Metabolic syndrome (MS) can promote the development of cardiovascular disease (CVD). The objective of this study was to examine the association of MS and its components with CVD, to further prevent and control CVD in Kazakhs.In the cohort study, a total of 2644 participants completed the baseline survey between April 2010 and December 2012.The follow-up survey was conducted from April 2016 to December 2016 and was completed by 2286 participants (86.46% follow-up rate). Cox regression was used to evaluate the association of each component and the number of combinations of MS components on the development of CVD.A total of 278 CVD patients were enrolled from rural residents of Xinjiang. The average age of the MS and non-MS groups was 46.33 and 38.71 years, respectively. Independent associations with CVD were found for elevated blood pressure (BP) (adjusted hazard ratio (HR) [aHR] = 1.50,95%confidence interval [CI]: 1.08–2.08), elevated waist circumference (WC) , and elevated triglycerides (TG) . Participants with one to 5 MS components had an increased HR for developing CVD, from 1.82to 8.59 (P for trend < 0.001), compared with those with no MS components. The risk of developing CVD increased when TG and WC coexisted ), when TG and BP coexisted , and when WC and BP coexisted ). However, no significant interactions were found between BP, WC, and TG.Elevations of BP, WC, and TG were independent risk factors for CVD in Kazakhs. Control of these factors is important to prevent CVD in this population. Cardiovascular disease (CVD) has become the leading cause of death in China . AlthougMetabolic syndrome (MS) is a highly prevalent constellation of vascular risk factors that include elevated blood pressure (BP), elevated blood glucose, obesity, and dyslipidemia . A meta-Xinjiang, a province in northwestern China, is a multiethnic settlement that includes nomadic Kazakh individuals. Owing to their special ethnicity, living environment, and genetic characteristics, there is a growing double epidemic of dyslipidemia and obesity in Kazakhs –10. To oA four-stage cluster random sampling was used to obtain a representative sample. In the first stage, according to the geographical distribution of the minority populations in Xinjiang, we selected Yili as the representative prefecture. Yili is approximately 4407 km (2739 miles) from Beijing. Approximately 98% of the population belong to the Kazakh minority. In the second stage, we randomly selected Xinyuan County from the Yili Prefecture. In the third stage, we randomly selected sex villages from Xinyuan County. In the final stage, we randomly selected residents ≥18 years of age who had resided in their village for at least 12 months. We excluded those with serious illness, unawareness, and unwillingness to cooperate, as well as pregnant women. A total of 2644 participants completed the baseline survey between April 2010 and December 2012. To improve the rate of follow-up, subjects were interviewed in-person and in household surveys. The follow-up survey was conducted from April 2016 to December 2016. The follow-up rate of 86.46% (2286 of the 2644 participants). The median follow-up period was 5.49 person-years . We then excluded 281 participants who had a history of CVD including coronary heart disease (CHD), stroke, and hypertension at baseline. Thus, as of December 2016, 2005 participants were eligible for the final analyses. Within the follow-up period, a total of 278 individuals developed , and BP according to standard methods . Blood sParticipants who had first-ever occurrence of stroke (ischemic or hemorrhagic), CHD, or hypertension during the follow-up period were diagnosed as having CVD. Subjects with CHD were hospitalized during the follow-up period if coronary intervention (cardiac catheterization or coronary bypass surgery) was performed, if there was angina (or nitroglycerin was initiated after cohort study), if myocardial infarction occurred, and because of congestive heart failure. Data regarding CVD events were obtained from the hospital medical records and questionnaire responses. If the same type of event occurred two or more times, the first occurrence was considered as the end event. CVD were determined based on self-reported questionnaire responses, medical insurance records, and local hospital discharge records from 2010 to 2016. Patients with self-reported manifestations of CVD findings required a certificate of diagnosis from a medical institution in their township during the investigation.According to the third report describing treatment of adult Americans as part of a modified United States National Cholesterol Education Program (2005 NCEP-ATP III) [Traditional risk factors used in the data analysis included age, sex, drinking (drinker/non-drinker), and family history of hypertension, diabetes, and CVD.P <  0.05 was considered statistically significant.The database construction and some statistical analyses have been previously described . Cox proTable As was expected, MS and the individual components of MS were significantly associated with the risk of developing CVD, independent of age, sex, drinking, and family history of hypertension, diabetes, and CVD. After adjusting for four other MS components and the traditional risk factors above, the elevated BP , elevated WC  and elevated TG  were still independently associated with CVD (Table P < 0.001), compared to those with no MS components. This trend persisted even after adjusting for age, sex, drinking, and family history of hypertension, diabetes, and CVD  to 8.59 (95%CI:4.43–16.67) , 1.27 (95% CI: 0.74–2.21), and 2.16 (95% CI: 1.54–3.04) for the subgroups without TG and with WC, with TG and without WC, and with TG and WC, respectively (Table Numerous studies have suggested that MS is a cluster of CVD risk factors –16. A mePresently, each component of MS appeared to be was associated with an increased prospective risk of CVD. Furthermore, as the number of MS components increased, the risk of CVD also increased, with a significant and cumulative-component response trend. These findings suggest the presence of a cumulative effect of MS components in the elevated risk of CVD. These are important findings because the relationships between clustering patterns of MS and CVD risk have not been thoroughly characterized in Kazakh populations. This synergistic association is also noteworthy as it provides valuable information for the establishment of appropriate policies in preventive CVD for the inhabitants of Xinjiang. This linear synergistic correlation has been previously reported in other ethnic groups , 22.The independent association of MS and its components in predicting CVD has become a subject of recent research interest. However, which MS component is more strongly associated with CVD remains unclear. In the Asia Pacific region, up to 66% of some subtypes of CVD can be attributed to hypertension . A more Current research found the elevations of BP, WC, and TG were independent risk factors for CVD in the Kazakh population. Suh et al.  found thHypertension is a risk factor for the development of atherosclerosis. Both clinical and experimental data show that elevated BP enhances the development of atherosclerosis. In fact, atherosclerosis tends to occur only in those parts of the vascular system subjected to high pressure . The eleWe analyzed further the interaction between TG and WC or between WC / TG and BP and explored whether their coexistence was an additional risk factor for CVD. When TG and WC coexisted, the aHR was 2.16, which indicated that the accumulation of TG and WC strengthens the CVD risk. Therefore, it is plausible that the coexistence of TG and WC constitutes the highest CVD risk. If so, the growing double epidemic of obesity and dyslipidemia among Kazakh people may greatly and rapidly increase their burden of developing CVD. Therefore, the dietary intervention play an important role in decreasing the incidence of CVD and improving the quality of life in the Kazakh population.This study is a large-scale prospective cohort study of a Kazakh population with a long follow-up period that reports the association of MS with CVD incidence. Our study had some limitations. First, the diagnosis of MS was based on a single measurement of its components at baseline, as was the case in other epidemiological studies . Second,Elevated BP, elevated WC, and elevated TG are independent risk factors for CVD in Kazakhs. Therefore, it is important to control BP, WC, and TG to prevention CVD in this population.


