsecond-pulsed dielectric barrier discharge (mspDBD) non-thermal plasma for treatment of Drosophila (both larvae and adults). Furthermore, we studied its effect on various parameters such as life span, survivability, fecundity, movement etc. We discovered that treatment at 50 Hz for 10 seconds was most efficacious in stimulating an effect without changing the life cycle or affecting the factors mentioned above. We also explored a physiological system in Drosophila modulated by ROS, i.e. hematopoietic system, and show that application of plasma can induce blood cell differentiation by increasing ROS levels in the glands.All flies used were either Canton-S or Hemolectin Gal4 GFP (obtained from Bloomington stock center).2. The plasma treatment dose and total energy delivered to the cells during treatment, was calculated by multiplying the energy per pulse by the plasma treatment duration and frequency. All samples were treated for 10 seconds and the frequencies of pulses were 50, 850, and 1000 Hz, which corresponds to 0.012, 0.02, and 0.24 J per fly, respectively.A mspDBD plasma was utilized for this study . Plasma D. melanogaster were treated as adults (day 1) and larvae (early second instar) for multiple lengths of time as well as at various frequencies  to determine the optimal frequency and time period for treatment in the experiment. A total of 30 adults were treated for each time period of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 seconds and then the number of survivors were recorded after 3 days. Media was changed after every three days to record their life span.Freshly hatched flies  were treated with plasma for 10 seconds and allowed to recover for 3 days in separate vials. One female and one male group (n = 5 groups) were mated in a vial and fecundity was tested at 3 different time points.A negative geotaxis assay was performed, 2 days after treatment, by counting the number of flies that cross 5 cm mark in 18 seconds after tapping them to the bottom of the vial (5 flies each tested for five times).Larvae were treated as second day instars. To ensure they were all at the same age, four plates were set up with plastic cages that allowed the flies to lay eggs for four hours. After 48 hours larvae were treated with plasma for the time periods of 5, 10, 15, 20, 25, 30, 45 and 50 seconds.w/v) paraformaldehyde (PFA). Samples were moved to PBS for 10 minutes and then washed in 0.1% (v/v) Triton-X 100 in PBS (PBS-T). Samples were incubated for 10 minutes in DAPI at 1:2000 dilutions in PBS. Tissues were washed in PBS-T for 10 minutes, and then mounted on slides with Mowiol (Sigma).Two days after mspDBD plasma treatment, larvae were dissected and stained with DAPI (Sigma). Lymph glands were dissected in phosphate buffered saline (PBS) and then fixed for 20 minutes in 4% (w/v) PFA for 3 minutes and then washed again in PBS twice for 2 minutes each time. The lymph gland were then mounted in PBS and immediately photographed under a Zeiss confocal microscope. ROS levels were quantified by measuring integrated intensities . Wh. Whvia JWhen plasma is generated, four major components are produced that can be delivered to biological targets, including electric fields, UV radiation, charged and neutral gas species   34] [35 [3534]  36]. Ho. Ho36]. in vivo model organism, which can be used to study various physiological processes, disease models and phenotypes.In summary, our study has standardized the technique for atmospheric pressure, floating electrode mspDBD plasma treatment in an S1 FigHistogram represents the average number of eggs laid by untreated and plasma treated flies. There is no significant difference in the number of eggs laid.(TIF)Click here for additional data file.


Background. Pharmacopuncture is a new form of acupuncture combining acupuncture with herbal medicine, and it has been used under various conditions in Korea. The aim of this study is to establish clinical evidence for the safety and efficacy of pharmacopuncture in Korea. Methods. We searched 9 databases and two relevant journals up to December 2014 using keywords, such as pharmacopuncture. All randomized, controlled trials evaluating pharmacopuncture under any conditions in Korea were considered. Results. Twenty-nine studies involving 1,211 participants were included. A meta-analysis of two studies on obesity showed that 5 to 8 weeks of pharmacopuncture reduced weight, waist circumference, and body mass index (BMI) more than normal saline injections. In the 5 studies of musculoskeletal conditions, 7 to 30 days of pharmacopuncture had additional effects on the reduction of pain intensity, and this benefit was maintained by limiting analyses to studies with a low risk of bias for randomization and/or allocation concealment. Conclusions. This systematic review suggests the potential of pharmacopuncture for obesity and musculoskeletal diseases. However, it is difficult to recommend pharmacopuncture as an evidence-based treatment because of methodological flaws and small sample sizes of the included studies. Further well-designed trials are needed to draw a definitive conclusion. Pharmacopuncture  is a new form of acupuncture treatment combining acupuncture and the injection of herbal medicine to the acupuncture points (acupoints). In Korea, pharmacopuncture was first officially introduced to the traditional Korean medicine (TKM) community in 1967 by Sang-Cheon Nam. While the conventional acupuncture treatment incorporates the physical stimulation of associated meridians and acupoints, pharmacopuncture adds chemical ingredients from therapeutic herbs with pharmacological effects  OR “herbal acupuncture” [All Fields] OR “aqua acupuncture” [All Fields] OR aquapuncture∗[All Fields] OR “acupoint injection” [All Fields]) AND  NOT Animals [MeSH] NOT Humans [MeSH]. These search terms were slightly modified for other databases. Trials conducted in Korea and published in English or Korean were sought.The search terms used for PubMed were as follows:   for the Studies which assessed the combined effects of pharmacopuncture plus other interventions  were also considered when the identical intervention was administered to both the pharmacopuncture group and the control group.For control groups, we considered placebo or sham, other interventions, and no interventions. Placebo or sham interventions were injections of normal saline or distilled water into the pharmacopuncture points or nonacupuncture points. Other interventions included acupuncture, herbal/western medicine, cupping, tuina, diet therapy, and physical therapy, including hot pack, transcutaneous electrical nerve stimulation (TENS), interferential current therapy (ICT), ultrasound, massage, and exercise.There was no restriction on the type of outcome measures, but they had to be relevant to the conditions. All the trials were conducted in Korea.Nonrandomized trials, animal or cell studies, literature research, and quasi-RCTs  were excluded. Trials including healthy participants were