Background and Aims: Non-invasive fibrosis staging is essential in metabolic associated fatty liver disease (MAFLD). Transient elastography (TE) is a well-established method for liver fibrosis assessment. We have previously shown that the macrophage marker sCD163 is an independent predictor for fibrosis in MAFLD. In the present study we tested whether the combination of macrophage markers and TE improves fibrosis prediction.Methods: We measured macrophage markers soluble (s)CD163 and mannose receptor (sMR) in two independent cohorts from Italy (n = 141) and Sweden (n = 70) with biopsy-proven MAFLD and available TE.Results: In the Italian cohort, TE and sCD163 showed similar moderate associations with liver fibrosis . TE had an area under the Receiver Operating Characteristics curve  for fibrosis; F ≥ 2 = 0.79 (0.72–0.86), F ≥ 3 = 0.81 (0.73–0.89), F4 = 0.95 (0.90–1.0). sCD163 also predicted fibrosis well . However, combining sCD163 and TE did not improve the AUROCs significantly . In the Swedish cohort, TE showed a closer association with fibrosis  than sCD163  and sMR . TE predicted fibrosis well , whereas sCD163 did not (best AUROC 0.75). sMR showed a better prediction , but the addition of sMR did not further improve the prediction of fibrosis by TE.Conclusion: In these cohorts of MAFLD patients, TE was superior to macrophage markers for fibrosis prediction and in contrast to our hypothesis the addition of these markers to TE did not improve its predictive capability. Metabolic associated fatty liver disease (MAFLD), a newly instituted and more appropriate term for non-alcoholic fatty liver disease (NAFLD) , is an iTransient elastography (TE) provides the value of liver stiffness as a measure of fibrosis. It has shown good accuracy in MAFLD, particularly for advanced fibrosis and cirrhosis detection , and hasMacrophages play an important role in MAFLD , and we We hypothesized that combining the macrophage markers sCD163 or sMR with TE would result in improvement of fibrosis prediction, and we tested this hypothesis in two independent cohorts of MAFLD patients with biopsy-proven disease.This cross-sectional study was performed in two established cohorts of MAFLD patients from liver centers in Italy and Sweden. The Italian cohort consisted of patients from the Division of Gastroenterology and Hepatology, Department of Medical Sciences, University of Torino, Italy, while the Swedish cohort comprised patients included at the Karolinska University Hospital, Stockholm, Sweden. Both of these cohorts have been described in previously published reports , 17.At both sites, all patients were referred for the investigation of abnormal liver tests or steatosis detected by ultrasound, and MAFLD was diagnosed by liver biopsy. In the Italian cohort, all patients had an alcohol intake of <20 g/day assessed by interviews with the patient or close family members, and in the Swedish cohort an intake of <30 g/day  an <20 g/day , assessed with phosphatidylethanol (PEth) and the alcohol-use disorders identification test (AUDIT) and the lifetime drinking history (LDH) questionnaires , 19.Liver disease of other etiology was excluded. The number of patients with biopsy-proven MAFLD and available TE was 141 in the Italian cohort and 70 in the Swedish cohort. At the time of liver biopsy, demographic and clinical data were recorded, including age, gender, ethnicity, height, weight, and waist circumference. Body Mass Index (BMI) was calculated. Diabetes was defined as hemoglobin A1c ≥ 48 mmol/mol, fasting blood glucose ≥7.0 mmol/L, previous diagnosis of diabetes or use of anti-diabetic drugs.At the time of biopsy, a fasting blood sample was obtained and routine biochemical tests were performed. Additional blood samples were drawn and frozen at −80°C for future research. All patients signed an informed consent form in accordance with the Helsinki Declaration. The acquisition, storage, and use of blood samples were approved by the Ethics Committee of the University Hospital San Giovanni Battista of Torino and by the Regional Ethics Committee of Stockholm (2011/13-31-/1 and 2018/134-32).Liver and hematological parameters, fasting glucose and insulin, triglycerides and cholesterol and its components were determined using standard assays and methods. All sCD163 and sMR measurements were performed at the Department of Clinical Biochemistry, Aarhus University Hospital.This was done in duplicate by in-house enzyme-linked immunosorbent assays (ELISAs) using a BEP-2000 ELISA-analyzer (Dade Behring) essentially as previously described . SolubleLiver biopsies were stained and examined locally by experienced pathologists as described by Kleiner et al.  and in aVibration controlled transient elastography  was performed within 2 weeks prior to the liver biopsy by expert operators in accordance with the instructions by the manufacturing company, including at least 3 h of fasting. The M probe was used as standard, and the XL probe as per the automatic probe selection tool or when the M probe failed.TE was expressed in kilopascal (kPa) and calculated as the median value of 10 successful acquisitions, defined by a success rate of >60%, and by an interquartile range <30%.2) + 1.13 × impaired glucose tolerance/diabetes mellitus  + 0.99 × aspartate aminotransferase/alanine aminotransferase – 0.013 × platelets (x109/L) – 0.66 × albumin (g/dL) (9/L) × square root )]  + 0.094 × body mass index (kg/mn (g/dL) . The FIBe(U/L))] .t-test was used for the comparison of normally distributed variables between the groups. For non-normally distributed data, the Mann-Whitney test was used. The relationship between sCD163/sMR and TE was analyzed by linear regression. Spearman's rank test was used to study the relationships of TE, sCD163, and sMR with histological scores. For differences in proportions, we used the χ2-test or Fisher's exact test.Student's SD and medians with interquartile ranges (IQR) or proportions. A p ≤ 0.05 was considered statistically significant. STATA version 14.0®StataCorp LP was used for data analysis.Multiple ordered logistic regression was used to assess the relationship between the histological fibrosis score and TE, sCD163, and sMR. This analysis provides odds ratios (OR) describing the increase in the odds for a given fibrosis stage in a patient who has a specific increase in a parameter compared with another patient. We chose to present the results corresponding to a 25% increase in TE, sCD163 and sMR based on our previous experience , 28 and Demographic, clinical, biochemical, and histological data for the patients from both cohorts are shown in p < 0.001), as shown in p < 0.001; p < 0.001; p < 0.001; p < 0.001; In the Italian cohort, sCD163 showed a moderate association with fibrosis  in the Italian cohort. In the same analysis including TE and sCD163 in patients from Sweden, TE showed a significant association  and sCD163 showed a trend . Similarly, the multivariate analysis with TE and sMR as the explanatory variables resulted in a significant association by TE  and a trend by sMR . When including both TE, sCD163, and sMR in