excluded.We did not include trials testing bee-venom pharmacopuncture or injection of conventional medicine because they did not investigate the chemical effects of herbal medicine. Trials comparing different types of pharmacopuncture were excluded because the efficacy of control intervention could not be determined.We reviewed all searched articles to evaluate their eligibility for inclusion. In case of uncertainties, authors were contacted for further information.After the selection of studies, we extracted the following data from the selected articles: author, year of publication, study design, participants , diseases or disorders, pharmacopuncture intervention, control intervention, outcome measures, main results, and adverse events . The outWe evaluated the ROB for the included studies according to the Cochrane Collaboration's ROB assessment tool .We rated ROB for each item using “Yes ,” “Unclear ,” or “No .” For patient blinding in studies with a placebo control, we assessed the study as having a low ROB when it clearly stated that patients were blinded. For the outcome assessor blinding, we judged that if it was clearly reported that the outcome assessor was blinded or the outcome measure was evaluated by blinded participants only, it was rated as having a low ROB. If the outcome measure was assessed by unblinded participants only, we rated it as having a high ROB. If the outcome measures were mixed with subjective and objective assessments and we could not obviously judge whether the outcome assessor was blinded or not, it was rated as having an unclear ROB. As for the incomplete outcome data reporting it was rated as having a low ROB if the number and reason for attrition were clearly reported in each group and were similar between groups and the percentage of withdrawals and drop-outs did not exceed 20% for short-term and 30% for long-term follow-up . If therMeta-analysis was performed using the Review Manager software . We used the mean difference (MD) and 95% confidence intervals (CI) to estimate the effect of an intervention for continuous outcomes using a random-effects model.If it was impossible to perform statistical pooling, studies were assigned to 1 of 4 categories to classify the result for interpretation. The comparison between two groups was based on the results of original study: (1) positive when the pharmacopuncture group was significantly better than the control group, P; (2) negative when the control group was significantly better than the pharmacopuncture group, N; (3) neutral when there was no significant difference between the groups, NS; and (4) not assessable when the results were complicated or the presented data were insufficient, NA.I2 test was used. An I2 value of 50% or more was considered to be an indicator of a substantial level of heterogeneity [To address the heterogeneity among the included studies, the ogeneity . Sensitiogeneity , 42. Forogeneity .n = 27; English: n = 2) were included in our review. Our search terms yielded 5,124 records: 49 from Ovid Medline or PubMed and 5,075 from domestic databases or relevant journals. After duplicated studies were removed, 3,030 records were screened. Based on the title and abstract, 2,929 records were excluded; 687 articles were inappropriate for the topic of this review; 2,105 were not clinical studies or were nonrandomized trials; and 137 trials did not satisfy the pharmacopuncture or control group criteria. Out of the remaining 101 studies, a total of 29 RCTs : data of 1,321 participants were included in the review. The number of participants in each group ranged from 10 to 37 in the pharmacopuncture group and from 9 to 46 in the control group. The median sample sizes per arm were 17 in the pharmacopuncture group and 18 in the control group.n = 15). Among them, there were 5 studies each for low back pain [n = 6), three studies of Bell's palsy [n = 3), diseases of the digestive system [n = 2), diseases of the circulatory system [n = 1), diseases of the genitourinary system [n = 1), and pregnancy, childbirth, and the puerperium [n = 1).The types of diseases/disorders were very heterogeneous. Thus, we classified them using ICD-10 codes. The most common disorders were diseases of the musculoskeletal system and connective tissue  [Scutellaria baicalensis, Coptis chinensis, Phellodendron amurense, and Gardenia jasminoides); four studies [Taraxacum officinale, Lonicera japonica, Rehmannia glutinosa, Forsythia viridissima, Coptis chinensis, Scutellaria baicalensis, Phellodendron amurense, and Gardenia jasminoides); another four studies [Gardenia jasminoides, Corydalis remota, Boswellia carteri, Commiphora myrrha, Prunus persica, Paeonia lactiflora, Salvia miltiorrhiza, and Caesalpinia sappan); and each used Ephedra sinica Stapf and Aconitum carmichaeli Debx. [ Calculus Bovis.Fel Ursi.Moschus (BUM) [ Panax ginseng plus BUM [Saposhnikovia divaricata Schiskin, Achyranthes bidentata Blume, Acanthopanax sessiliflorum Seem, Cibotium barometz J. Smith, Glycine max Merrill, and Eucommia ulmoides Oliver)) [The types of pharmacopuncture were highly variable. Out of the 29 included studies, 12 tested monoherbal medicine pharmacopuncture: six studies , 36, 37  studies , 29 usedscens L. , scolopescens L. , and rooh. (UDP)  were useh. (UDP) , 25, 32  studies , 28, 31  studies , 33, 35 li Debx. , Calculuus (BUM) , Panax gOliver)) .Participants received fixed , partially individualized (using a fixed set of points to be given with a set of points to be used flexibly), or individualized pharmacopuncture treatment (each participant received a tailored treatment). Out of the 29 studies, 18 used fixed , 35–37, The number of pharmacopuncture sessions ranged from 3 to 16 over 6 days to 8 weeks.Regarding pharmacopuncture points used in the studies, 19 studies , 36, 37 As for the extraction methods of pharmacopuncture, 6 studies , 27, 30 In total, 24 studies mentioned the type of injector: 16 studies , 35–37 uEach amount of injection ranged from 0.05 mL to 1 mL, and the total amount of injection ranged from 0.2 mL to 5 mL. Only one study  did not Seven studies , 30, 36 The angle of injection was reported in only three studies: two studies , 27 usedIn this review, control procedures were classified into four types. First, pharmacopuncture was compared with normal saline , 35–37 oOutcome measures reported in the included studies were very diverse because of the various types of focused diseases. Intensity of discomfort  was investigated in 20 trials , 35, 37.The majority of the included trials were assessed as having a high ROB. Details of the ROB assessments are presented in Twelve out of the 25 studies reported adequate methods of sequence generation, such as using a random number table, computer random number generator, randomization code, or coin toss , 29, 36.The participant, practitioner, and outcome assessor each were blinded in only two trials , 30. DouIn terms of addressing incomplete outcome data, 13 studies , 34, 36 As for the selective outcome reporting, we could not locate and compare the protocols of any of the included studies. Therefore, we judged the ROB based on the described methods in each study. One study  had a hiThe key outcomes from the included studies are provided in n = 5) [n = 5) [n = 3) [n = 3) [Low back pain (n = 5) , 31, 33, [n = 5) , 28, 29, [n = 3) , and Bel [n = 3) , 14, 17 A total of 10 studies were available for statistical pooling Figures  and 3. A Capsicum frutescens L. or Ephedra sinica Stapf-Aconitum carmichaeli Debx. pharmacopuncture significantly reduced weight, waist circumference, and BMI compared with the normal saline injection group by 1.36 kg, 4.59 cm, and 0.52 kg/m2, respectively, immediately after treatment  = 1, p = 0.28, and I2 = 14%; χ2 = 1.27, df = 1, p = 0.26, and I2 = 21%; χ2 = 0.35, df = 1, p = 0.55, and I2 = 0%). Another study [Among the three studies on obesity, two studies , 11 showI2 = 10%). Three studies that compared pharmacopuncture with tuina manual therapy [I2 = 15%).In five studies on musculoskeletal diseases , 26–29,  therapy –25 repor Hominis Placenta Pharmacopuncture with acupuncture showed no significant difference between the groups. In shoulder pain caused by stroke [As statistical pooling was impossible in the other seven trials, detailed results were described as follows. Two trials on HNP of the L-spine , 33 showy stroke , 35, they stroke , acupunc Hominis Placenta Pharmacopuncture was significantly better than normal saline injection [The results of pharmacopuncture were composited for each disease. For headache, both Carthami-Semen and Hwangryunhaedok-tang pharmacopuncture improved symptoms compared with normal saline injection , 15. Fornjection . Howevernjection , 14. Onenjection . Hominis Placenta Pharmacopuncture compared with distilled water injection was not assessable in leg spasticity of stroke patients due to the complexity of the results. For modified Ashworth scale (MAS), H-reflex/M-response ratio (H/M ratio), and Berge balance scale (BBS), there were no significant differences between groups, while the time up and go (TUG) in pharmacopuncture group was significantly lower than in distilled water injection group [The effect ofon group . Hominis Placenta Pharmacopuncture had a similar effect to acupuncture in dyspepsia [One study ofyspepsia . The effyspepsia . Hominis Placenta Pharmacopuncture for dysmenorrhea showed a similar effect to normal saline injection [One study onnjection . Hominis Placenta Pharmacopuncture had complicated results compared with normal saline injection [ Hominis Placenta Pharmacopuncture group showed significant higher VAS than normal saline injection group before the treatment (p = 0.023). After treatment, two groups reported similar thirst symptom (p = 0.510) without correcting the baseline value. Therefore, we could not assess the results.njection . There w Capsicum frutescens L. with normal saline and reported moderate AEs related to anesthesia cream or pharmacopuncture . In each of the two studies by Park et al. [Only five studies reported AEs. In the study by Seo , they cok et al. , 20, thek et al.  comparedk et al.  and Kim k et al.  reportedk et al. , 31–36 dWe performed sensitivity analyses by excluding studies with predefined less desirable characteristics, and the results from the musculoskeletal studies were robust.I2 = 0%). One study with adequate random sequence generation and/or allocation concealment did not provide enough information; thus, the effect of pharmacopuncture compared with normal saline could not be assessed in shoulder pain caused by stroke [When the analyses were limited to two studies with a low ROB for random sequence generation and/or allocation concealment , 29, phay stroke .There was no study with ≥40 participants per arm.Our review on pharmacopuncture aimed to establish the evidence of pharmacopuncture treatment of any disease. The analyses of two trials on obesity , 11 demoMost of the included studies had methodological weaknesses. Thirteen out of 25 studies , 30, 36 There were some limitations in this review. Our review only included trials conducted in Korea and published in Korean or English. Therefore, we could not necessarily remove a potential language bias. Egger et al.  reportedThe included trials were mostly of poor quality; thus, the reported data are likely to be overestimated. In addition, most of the included studies were small. Median sample sizes per arm were 17 in the pharmacopuncture group and 18 in the control group. The effect size of small studies may have been inflated due to poor methodological design and conduct . Moore eThe efficacy of the treatment used for the control group, such as acupuncture, herbal medicine, and tuina manual therapy, was not yet established; therefore, we could not attribute “positive” results solely to the effectiveness of pharmacopuncture. Additionally, clinically meaningful information on follow-up results was sparse in the majority of the included trials. Therefore, the available evidence prevented us from further examining how long its benefit was maintained.As we included all conditions/diseases, the focus of our review may seem blurred. However, this review provides an overview of the entire primary pharmacopuncture researches conducted in Korea. The results help to set priorities and directions for future research on pharmacopuncture.Although this review represented the applicability of pharmacopuncture, the standardisation of pharmacopuncture intervention was not performed. Thus, in the future, it is absolutely necessary to standardise it to apply pharmacopuncture in routine clinical practice. The degree of pharmacopuncture stimulation could be influenced by the following factors: (1) pharmacopuncture types; (2) concentration and extraction methods of pharmacopuncture; (3) amount, depth, and angle of injection; (4) syringe types, including thickness and length; (5) pharmacopuncture points; and (6) number of sessions based on the STRICTA guidelines . CurrentHowever, the standardisation of these factors has yet to be completed, and there is no firmly established research method for pharmacopuncture studies; therefore, pharmacopuncture interventions of the included trials were very heterogeneous. In addition, future studies should include not only a test of the efficacy and safety of pharmacopuncture but also an examination of the validity of the intervention based on the standardised guidelines.The results of this review demonstrate the effectiveness of pharmacopuncture for the treatment of obesity and musculoskeletal diseases compared with normal saline injections and other interventions, respectively; however, given the methodological flaws and small sample sizes, the available evidence is insufficient to recommend pharmacopuncture as an evidence-based treatment option. In the future, the standardisation of pharmacopuncture intervention and the adequate reporting of pharmacopuncture intervention in accordance with STRICTA guidelines are needed.