the analysis in the Swedish cohort, TE remained significantly associated with fibrosis , whereas sCD163 and sMR lost significance .In the multiple ordered logistic regression analysis, both TE and sCD163 were significantly associated with the stage of fibrosis . We thus determined the predictive characteristics of TE and the combined model (TE + sCD163) for advanced fibrosis (F ≥ 3) based on its importance for long-term outcome in MAFLD . We usedIn the Swedish cohort, the AUROCs of sCD163 and sMR were moderately good, whereas TE performed even better. Adding sCD163, sMR or both of these markers to TE failed to change its AUROCs , for whiBesides fibrosis, the presence of necroinflammatory activity and steatohepatitis may be determinants of progressive disease and pharmacological response, and a combination of at least significant (≥F2) fibrosis, steatohepatitis and NAFLD activity score (NAS) ≥4 has been proposed to identify patients at risk and inclusion into clinical trials . In the p = 0.43] in the Italian cohort. In the Swedish cohort, this was the case for both sCD163  and sMR . Furthermore, looking at the histological grades of steatosis, lobular inflammation and hepatocyte ballooning, sCD163 was only significantly associated with steatosis in the Italian cohort, whereas sMR tended to associate with ballooning in the Swedish cohort. TE showed no significant correlation with any histological measures other than fibrosis in any cohort  vs. 1.52 (1.15–2.24) mg/L, y cohort .We calculated the established markers of liver fibrosis NAFLD Fibrosis Score and FIB-4 in both cohorts. In the Italian cohort, FIB-4 and NFS had higher AUROCs for F ≥ 2 compared with sCD163, whereas sCD163 had higher AUROCs for F ≥ 3 and F4 stages . A combiIn the Swedish cohort, FIB-4 had higher AUROCs than both sCD163 and sMR for all fibrosis stages. Combining FIB-4 with sMR provided higher AUROCs for F ≥ 2 and F ≥ 3 stages than both these markers, whereas combinations with sCD163 did not result in additional predictive value.NFS showed moderate prediction for fibrosis in the Swedish cohort, however, a combination of sCD163 and NFS had an AUROC for F ≥ 2 higher than both these markers alone .In this study of two independent cohorts of patients with biopsy-proven MAFLD and available TE, we measured macrophage markers sCD163 and sMR and combined them with TE hypothesizing that this would improve the prediction of fibrosis. The markers independently predicted fibrosis moderately well, but in contrast to our hypothesis, adding sCD163 and sMR to TE did not significantly improve its predictive capability.Our study has strengths and limitations. The key strength was the well-characterized patients with histologically classified disease in two independent cohorts. Robust well-established and validated methods including the XL probe were used for the measurement of liver stiffness and for macrophage markers. The most significant limitation was the lower number of patients in the Swedish cohort, which inherently could raise the issue of inadequate power, however, as the combinations of TE with macrophage markers had generally lower AUROCs than TE alone, we do not believe that a larger number of patients in this cohort would have resulted in a better performance of the combined models. A more general drawback may be the cross-sectional design of the study and its focus on liver histology. Liver biopsy is an imperfect gold standard due to potential sampling error and interobserver variability, and the possible resulting bias is not easily predictable.Combinations of elastography with biochemical markers and composite scores have been explored in MAFLD before. Gaia et al. showed no improvement in the fibrosis prediction of TE with the addition of ultrasonographic and biochemical measures in a smaller study . A largeIn our study, sCD163 and sMR predicted fibrosis with moderate accuracy. Furthermore, both sCD163 and sMR showed independent associations with fibrosis in the multiple ordered logistic regression analysis with TE as a covariate. These findings supported our hypothesis and held promise of an add-on value of the macrophage markers for fibrosis prediction when combined with TE. Of note, in the Italian cohort the combination of sCD163 and TE did in fact result in numerically higher AUROCs than TE alone, as well as higher NPV and PPV for advanced fibrosis. However, the increase in the AUROCs was not significant and while the NPV of the combined model was strong at 92%, TE in itself already had an adequate NPV at 88%. At the same time, the PPV of the combined model remained low at 62%, a value not fit to rule in advanced fibrosis. In the Swedish cohort, the AUROCs of the combined models were even slightly lower compared to TE. Thus, based on our data, sCD163 and sMR did not contribute to the detection of advanced fibrosis beyond TE.Several explanations for this result can be considered. In line with earlier studies, combining sCD163 and TE led to a higher PPV in the Italian cohort, which however was too low for practical use. This may partly be explained by the distribution of fibrosis in the Italian cohort with only 23% having fibrosis stage 3 or higher. The prevalence of disease affects predictive values, and in this case, favors NPV over PPV. Thus, sCD163 may have had a more pronounced impact on PPV in a cohort with more frequent advanced fibrosis. However, the distribution of fibrosis stages does not explain the similar AUROCs of TE and combined models in the patients from the Swedish cohort, as they had more severe fibrosis. The lack of an improvement of TE prediction by addition of sCD163 and sMR may be rooted in the nature of these biomarkers. sCD163 and sMR were not developed with the purpose of fibrosis detection, but are functional markers of macrophage activation, and have been associated with liver fibrosis reflecting the roles of macrophages in liver disease and fibrogenesis . In contBoth sCD163 and sMR failed to help detect the combination of significant fibrosis, steatohepatitis and necroinflammation suggested as the FAST algorithm to identify patients with progressive disease suitable for clinical trials , and theIn our study, the AUROCs of sCD163 were higher compared with NFS and FIB-4 in the Italian cohort, whereas FIB-4 was superior to sCD163 and sMR in the Swedish cohort. Interestingly, in several instances the combinations of the macrophage markers with NFS/FIB-4 showed AUROC values higher than the single markers, which suggests that sCD163 and sMR may be valuable as add-ons to the biomarker-based screening of MAFLD patients in the setting of primary care. However, this should be further tested in primary care cohorts more suitable for this purpose due to the expectedly lower prevalence of advanced fibrosis compared with our cohorts of patients referred to tertiary centers.per se, and other more fibrosis specific markers may be more useful and could be explored in future studies.In conclusion, despite significant independent associations with fibrosis in two cohorts of patient with MAFLD, macrophage markers sCD163 and