An endometrial polyp is a frequently encountered gynecologic disease with abnormal uterine bleeding and infertility being the two common presenting problems, and hysteroscopic polypectomy is an effective method to remove them. The postoperative polyp recurrence might result in reappearance of abnormal uterine bleeding or infertility, whereas factors influencing the postoperative recurrence potential have limited data.This case-series report included 168 premenopausal women who suffered from endometrial polyps and underwent hysteroscopic polypectomy. All of them were awaiting a future pregnancy. Office hysteroscopy was done before and after hysteroscopic polypectomy, in which preoperative hysteroscopy examined the number, type, and location of endometrial polyps, and postoperative hysteroscopy checked the polyp recurrence. Surgical indications, either infertility or the presentation of abnormal uterine bleeding, and follow-up duration were recorded.P = 0.015) and longer duration of follow-up (P = 0.004) were significantly associated with an increased risk of postoperative polyp recurrence. The type of endometrial polyps was not correlated with polyp recurrence potential, whereas pedunculated type endometrial polyps were closely related to the presentation of abnormal uterine bleeding (P = 0.001).Seventy-three out of 168 (43%) women had polyp recurrence after hysteroscopic polypectomy. Multivariate logistic regression analysis revealed that more endometrial polyps (A higher number of endometrial polyps and longer follow-up duration are associated with a greater potential of polyp recurrence after hysteroscopic polypectomy. Endometrial polyps are benign localized overgrowths of endometrial tissue, composed of glands, stroma, and blood vessels covered by epithelium . It is aEndometrial polyps can be diagnosed by ultrasound, sonohysterography, hysteroscopy and curettage. Among them, hysteroscopy is superior to the other three methods because it is able to detect the number, type, and location of endometrial polyps. After diagnosis, hysteroscopic polypectomy is now the gold standard for treatment. It is undertaken under direct visualization to completely remove the polyps with adjacent endometrium left intact . For infStudies demonstrated the postoperative recurrence rates of endometrial polyps to range from 2.5% to 43.6%, depending on the follow-up duration and the nature of polyps ,9. HyperInfertile women are more likely to suffer from endometrial polyps , which sWe enrolled premenopausal women who had endometrial polyps and underwent hysteroscopic polypectomy. There was endogenous estrogen produced by these women. The diagnosis was endometrial polyp without hyperplasia exclusively made by histopathologic examination, in which there were irregularly dilated endometrial glands and thick-walled vessels scattered in fibrotic stroma. These women were awaiting a future pregnancy, though some of them did not plan a pregnancy at that time. Only those who underwent office hysteroscopy both pre- and postoperatively were included. Finally there were 168 women in this retrospective case-series report.Their age, gravida, parity, surgical indications, and follow-up duration were recorded. Infertility was defined as inability of a couple having frequent intercourse and not using contraception to conceive within one year. Abnormal uterine bleeding was defined as prolonged menstrual bleeding more than seven days or the presentation of any intermenstrual bleeding. This study was approved by the Institutional Review Board (IRB) of the National Taiwan University Hospital. Since this descriptive study was done by reviewing medical records, obtaining the informed consent from participates was not mandatory according to the rules of IRB. The patient records/information was anonymized and de-identified prior to analysis.Office hysteroscopy was done in the follicular phase before hysteroscopic surgery for diagnosis and localization of endometrial polyps. It was carried out with Hysterovideoscope HYF type V . This flexible, single-flow hysterovideoscope has an outer diameter of 3.8 mm and a 120° field of view. The procedure has been described previously ,14. In bThe number of endometrial polyps was counted, and their types, either pedunculated or sessile, were observed. The type of endometrial polyps was determined based on the angle that is made between the polyp and the adjacent uterine wall. Pedunculated type polyps were defined with the angle of polyp surface to endometrium < 90 degrees , whereasHysteroscopic polypectomy was carried out in the follicular phase of the subsequent menstrual cycle. Operations were done exclusively by the same physician (JH Yang) with a 12-degree resectoscope bearing an outer diameter of 8 mm  ,14,15. TU test for continuous variables, and Fisher’s exact test for categorical variables. Univariate logistic regression analysis was done to examine the association between the risk of polyp recurrence and polyp number, as well as between the risk of polyp recurrence and follow-up duration. Multivariate logistic regression analysis was performed, in which recurrence of endometrial polyp was used as the dependent variable, while age, type of endometrial polyp, polyp number, and follow-up duration were used as independent variables.Data were expressed as either median (25%-75%) or mean ± SD. Differences between the groups were compared using Mann-Whitney P value < 0.05 was considered statistically significant. All statistical analyses were performed using the PC version of the Statistical Analysis System  and the Statistical Program for Social Sciences .Since 25%, 50%, and 75% values of the polyp number in all subjects were 1, 3, and 5 respectively, we then subgrouped them into n = 1, between 2 and 3, between 4 and 5, and ≥ 6. Cochran-Armitage trend test and multilinear regression analysis with dummy variables were carried out to examine the trend of polyp recurrence risk with the incremental levels of follow-up duration and polyp number. Kruskal-Wallis test was done to examine differences among three groups. A Clinical characteristics of 168 women in this study are shown in P = 0.010). The follow-up duration ranged from 2 to 73 months with a mean value of 18.8 months, and women with polyp recurrence had longer follow-up duration than those without polyp recurrence .Pedunculated and sessile type polyps accounted for 24% and 52% of the cases respectively, and another 23% women had both types of endometrial polyps. The number of endometrial polyps were between 1 and 15 with a mean value of 3.6, in which women with a polyp recurrence had a higher number than those without polyp recurrence . When we used polyp number = 1 as the benchmark, the risks of polyp recurrence were 1.01, 1.83, and 2.62 respectively in women having a polyp number 2–3, 4–5, and ≥ 6. After adjustment for age, polyp type, and follow-up duration, the risk of polyp recurrence in relation to the number of endometrial polyps was even higher   .P = 0.005 by Cochran-Armitage trend test). As we used follow-up duration < 1 year as the benchmark, the risks of polyp recurrence were 1.14, 1.8, and 3.25 respectively in women having follow-up duration 1–1.9 years, 2–2.9 years, and ≥ 3 years. After adjustment for age, polyp type, and polyp number, the risk of polyp recurrence in relation to the follow-up duration was even higher   .This is a study investigating the factors influencing the postoperative recurrence potential of benign endometrial polyps. After considering all the variables including age, polyp type, polyp number and follow-up duration, the multivariate regression model revealed that polyp number and follow-up duration, but not age and polyp type, were significantly associated with the risk of postoperative polyp