sMR did not improve the prediction of fibrosis by TE in combined models. This may be attributed to the nature of these markers as related to macrophage activation and not fibrosis The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the University Hospital San Giovanni Battista of Torino and Regional Ethics Committee of Stockholm. The patients/participants provided their written informed consent to participate in this study.KK: study design, conception, data analysis, interpretation, and manuscript preparation. CR, RY, AA, and HH: study subject inclusion, data acquisition, interpretation, and critical revision of the manuscript for intellectual content. HM: data analysis, interpretation, and critical revision of the manuscript for intellectual content. PS and EB: study design, conception, administrative support, data interpretation, and critical revision of the manuscript for intellectual content. HG: study design, conception, obtained funding, data interpretation, study supervision, and critical revision of the manuscript for intellectual content. All authors have approved the submitted manuscript.RY was employed by the company Boehringer Ingelheim International, Gesellschaft mit beschränkter Haftung, Ingelheim, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Osteosarcoma (OS) is the most common primary malignant tumor of the skeletal system in the clinic. It mainly occurs in adolescent patients and the pathogenesis of the disease is very complicated. The distant metastasis may occur in the early stage, and the prognosis is poor. MicroRNAs (miRNAs) are non-coding RNAs of about 18–25 nt in length that are involved in post-transcriptional regulation of genes. miRNAs can regulate target gene expression by promoting the degradation of target mRNAs or inhibiting the translation process, thereby the proliferation of OS cells can be inhibited and the apoptosis can be promoted; in this way, miRNAs can affect the metabolism of OS cells and can also participate in the occurrence, invasion, metastasis, and recurrence of OS. Some miRNAs have already been found to be closely related to the prognosis of patients with OS. Unlike other reviews, this review summarizes the miRNA molecules closely related to the development, diagnosis, prognosis, and treatment of OS in recent years. The expression and influence of miRNA molecule on OS were discussed in detail, and the related research progress was summarized to provide a new research direction for early diagnosis and treatment of OS. The current treatment is mainly surgery combined with neoadjuvant chemotherapy. However, surgery cannot effectively control the metastasis of tumors, and some patients are still not sensitive to chemotherapy. These problems, which led to the lung metastasis of tumors, have become major problems in the treatment of OS; so, improving the existing treatment concepts and looking for further new treatment technologies are imperative. miRNAs are endogenous single-stranded small RNAs of about 18–25 nt in length, which are involved in post-transcriptional regulation of genes, and have important effects on gene expression, biological development, and behavior  found th2The occurrence and development of OS is affected by many factors including genetics, inflammation, and environment. Other factors include age, gender, ethnicity, and preexisting bone abnormalities. It is a very complicated pathophysiological process. Changes in miRNA expression profiles are involved in the development and progression of OS through a variety of complex direct or indirect regulatory actions. Some miRNAs promote the proliferation and differentiation of OS cells by significantly upregulating expression in OS . The stuThere are also significant expression reductions in various miRNAs in OS, which may regulate the development and progression of OS through some important negative regulatory mechanisms. Such as Sun et al.  added diNovello et al.  have fou3At present, the clinical diagnosis of OS mainly depends on the imaging examination and tissue biopsy. However, with the in-depth study of miRNA function mechanism in OS, the change of miRNA in blood circulation can also be used as a non-invasive auxiliary index for diagnosis and prognosis of OS. After the wide application of clinical chemotherapy, the long-term survival rate of OS patients has been significantly improved ,16. TumoP = 0.001), tumor volume (P = 0.01), and distant metastasis (P = 0.001) showed a significant positive correlation, and multivariate analysis showed that serum high miRNA-191 levels were independent risk factors for short-term mortality (P = 0.01). de Azevedo et al. [P = 0.01) and high tumor clinical stage (P = 0.006). The AUC for the diagnosis of OS was 0.844, and patients with high miRNA-221 levels had a worse prognosis. Another study included 166 patients with OS. The results showed that miRNA-27a differentially diagnosed OS and healthy controls with an AUC of 0.867, and high miRNA-27a expression levels with higher clinical stage (P = 0.001), distant metastasis (P = 0.01), and poor response to chemotherapy (P = 0.008) [n = 114), where the serum miRNA-300 levels in OS patients were significantly higher than those in healthy patients, and the AUC for diagnosis of OS was 0.884, and multivariate regression analysis showed that miRNA-300 , clinical stage , and distant metastasis  were independent prognostic factors affecting the overall survival of OS patients. In addition, such as studies by Lian et al. [An area under the curve (AUC) of 0.8606 for diagnosis of OS and a significant reduction in miRNA-199a-5p after OS surgery  can be u= 0.008) . Liu et n et al.  have shoP = 0.001), and miRNA-126 was associated with higher TNM staging (P < 0.001), and Kaplan–Meier analysis showed that patients with a lower miRNA-126 expression had a shorter overall survival time (P = 0.008)  .4In recent years, many studies have shown that miRNA molecules not only participate in the regulation of OS growth microenvironment, tumor resistance, and tumor cell exocytosis, but can also be used to prepare nanoparticles and target OS. The analysis of the mechanism of action of miRNA molecules in the above events will help provide new ways and means for the clinical treatment of OS. The following are introduced to participate in the regulation of OS growth microenvironment, tumor resistance, tumor cell exocytosis function, and miRNA molecules for nanotargeted therapy and their functional characteristics.4.1Many miRNAs are upregulated or downregulated in different types of tumor microenvironments, effectively regulating the expression of these miRNAs and inhibiting tumor growth. Xu et al.  found thYe et al.  observed4.2Long-term use of certain chemotherapeutic drugs in patients with OS is easy to induce gene mutations, resulting in drug resistance, and thereby increasing the difficulty of treatment of OS. In response to this problem, many scholars have studied the mechanism of OS resistance and the key miRNA molecules involved in the regulation of drug resistance. Zou et al.  found thIn summary, miRNA molecules are involved in the drug resistance behavior of OS. They can not only be used as a novel biomarker for judging whether or not OS produces drug resistance, but can also regulate the drug resistance behavior of OS and become an important treatment target for OS ,35,36,384.3miRNAs in OS exosomes can be released into body fluids , which can serve as potential biomarkers for disease. Fujiwara et al.  collecte4.4With the rapid development of nanotechnology, the application of nanoparticles to carry miRNAs, through the nanosize effect (EPR effect), has become possible to achieve targeted therapy of tumors. Zhang et al.  found th5In summary, miRNAs play an important role in the development, invasion, metastasis, and prognosis of OS. The differential expression of miRNAs in OS cells provides a new target for the early diagnosis and treatment of OS. The study of drugs affects the expression level of miRNAs in OS cells, which in turn affects the progression and prognosis of tumors, and provides a new direction for the study of molecular targeted drugs for the treatment of OS –46. Howe


The 2015 MERS outbreak in South Korea was the largest event outside of the Middle East. Under such circumstances, individuals tend to resort to non-conventional solutions such as complementary and alternative medicine (CAM) to manage health. Thus, this study aims to examine characteristics of CAM use among outpatients in a community hospital setting during the 2015 MERS outbreak and to assess potential predictors of CAM use during the epidemic.p-value of less than 0.05 was considered statistically significant for all analyses.A cross-sectional study was conducted among 331 patients (response rate: 82.75%) at a community hospital located in Seoul, South Korea. The survey instrument included 36 questions on the use of CAM, demographic characteristics, health status, and respondents’ perceptions and concerns about MERS infection. Chi-square test and logistic regression were conducted for data analysis using SPSS ver. 21.0., and a 76.1% of respondents used one or more types of CAM modalities during the MERS outbreak. Consumption of easily accessible modalities such as multivitamin (51.2%) and food products (32.1%) was most popular, and the majority of CAM users relied on mass media (52.4%) and the internet (27.4%) to obtain information on CAM. The use of CAM was associated with age between 40 and 49, age over 50, prior CAM use, and dissatisfaction with the government response to the MERS outbreak.CAM was commonly used by outpatients during the 2015 MERS outbreak in Korea, and mass media was the main source of information. Establishing a media platform is of paramount importance to provide reliable information and ensure the safety of its use. Recent emerging infectious diseases such as Severe Acute Respiratory Syndrome (SARS), Avian Influenza, and Middle East Respiratory Syndrome related coronavirus (MERS-CoV) continue to be a major public health concern . The 201As such, individuals exposed to risks and uncertainties tend to seek self-care measures and resort to non-conventional solutions such as complementary and alternative medicine (CAM) in order to demonstrate their autonomy, mitigate the risk, and make the situation more manageable –15. AlsoIn Korea, the use of CAM is widespread and culturally embedded as traditional Korean medicine (TKM) is recognized as a part of mainstream medicine in the Korean healthcare system. TKM is easily accessible in communities as TKM providers are available in all three levels  of healthcare facilities. Some of its modalities, such as acupuncture, moxibustion, cupping, and herbal preparations, have been covered by the national health insurance since 1987, and patients can choose between TKM and conventional medicine based on their preferences . TherefoHowever, previous studies on CAM use during epidemics focus on the clinical efficacy of CAM modalities –25. ThosA descriptive cross-sectional study was conducted among patients attending the outpatient departments at a community hospital located in Seoul, South Korea. All outpatients over the age of 18 were eligible to participate in the survey. Exclusion criteria were those who were admitted to the inpatient unit at the time of the survey, unable to speak or understand the survey questionnaire.z0.025 is 1.96 which corresponds to the confidence interval of 95%. This equation gave the minimum sample size as 328, and we distributed a total of 400 survey questionnaires assuming a 20% non-response rate.The formula for sample size determination using confidence interval (CI) of proportion was used to calculate the sample size:  studies , 31, d iThe semi-structured survey questionnaire was developed in the Korean language  and the level of self-perceived interest in health-promoting behavior of respondents . The second section contained 11 questions regarding respondents’ perceived danger and severity of MERS outbreak , concerns about MERS infection , the opinions on the trust in government response during the MERS outbreak, and source of information about MERS outbreak. The third part consisted of seven questions on CAM use , and the last section had nine questions on respondents’ sociodemographic characteristics such as age, gender, marital status, level of education, occupation, level of income, religion, and area of residence.The survey was conducted from November 26th to December 2nd, 2015. Two surveyors were trained and recruited for data collection, and the information about the research was given verbally to each respondent. The respondents were asked to complete the questionnaire by themselves, but if respondents did not understand certain questions, they were able to ask the surveyors for clarification. All participants were assured of their confidentiality and completed IRB-approved written informed consent before the survey. A total of 400 self-completed questionnaires were distributed, but 69 participants did not return or complete the questionnaire (response rate: 82.75%); therefore, the data of 331 respondents were included in the final analysis.p-value of less than 0.05 was considered statistically significant for all analyses.The collected data of 331 respondents were analyzed using the Statistical Package for Social Sciences (SPSS) version 21.0. Descriptive statistics were used to examine sociodemographic and health-related characteristics of respondents. Pearson’s Chi-square test was performed to identify differences in sociodemographic characteristics and perceptions and concerns experienced during the MERS outbreak between CAM users and non-users. Lastly, multivariate logistic regression analysis was conducted to determine potential predictors of CAM use during the MERS outbreak. The significant factors from previous chi-square test  were subjected to the regression analysis. A The ethical approval of the study was obtained from the Institutional Review Board on Human Subjects Research and Ethics Committees, Hanyang University, Seoul, Korea (HYI-15-210-1).Table p = 0.003), age (p = 0.001), marital status (p = 0.035), occupation (p = 0.001), religion (p = 0.006), smoking status (p = 0.003), alcohol consumption (p = 0.041), and prior use of CAM (p < 0.001) between CAM users and non-users were observed76.1% of respondents have used at least one of the listed CAM modalities during the MERS outbreak, and significant differences in gender , followed by to prevent disease and maintain/promote health (53.2%). Also, 72.6% of CAM users were willing to recommend CAM to others. As the reason for non-use of CAM among 79 non-CAM users, 27.8% responded that they do not believe that CAM is relevant to disease treatment, and 26.6% reported that they do not trust in the effectiveness of CAM  and self-perceived danger of the MERS outbreak (p = 0.017) was significantly greater than that of non-CAM users. A more significant proportion of CAM users (48.8%) regarded government response to the MERS outbreak as inappropriate (p = 0.020), and CAM users practiced more hand-washing (p = 0.011) and refrained from physical contact with others (p = 0.010) compared to the non-users.CAM users’ concern for the lack of definitive treatment for MERS infection  and the internet, which were the least popular among patients with chronic disease and non-airborne infectious disease , 47–49. Lastly, several factors were identified as potential predictors of CAM use during the MERS outbreak. As for sociodemographic characteristics, previous studies found female gender, younger age, and tertiary education level to be associated with CAM use among people with risk of disease development , 55. HowLimitations of this study include recall bias and lack of variability in research locations. The survey was implemented in November and December of 2015, which was near the end of the MERS epidemic in Korea, so recall bias may affect the result. Also, the participants of the study were selected from patients visiting outpatient departments of a community hospital in Seoul; therefore, the result of the study may not fully represent those of other people in Korea. Lastly, cross-sectional studies can only infer the association between CAM use and potential predictor variables.During the 2015 MERS outbreak in Korea, the prevalence of CAM use was considerably high among patients with older age, prior CAM use, and dissatisfaction with the government response to the MERS outbreak. Also, the majority of respondents reported the mass media and the internet as the main source of CAM and disease-related information. In the era where emerging infectious diseases continue to be a major public health concern, the effective use of mass media and information and communication technology should be considered to achieve efficient delivery of health information and to promote behavior changes in situations of vulnerability. Therefore, public health authorities should build an integrated platform that provides proven and reliable information during public health emergencies to protect the health of citizens.Additional file 1. Survey questionnaire. Survey questionnaire developed to examine the pattern of CAM use among Koreans during the 2015 MERS outbreak in Korea.


The area under the receiver operating characteristic (ROC) curve was 0.978 (p < 0.001), with the cut-off set to 1.37 mg/mL, which was characterized by 80% of sensitivity and 100% of specificity. The high sensitivity and specificity demonstrate the screening utility of maternal ApoE concentration in prenatal fetal DS screening.Prenatal screening for Down syndrome (DS) is based on both noninvasive and invasive methods. Noninvasive, cell-free fetal DNA genetic tests are expensive, whereas biochemical methods remain imprecise. Amniocentesis is the most frequently used invasive diagnosis procedure, characterized by 99.8% diagnostic efficiency and less than 1% risk of miscarriage. The aim of this study was to evaluate the screening value of apolipoprotein E (ApoE) as a potential noninvasive biomarker for prenatal DS assessment. This study was conducted on a group of female patients who decided to undergo routine amniocentesis between the 15th and 18th week of pregnancy at the Department of Reproduction and Gynecological Endocrinology of the Medical University of Bialystok, Poland. For the purpose of this study, 20 women with DS fetuses were selected as the study group, and 20 healthy pregnant women with euploid fetus karyotypes as the control group. The plasma levels of ApoE were significantly higher in the study group compared to healthy subjects ( Trisomy 21, also known as Down syndrome (DS), is one of the most frequently occurring chromosomal disorders worldwide . TrisomyApolipoprotein E (ApoE), a glycoprotein with a linear polypeptide chain rich in arginine, is a hydrophilic component of high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), lipoproteins, and chylomicrons . It is mNowadays, prenatal screening of DS is based on both noninvasive methods, which estimate the risk of DS-affected pregnancy, and invasive techniques, which verify the presence of chromosomal aberrations. Serum screening and ultrasounds are intended to identify women with pregnancies at high risk of chromosomal abnormalities. However, diagnostic testing is indicated in international guidelines, mainly focusing on individual risk assessment based on historical, biochemical, and biophysical variables . AmnioceThe aim of the study was to evaluate the screening usefulness of maternal ApoE measurement as a potential noninvasive marker in prenatal diagnostics of DS.The study and control groups consisted of women who underwent routine amniocentesis between the 15th and 18th weeks of gestation at the Department of Reproduction and Gynecological Endocrinology of the Medical University of Bialystok, Poland. The indication for amniocentesis was an increased risk of chromosomal aberrations in noninvasive prenatal screening or patient age above 35 years. Exclusion criteria were as follows: chronic or acute diseases, hormonal treatment, anti-inflammatory treatment, high-risk pregnancy, or preterm delivery in the patient’s medical history. All participants were informed about potential risks prior to the procedure and received relevant information regarding the study. Study participants were matched according to age, ethnicity, socioeconomic status, the course of pregnancy, body mass index (BMI), and the number of pregnancies with marked episodes of pregnancy pathology. The patient recruitment period started in 2017 and lasted 2 years. Following karyotype test result analysis, 20 women carrying fetuses with DS and 20 women with euploid (non-DS) fetuses were enrolled in the study. All patients were submitted to amniocentesis within a determined period, then randomized. An adequate sample size to detect a difference was demonstrated using power analysis [p > 0.05). Thus, the groups were compared using a parametric two-way ANOVA test; p < 0.05 was considered statistically significant. In addition, the receiver operating characteristic (ROC) curves were determined using a medical package included in the Statistica program. Diagnostic sensitivity and specificity were calculated using a cut-off value