recurrence. Our study focuses on benign endometrial polyps, and their recurrence potential increases as the follow-up duration is longer. This positive association between the recurrence rate and follow-up period is also found in endometriosis, in which the 2-year recurrence rate is 21.5% postoperatively, whereas the 5-year recurrence rate is as high as 40–50% .The type of endometrial polyps, either pedunculated or sessile, does not play a role in the postoperative recurrence potential, whereas pedunculated polyps are strongly associated with the presentation of AUB. The uterine cavity is a potential but collapsed cavity in normal conditions. Compared with sessile polyps, the surface of pedunculated endometrial polyps is prone to touch the endometrium of the opposite uterine wall, and AUB occurs consequently. The intracavitary bleeding might therefore interfere with embryo implantation .The postoperative polyp recurrence rate was 43% in this study, higher than those reported previously ,9. ThereSecondly, we performed hysteroscopic polypectomy without the application of diathermy. All visible endometrial polyps were bluntly removed with the cutting-loop under direct hysteroscopic vision. The reason why we did not use diathermy was to avoid deep injury to the endometrium or even myometrium as well as to prevent the formation of intrauterine adhesions, both of which might be unfavorable to future reproduction. Accordingly, the stalk and base of endometrial polyps might not be completely removed, which enhanced the risk of polyp recurrence. Comparatively, hysteroscopic resection using monopolar energy is highly effective in the treatment of endometrial polyps with a recurrence rate of 0–4.5% ,8. ThirdThe location of recurrent polyps was also evaluated with hysteroscopy in this study, and results revealed that a higher number of endometrial polyps were associated with an increased postoperative recurrence potential of polyps at the same location . AlthougHysteroscopic polypectomy is presently the gold standard for treatment of endometrial polyps. It is undertaken under direct vision to completely remove the polyps while preserving the adjacent endometrium . For infThe number of endometrial polyps and follow-up duration, but not the type of polyps, are major factors that determine the recurrence potential after hysteroscopic polypectomy. A higher number of endometrial polyps and longer follow-up duration are associated with a greater potential of polyp recurrence.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


Poor oral health is still a major burden for populations throughout the world, particularly in developing countries. The aim of this study was investigate oral health behaviour  and associated factors in low, middle and high income countries. Using anonymous questionnaires, data were collected from 19,560 undergraduate university students  from 27 universities in 26 countries across Asia, Africa and the Americas. Results indicate that 67.2% of students reported to brush their teeth twice or more times a day, 28.8% about once a day and 4.0% never. Regarding dental check-up visit, 16.3% reported twice a year, 25.6% once a year, 33.9% rarely and 24.3% never. In a multivariate logistic regression analysis, being a male, coming from a wealthy or quite well off family background, living in low income or lower middle income, weak beliefs in the importance of regular tooth brushing, depression and PTSD symptoms, tobacco use and frequent gambling, low physical activity, and low daily meal and snacks frequency were associated with inadequate tooth brushing (<twice daily). Further, being a male, older age, coming from a not well off or poor family background, living in low income or lower middle income, weak beliefs in the importance of regular tooth brushing, PTSD symptoms, illicit drug use, low physical activity, and low daily snacks frequency, skipping breakfast and inadequate fruit and vegetables consumption were associated with less than one annual dental care visit. Oral health behaviour among the students was found to be low. Various risk factors identified can be used to guide interventions to improve oral health behaviour among university students. Healthy habits and good oral hygiene, including twice daily tooth brushing, are critical in preventing gum disease and maintaining good oral health . The preVarious factors have been found to be associated with suboptimal tooth brushing among young people, including sociodemographics such as men ,14,15,16Few studies have been conducted on oral health in university students, in particular in low and middle income counries. Therefore, the aim of this study was to investigate oral health behaviour  and associated factors in low, middle and high income countries. The two hypotheses being that (1) male university students from lower socioeconomic backgrounds, having low physical activity, having more addictive risk behaviours, poorer dietary behaviour and poorer mental health are more likely to have poor tooth brushing behaviour, and (2) male university students from lower socioeconomic backgrounds and risk behaviours are more likely to have less than annual or no dental visits. This study adds to the existing research, in particular in the group of university students who are in a period of transition to take on personal responsibility for oral health behaviours and who will be future leaders and role models in their societies.This cross-sectional study was carried out with a network of collaborators in participating countries (see Acknowledgments). The anonymous, self-administered questionnaire used for data collection was developed in English, then translated and back-translated into languages  of the participating countries. The study was initiated through personal academic contacts of the principal investigators. These collaborators arranged for data to be collected from intended 400 male and 400 female undergraduate university students aged 16–30 years by trained research assistants in 2013 in one or two universities in their respective countries. The 27 universities involved were located in the capital cities or other major cities in the participating countries. Research assistants working in the participating universities asked classes of undergraduate students to complete the questionnaire at the end of a teaching class. Classes were selected through a stratified random sample procedure. The students who completed the survey varied in the number of years for which they had attended the university. A variety of majors were involved, including education, humanities and arts, social sciences, business and law, science, engineering, manufacturing and construction, agriculture, health and welfare and services. Informed consent was obtained from participating students, and the study was conducted in 2013. Participation rates were in most countries over 90%. Ethics approvals were obtained from institutional review boards from all participating institutions.Oral health behaviour was assessed with two questions, (1) “Do you brush your teeth? … Twice or more a day, about once a day, less than once a day, and seldom or never”  and (2) Socio-demographic questions included age, gender, and socioeconomic background were assessed by rating their family background as wealthy , quite well off (within the 50% to 75% range for their country), not very well off (within the 25% to 50% range from “country”), or quite poor  .Health beliefs in the importance of brushing teeth regularly were assessed with one question. The response option ranged from 1 = of very low importance to 10 = of very high importance .Centres for Epidemiologic Studies Depression Scale (CES-D). We assessed depressive symptoms using the 10-item version of the CES-D [he CES-D . Scoringhe CES-D . The CroPost traumatic stress disorder (PTSD). Breslau’s 7-item screener was used to identify PTSD symptoms in the past month [st month . Items ast month . The CroTobacco use was assessed with the question: Do you currently use one or more of the following tobacco products ? Response options were “yes” or “no” [ or “no” .Binge drinking was assessed with one item, “How often do you have (for men) five or more and (for women) four or more drinks on one occasion?” Response options ranged from 1 = never to 5 = daily or almost daily [st daily .Illicit drug use was assessed with the question, “How often have you taken drugs in the past 12 months; other than prescribed by the health care provider.”South Oaks Gambling Screen (SOGS), a standardized measure of pathological gambling and gambling behaviours in their lifetime [The lifetime  was usedPhysical activity was assessed using the self-administered International Physical Activity Questionnaire (IPAQ) short version, for the last 7 days (IPAQ-S7S). We used the instructions given in the IPAQ manual for reliability and validity, which is detailed elsewhere [lsewhere . We catelsewhere  as low, Fruit and vegetable (FV) consumption was assessed with two questions ‘‘How many servings of fruit do you eat on a typical day?’’and ‘‘How many servings of vegetables do you eat on a typical day?’’ using the 24-h dietary recall data as the gold standard . Insuffip < 0.05 level in bivariate analyses were included in the multivariate model. p < 0.05 was considered significant. Variance inflation factor (VIF) and tolerance values for each model indicate multicollinearity was not a concern in any of the multivariate analyses. Since the study used a clustered design, country was included as a clustering variable in the regression models.The data were analysed using STATA 11.00 . Descriptive statistics were used for reporting the proportion of oral health behaviour. We used univariate logistic regression, followed by multivariate unconditional logistic regression to obtain adjusted odds ratios (AOR) and associated 95% confidence intervals to assess the association between sociodemographic variables, health beliefs, addictive risk behaviour, low physical activity, poor mental health, dietary behaviour and oral health behaviour . All variables statistically significant at the In all, 21,639 university students were recruited, 20,222 agreed to participate and for 19,560 complete questionnaires of students in the age range of 16–30 years were available. The final sample included 19,560 university students (41.4% men and 58.6% women), with a mean age of 20.8 years (SD = 2.8). Overall, 67.2% reported to brush their teeth twice or more times a day, 28.8% about once a day and 4.0% never. Regarding dental check-up visit, 16.3% reported twice a year, 25.6% once a year, 33.9% rarely and 24.3% never. The overall prevalence of recommended tooth brushing frequency (twice or more a day) among university students differed by country, ranging from below 30% in Nigeria, India and Egypt, to about 90 or more percent in Thailand, Indonesia and Venezuela. Likewise, the dental check-up visit (at least once a year) differed by country, ranging from below 25% in Egypt, Ivory Coast, Nigeria, and South Africa to above 70% in Columbia, Venezuela, and Russia. The belief in the importance of tooth brushing was generally positively evaluated, with a mean of 8.7, on a scale from 1 to 10. However, university students from several countries had a weak belief in the importance of tooth brushing, e.g., Tunisia (5.7), Nigeria (6.5) and Egypt (7.2) see .In multivariate logistic regression analysis, being a male, coming from a wealthy or quite well off family background, living in low income or lower middle income, weak beliefs in the importance of regular tooth brushing, poor mental health (depression and PTSD symptoms), addictive risk behaviour (current tobacco use and frequent gambling), low physical activity, and dietary behaviour  were associated with inadequate tooth brushing (<twice daily). Further, being a male, older age, coming from a not well off or poor family background, living in low income or lower middle income, weak beliefs in the importance of regular tooth brushing, poor mental health (PTSD symptoms), addictive risk behaviour (past year illicit drug use), low physical activity, and dietary behaviour  were associated with less than one annual dental care visit see .The study found, among a large sample of university students from 26 low, middle and high income countries across Asia, Africa and the Americas, a large group of 32.8% engaged in inadequate or no tooth brushing, and 58.2% rarely or never had gone for a dental check-up visit. These findings seem to confirm low rates of tooth brushing and dental attendance in developing economy countries ,5,8,9,10However, the study found a large country variation in the overall prevalence of inadequate tooth brushing and less than annual dental visits, which in part confirms findings from previous studies of poor oral health practices in India, Nigeria, South Africa, and Turkey ,4,9,42, In concordance with a number of other studies ,25,26,27Further, the study found that living in a low income or lower middle income was associated with poor oral health behaviour , the same was true regarding coming from a not well off or poor family background and poor dental attendance, while coming from a wealthy or quite well off family background was associated with poor tooth brushing behaviour. Previous studies have generally found that lower socioeconomic background was associated with a poorer oral health behaviour  ,19,26,28Unexpectedly, students from more affluent economic backgrounds were less likely to brush their teeth twice of more a day. It is possible that in this group other factors such as lack of knowledge and cultural values about the importance of tooth brushing impact on poor oral health behaviour . UniversAfter adjusting for relevant socio-demographic variables, the current study findings indicate associations between poor oral health behaviour and weak beliefs in the importance of regular tooth brushing, poor mental health, and health risk behaviours, including addictive risk behaviours, low physical activity, inadequate dietary behaviours (skipping breakfast and insufficient fruit and vegetable consumption), which is largely consistent with findings of previous research ,23,25,29This study had several limitations. The study was cross-sectional, so causal conclusions cannot be drawn. The investigation was carried out with students from one or two universities in each country, and inclusion of other centres could have resulted in different results. University students are not representative of young adults in general, and the oral health behaviour levels, sociodemographic and health risk behaviour variables may be different in other sectors of the population. A further limitation of the study was that all information collected in the study was based on self-reporting. It is possible that rates of oral health behaviours were under or over reported. However, a previous study found that the self-reported dental care use was valid for some service types . FurtherThis study confirms low rates of tooth brushing and dental attendance among university students in different cultures across Africa, Asia and the Americas. Various factors identified, such as socioeconomic status, weak beliefs in the importance of regular tooth brushing and co-occurrence of general health risk behaviour can be used to guide interventions to improve oral health behaviour among university students. Oral health promotion programmes targeting the young adult population could help to improve their attitude and oral health behaviour to achieve good oral health.