that was calculated by the Youden’s index (as a criterion for selecting the optimum cut-off point) [Plasma ApoE concentration was determined using an enzyme-linked immunosorbent assay (ELISA) (ELISA Kit for apolipoprotein E (ApoE); Cloud-Clone Corp., Wuhan, Hubei 430056, China, SEA704Hu) according to the manufacturer’s protocol and observing the principles of internal laboratory control for the performed determinations. Samples and controls were measured in the same run using the blind analysis method. Duplicate samples were assessed and the average of the two results was calculated . Statistf point) .The procedures were approved by the Local Ethics Committee of the Medical University of Bialystok, Poland, and written informed consent was obtained from each participant (R-I-002/36/2014).In this research, the first participant was entered on 1 January 2015. The last participant was randomized on 12 April 2019, at the end of the trial. A total value of 100 pregnant women were screened, and 40 were randomized and enrolled for the subsequent analysis.p < 0.001). The comparison of ApoE concentrations measured in the study vs. control group is presented in Basic statistics that were measured in the study, control, and total group, such as average ApoE concentration values, minimum and maximum values, and standard error, are presented in To determine the diagnostic utility of the ApoE test, the ROC curve was calculated as an illustration of the relationship between sensitivity and specificity . The cutp < 0.001).To evaluate the clinical applicability of ApoE as a prenatal screening tool, the area under the ROC curve (AUC) was evaluated. The AUC value was 0.978, which was significantly higher in comparison to AUC 0.5, which is the threshold of the diagnostic usefulness of a test  and child neurodevelopment, while considering genetic ApoE polymorphism. The study revealed that the presence of the APOE ε4 allele combined with Hg exposure resulted in a decline in cognitive performance in the studied children . Pinto eIn our study, plasma ApoE levels were considered low, but comparable to those found in the study by Kaneva et al., where the shift in plasma ApoE toward lower levels in European residents as a result of specific features of lipid metabolism was demonstrated . We alsoThe study performed by Rindler et al. identified the placental ApoE synthesis as the major maternal lipid profile modifier during pregnancy. ApoE has been suggested to play a supportive role in regulating maternal and fetal homeostasis ,35,36. AIn our study, women with a confirmed DS pregnancy had significantly higher plasma ApoE concentrations compared with healthy subjects. The traceability of commonly used biochemical noninvasive tests can be characterized by PAPP-A tests, with sensitivity estimated at 90% with 5% false-positive results, and the triple test , whose sensitivity is estimated at 60–70% ,12,13,14Our study demonstrated the relationship between maternal ApoE and fetal DS occurrence, and showed that ApoE can be used as a predictive marker of this disease, but further studies are required. The discovery of dysregulated metabolic pathways could lead to the establishment of new diagnostic targets, which may enable optimal early diagnosis, resulting in improved therapy application and enhanced quality of life.


Legionella pneumophila isolates from the 1980s to the present day, deriving data from clinical and/or water samples from studies carried out all over the world. Eighty-nine papers meeting the inclusion criteria, i.e., “Legionella pneumophila” and “resistance to antibiotics”, were evaluated according to pre-defined validity criteria. Sixty articles referred to clinical isolates, and 18 articles reported water-related L. pneumophila isolates, while 11 articles included both clinical and water isolates. Several methods have been proposed as suitable for the determination of MICs, such as the E-test, broth and agar dilution, and disk diffusion methods, in vivo and in vitro, using various media. The E-test method proposed by the European Society of Clinical Microbiology and Infectious Diseases (EUCAST) seems to be the second most frequently used method overall, but it is the preferred method in the most recent publications (2000–2019) for the interpretation criteria. Erythromycin has been proved to be the preference for resistance testing over the years. However, in the last 19 years, the antibiotics ciprofloxacin (CIP), erythromycin (ERM), levofloxacin (LEV) and azithromycin (AZM) were the ones that saw an increase in their use. A decrease in the sensitivity to antibiotics was identified in approximately half of the reviewed articles.The current systematic review investigates the antibiotic susceptibility pattern of  The extraordinary genetic capacities of microbes have benefitted from man’s overuse of antibiotics to exploit every source of resistance genes . Multidr problem .Pseudomonas aeruginosa being the bacterium that has been approached as a priority [An increasing awareness of the antibiotic-resistance crisis related to drinking water consumption seems to be growing in the scientific community, even though the number of relevant papers in the literature is not high. Antimicrobial resistance in bacterial pathogens (ARBs) of water microbiomes and ARGs in drinking water pose a significant public health concern when they transfer antibiotic resistance to human pathogens, a situation which is still unclear . The effpriority .Legionella pneumophila is a facultative intracellular pathogen known to cause community-acquired pneumonia (CAP). Monitoring the emerging issue of the antibiotic resistance of L. pneumophila isolates is a recent issue in the treatment of Legionnaires’ disease (LD) [ase (LD) . The antase (LD) , which hase (LD) ,19,20,21L. pneumophila isolates derived from clinical and/or water samples in accordance with (a) chronology, (b) geographical distribution, (c) the groups of antibiotics and (d) the methods used and the corresponding interpretation for the identification of microbial susceptibility.The aim of this systematic review is to focus on the antibiotic susceptibility pattern of L. pneumophila and susceptibility to antibiotics. The database search was performed between October and December 2019. The search was performed using various key terms and their variations in combination using a Boolean search technique ” were preset as “inclusion criteria” in the review. Papers meeting the inclusion criteria were evaluated according to pre-defined validity criteria  of the authors; each author was responsible for one or two database searches, which were repeated twice for reasons of reproducibility (the same number of articles emerged). Additionally, articles in compliance with the set were evaluated by all of them for reasons of reliability. The data extracted from the eligible studies are presented in The analysis of the extracted data was performed using the standard Excel worksheets and the OriginLab 2020 software . The graphs  were basIn deciding which antibiotics should be selected to work with, the following