Panax notoginseng) growth is often hampered by replant failure. In this study, we aimed to examine the role of autotoxicity in Sanqi replant failures and assess the role of ginsenosides in autotoxicity.Sanqi ginseng (The autotoxicities were measured using seedling emergence bioassays and root cell vigor staining. The ginsenosides in the roots, soils, and root exudates were identified with HPLC-MS.1, Rg1, Re, Rb1, Rb3, Rg2, and Rd, were identified in the roots and consecutively cultivated soil. The ginsenosides, Rg1, Re, Rg2, and Rd, were identified in the root exudates. Furthermore, the ginsenosides, R1, Rg1, Re, Rg2, and Rd, caused autotoxicity against seedling emergence and growth and root cell vigor at a concentration of 1.0 µg/mL.The seedling emergence and survival rate decreased significantly with the continuous number of planting years from one to three years. The root exudates, root extracts, and extracts from consecutively cultivated soils also showed significant autotoxicity against seedling emergence and growth. Ginsenosides, including ROur results demonstrated that autotoxicity results in replant failure of Sanqi ginseng. While Sanqi ginseng consecutively cultivated, some ginsenosides can accumulate in rhizosphere soils through root exudates or root decomposition, which impedes seedling emergence and growth. Panax notoginseng (Burk.) F. H. Chen], a member of the Araliaceae family, is one of the most important herbal medicines in China and is in high demand -) relative to the authentic standards  were clustered into one group and that other treatments  without significant autotoxicity were clustered into another group. The control was separate from all of the treatments. These results clearly indicated that the ginsenosides  present in the roots, cultivated soils and root exudates exerted important autotoxic effects on the seed germination and seedling growth at a concentration of 1.0 μg mL-1.The data from all of the treatments were analyzed using PCA to identify the primary factors causing the autotoxicity of Sanqi ginseng. Based on the PCA analysis, we observed that the control and treatments formed three different groups . The fir1, Rg1, Re, Rb1, Rg2 and Rd at a concentration of 1.0 μg mL-1 for 24 h . This finding is consistent with previous findings [1 in the plow layer of the soil from the Sanqi ginseng fields. In addition, Nicol et al. (2003) confirmed the presence of the Rb1, Rb2, Rc, Rd, Re, F11, and Rg1 ginsenosides in the soil extracts of American ginseng using HPLC-MS [1, Rb2, Rc, Rd, Re and Rg1) that were recovered from the root-associated soil were present in the root exudates [1, Re, Rg2, and Rd ginsenosides in the root exudates using HPLC-MS data. This finding could be explained by the small concentrations of these compounds in the root exudates or by the limitations of our detection method. Overall, it is likely that the ginsenosides found in the soils were partly derived from the root exudates or from root decomposition.Ginsenosides can accumulate in the rhizosphere soil of Sanqi ginseng from the decomposition of plant residues and from root exudates. In this study, we confirmed that eight major ginsenosides, namely Rfindings . More im HPLC-MS . These dexudates . In our -1 (dry weight) in the soil that was continuously cultivated for one to three years. Thus, we selected a ginsenoside concentration of 1.0 μg/mL to test the autotoxicity. These results indicated that the R1, Rg1, Re, Rg2, and Rd ginsenosides exerted autotoxic effects on seed germination and root cell vigor at a concentration of 1.0 μg/mL. The PCA analysis indicated that the autotoxicity of the R1, Rg1, Re, Rg2 and Rd ginsenosides partly overlapped with the autotoxicity shown in the extracts from the consecutively cultivated soil and fibrous roots. However, we found that the inhibitory effects of each ginsenoside were not as potent as the extracts from the soils and roots when tested separately. This finding may be explained by the coexistence of these ginsenosides in the soils, which will cause a stronger autotoxic effect when the ginsenosides function synergistically in natural settings. Similar results have also been reported for other plant species [Some ginsenosides showed autotoxicity against seed germination and seedling growth at rhizosphere-relevant concentrations. In this study, we found that the content of a single ginsenoside varied from 0.12 to 1.71 μg g species , 42. In  species . Thus, fP. quinquefolium and P. notoginseng pathogens, such as Cylindrocarpon destructans, Fusarium solani, Phytophthora cactorum, and Pythium irregulare [Fusarium oxysporum and autotoxic cinnamic acid may enhance soil sickness [Ginsenosides are primary components of ginseng and play a multi-purpose ecological role. Like other saponins, ginsenosides are fungitoxic in plants and act as a host for chemical defenses at high concentrations . In the regulare –30. If tsickness –45. ThusAutotoxicity is a cause of replant failure in Sanqi ginseng. Some ginsenosides can accumulate in soils through root exudates or root decomposition and show autotoxicity against seed germination and seedling growth at soil-relevant concentrations. These results provide new insights into the allelopathic mechanisms involved in Sanqi ginseng replant failure.S1 Dataset(ZIP)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.


Nevertheless, only a small proportion of individuals develop cirrhosis, however the large proportion of the population affected by NAFLD has led to predictions that NAFLD will become a leading cause of end stage liver disease, hepatocellular carcinoma (HCC), and indication for liver transplantation. HCC may arise in non-cirrhotic liver in the setting of NAFLD and is associated with the presence of the metabolic syndrome (MetS) and male gender. The MetS and its components also play a key role in the histological progression of NAFLD, however other genetic and environmental factors may also influence the natural history. The importance of NAFLD in terms of overall survival extends beyond the liver where cardiovascular disease and malignancy represents additional important causes of death.Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease in the world, paralleling the epidemic of obesity and Type 2 diabetes mellitus (T2DM). NAFLD exhibits a histological spectrum, ranging from “bland steatosis” to the more aggressive necro-inflammatory form, non-alcoholic steatohepatitis (NASH) which may accumulate fibrosis to result in cirrhosis. Emerging data suggests fibrosis, rather than NASH  The prevalence of non-alcoholic fatty liver disease (NAFLD) parallels that of obesity, which has steadily risen throughout the world over the past thirty years . The natNon-alcoholic fatty liver disease (NAFLD) encompasses a histological spectrum from non-alcoholic fatty liver (NAFL), which is characterized by steatosis with no or minor inflammation, to non-alcoholic steatohepatitis (NASH) where inflammation and ballooning is present, with or without fibrosis. The natural history of NASH tends to parallels the more aggressive histological picture, with prospective cohort studies demonstrating a higher rate of morbidity and mortality compared to NAFL, particularly when fibrosis is present ,11. NeveAt least 12 studies have analysed the progression of steatosis, steatohepatitis, and fibrosis in NAFLD cohorts by utilizing paired liver biopsies ,18,19,20No consistent relationship between sex and fibrosis has been found in NASH, with cross-sectional studies reporting conflicting findings ,26. The Hispanic patients have an increased prevalence of NAFLD compared to Caucasians; however, there appears to be no difference in degree of liver injury between these ethnic groups ,30. In cPNPLA3 and TM6SF2 genes are common in the general population with minor allele frequencies of 20%–50% and 10%, respectively [i.e., NASH and fibrosis) [IFNL4 gene, which is associated with response to interferon based treatment in chronic hepatitis C, has also been associated with fibrosis in NAFLD and has been amalgamated into a predictive score in conjunction with other clinical factors [Polymorphisms in the ectively . The rs7ibrosis) ,36,37,38ibrosis) . Similar factors .Cross-sectional studies have demonstrated increasing age to be consistently associated with more severe fibrosis in NASH patients; however, this may reflect the cumulative sum of metabolic exposures and longer duration of NAFL/NASH in these populations ,40,41. IDiabetes and obesity have demonstrated to be predictive of a higher rate of fibrosis progression in some but not all longitudinal studies ,9,12,22.The degree of hepatic steatosis does not appear to predict disease progression in NASH. The degree of inflammation however, has been associated with progression to advanced fibrosis in a meta-analysis, but not in any single cohort study .Overall, the risk of progression to cirrhosis and decompensation in NAFLD patients is low with a population based study demonstrating an incidence of 3.1% for both end-points over a mean 7.6 year follow-up . NeverthThe risk of progression to end-stage liver disease is influenced by the severity of underlying liver histology; the majority of patients with NAFLD have simple steatosis, however, up to 30% of patients may have NASH  and are et al. noted that a large proportion of patients with cryptogenic cirrhosis had been exposed to metabolic risk factors [Once cirrhosis has developed, the risk of developing a major complication of portal hypertension is 17%, 23%, and 52% at one, three, and 10 years, respectively . The sur factors  and almo factors .Compared with individuals of the general population of the same age and gender, those with NAFLD have a lower than expected survival, at a standardized mortality ratio from 1.34 to 1.69 according to American and Swedish studies ,12. The NAFLD is associated with a significantly higher overall mortality compared to the age and sex-matched general population, which in part is likely related to excess vascular as well as liver-related death. Cross-sectional population-based studies and meta-analysis have demonstrated NAFLD to be independently associated with predictors of cardiovascular disease including endothelial dysfunction, arterial stiffness and myocardial dysfunction ,57,58,59vs. 7.5%) over a mean follow-up period of 13.7 years [Supporting these observations, analysis of over 11,000 participants in the NAHNES study conducted between 1989 and 2004 with median follow up of 14.5 years, demonstrated increased (69%) overall mortality in NAFLD patients with advanced fibrosis assessed by means of NAFLD fibrosis score, APRI and FIB 4. The increase in mortality in this subgroup was largely driven by cardiovascular disease (CVD) (adjusted hazard ratio 2.7 to 3.5) . Other c.7 years .et al. [i.e., non-NASH) increased their activity score and 28% had fibrosis progression at three years. Fibrosis progression was seen in 20% to 30% of patients with both low and high NAS scores. Twenty-three per cent of patients with simple steatosis developed NASH in 3 years.A pioneer research published in 2006 compared the levels of serum concentrations of transforming growth factor-beta1 (TGF-β1) a marker of fibrosis, and ferritin between NAFL, and NASH patients . No diffet al.  reportedet al. [A retrospective study analysing a database of 70 NAFLD patients with paired biopsies showed that patients with NAFL can evolve towards well-defined steatohepatitis, and in some of them, bridging fibrosis after a follow-up of less than 5 years. The presence of mild lobular inflammation or any amount of fibrosis substantially increased the risk of histological progression in the mid-term while those with steatosis alone are at lowest risk . More reet al.  in the Det al. conducted a systematic review and meta-analysis of 11 studies involving 411 patients with paired liver biopsies [vs. 14.3 years, respectively). Collectively, these studies suggest that overall NAFL has a more indolent rate of progression than NASH; however, there is considerable heterogeneity, with one quarter of NAFL patients developing bridging fibrosis over a relatively short time period. Currently, reliable histological and clinical predictors of disease progression are lacking, however it appears that worsening metabolic disease  frequently parallels the histological progression [Lastly, Singh biopsies . Patientgression ,9.The prognosis of an individual patient with NAFLD is highly variable. A greater likelihood of progressive disease was initially described in those patients with NASH, which is often defined according to the NAFLD activity score (NAS score). The NAS is the unbalanced sum of steatosis, ballooning, and lobular inflammation , and waset al. of 209 NAFLD patients with a median follow up of 12 years found that advanced fibrosis was the only histological lesion independently associated with liver-related mortality  [p < 0.001). In contrast patients with a high NAS (5–8) without severe fibrosis did not have increase mortality compared with reference population. Finally, Angulo et al. conducted an international multicentre cohort study to determine the long term prognostic significance of histologic features of NAFLD [Recent evidence coming from prospective cohort studies suggest that fibrosis predicts liver and non-liver related mortality more reliably than NAS or its individual components ,65,66. A.5–21.4) . OverallPNPLA3 rs738409 variant [HCC is the six most common cancer worldwide, the third most common cause of cancer related death and has a globally rising incidence ,68. Seve variant .NASH was found to be the third most common risk factor for HCC in a U.S. veterans population of 1500 with HCC diagnosed over a six year period . Neverthvs. 4.0%, p = 0.09) [The cumulative incidence of HCC in NASH cirrhosis ranges between 2.4% and 12.8% over a 3.2–7.2 year period, and the cumulative HCC mortality in NAFLD/NASH cohorts is 0%–3% over 5.6–21 years . A large = 0.09) . These f = 0.09) ,78. All  = 0.09) ,79. Inte = 0.09) .Once HCC develops in NAFLD cirrhotic patients, survival appears to be shorter survival than patients with HCV-HCC . This maThe development of HCC in non-cirrhotic patients with NAFLD is increasingly reported with cross-sectional studies demonstrating between 15% and 50% of cases being diagnosed without cirrhosis ,81,85,86Not surprisingly, HCC associated with non-cirrhotic NAFLD is less likely to be detected during surveillance and thus is more likely to be more advanced when compared to HCC in cirrhosis patients ,81,84. NPNPLA3 and TM6SF2 genes, influence the severity of underlying liver histology and, thus, are likely to impact on risk of developing cirrhosis and HCC. Recent studies have demonstrated NAFL in addition to NASH, may lead to progressive fibrosis and have emphasized the importance of fibrosis level in determining future mortality risk. A greater understanding of the factors that alter the natural history of NAFLD will lead to better prognostication and targeting of NAFLD populations at greatest risk for specific therapies.NAFLD is common in the general population, however the natural history and impact on patient morbidity and mortality is widely divergent. Metabolic factors, such as diabetes, obesity, and hypertension, as well as common genetic polymorphisms in the