criteria were considered: (a) the most cited antibiotics in the revised articles included in the study  raw dataAs for the methods of assessment for microbial susceptibility, six main categories were identified: agar dilution, E-testing, plating, broth dilution, microdilution and disk testing  values for each antibiotic. These were the ECOFF value guidelines proposed but not yet officially established by the European Society of Clinical Microbiology and Infectious Diseases (EUCAST) and the ones proposed by Bruin et al. [Two horizontal lines for n et al. , a refern et al.  has alson et al.  and Bruin et al.  suggest n et al. . The EUCn et al. . During n et al. ,33. ThisThe study selection process is presented in Legionella isolates that originated from two major outbreaks, both of which occurred in the USA , 23 articles were published in the 1990s (25%), and 11 articles were from the 1980s (11%). The oldest article identified in the present search was by Thornsberry et al. , who rep the USA  raw data the USA  raw dataL. pneumophila isolates (20%) while 11 articles included both clinical and water isolates (12.4%). The above results are subject to the restriction that clinical articles were more abundant than environmental ones (60 vs. 18); this fact is a limitation within the research and, therefore, of this review.Sixty articles referred to clinical isolates (67.4%) and 18 articles reported water-related L. pneumophila strains were related to CAP. There are fewer studies than expected considering the spread and the severity of the disease as well as the antibiotic scheme applied for therapy. In some papers dealing with both clinical and environmental strains, there is no discrimination between the two types of strains. One of the reasons for this discrepancy is that L. pneumophila usually causes a non-productive pneumonia, and it is difficult to obtain respiratory secretions for the isolation of the pathogen before the administration of any antibiotic [L. pneumophila, the determination of the minimum extracellular concentration inhibiting intracellular multiplication could be necessary next to conventional MIC determinations.The type of specimen, in most cases being Broncho Alveolar Lavages/BAL and secretions from Respiratory Tract Infections/RTIs for the clinical isolates, was reported in 27 articles , and all of them were related to patients with CAP  raw datatibiotic . Neverthtibiotic  evaluateL. pneumophila [L. pneumophila isolates were inhibited in the following decreasing order: doxycycline > tigecycline > cefotaxime. Moreover, the MICs of azithromycin, ciprofloxacin, levofloxacin, moxifloxacin and tigecycline were significantly lower for L. pneumophila Serogroups 2–15 than for L. pneumophila Serogroup 1. In another Italian study [Legionella strains were inhibited by low concentrations of macrolides and fluoroquinolones, while between the macrolides, clarithromycin was, overall, the most active drug compared to azithromycin. Again, differences in the MICs between L. pneumophila Serogroup 1 and Serogroups 2–15 were detected. Xiong et al. have reported the increased effectiveness of levofloxacin [L. pneumophila strains [The site of sampling for the water-related isolates was reported in the 79.3% of the relevant articles 23/29,  raw dataumophila . In mostumophila , severalan study , the autan study ,42,43,44floxacin , followefloxacin . Contrarfloxacin  who revefloxacin . Similarfloxacin  where tefloxacin  studied  strains ,50.L. pneumophila isolate set. From the above-extracted data, it can be concluded that—until recently, when a protocol was proposed by the EUCAST [L. pneumophila’s microbial resistance, as well as common criteria for interpreting the results , has led many researchers to select whatever published protocol is appropriate for each study. Due to this variety in the application of the methods, it is difficult to compare and correlate the published data. This serves to enhance the difficulty of studying the microbial susceptibility of L. pneumophila. This limitation has been cited in the published literature. For example, a study was carried out by Sharaby et al. [Several methods have been proposed as suitable for the determination of MICs, such as the E-test, broth and agar dilution, and disk diffusion methods, in vivo and in vitro. Nonetheless, none of these methods is considered as a gold standard . All 89 e EUCAST —the lacky et al. , where iL. pneumophila’s antimicrobial susceptibility. However, in seven articles, two or three different culture media were used in order (a) to compare the results (https://mic.eucast.org/Eucast2/SearchController/search.jsp?action=performSearch&BeginIndex=0&Micdif=mic&NumberIndex=50&Antib=1&Specium=230). The difficulties in determining the MICs for clinical strains have been well explained. This arises from the fact that the acquisition of a suitable specimen from a patient before the administration of antibiotics is not always feasible [Legionella [L. pneumophila. However, as the committee states, it should be used as guidance for antibiotic resistance rather than for the guidance of treatment [In 80 articles, only one medium was used for the identification of  results  raw datafeasible . Combinegionella ,43,46,47gionella  of each reatment .L. pneumophila’s antimicrobial sensitivity from all the reported methods are presented in L. pneumophila isolates    raw datand 1990s  raw dataL. pneumophila? Historically—since the discovery of the progenitor macrolide, erythromycin, in 1950—many derivatives have been synthesized. This has led to compounds with better bioavailability and acid stability and improved pharmacokinetics. These efforts led to the second generation of macrolides, including well-known members such as azithromycin and clarithromycin. Subsequently, to address increasing antibiotic resistance, a third generation of macrolides displaying improved activity against many macrolide resistant strains was developed. However, these improvements were accompanied by serious side effects, leading to disappointment and causing many researchers to stop working on macrolide derivatives once treatments had reached the end [Legionella’s microbial susceptibility follows the routine clinical practice as prescribed by each patient’s doctor. As a result, they do not test for susceptibility according to standard and approved protocols. The selection of the antibiotics tested according to the provenance of the isolates is of interest. As for resistance testing in isolates from water samples, ciprofloxacin seems to be the first choice , followed by erythromycin (66%), azithromycin (55%) and rifampicin (55%). Levofloxacin and clarithromycin were both reported in nine articles (50%), and doxycycline, in seven (39%)   that was applied by each research group.As for the geographical distribution of the use of antibiotics, in Europe, ciprofloxacin seems to be the antibiotic of choice . Meanwhile, erythromycin was most commonly cited in articles from the USA  and Asia   and macrolides (clarithromycin and azithromycin) have potent antimicrobial activity against The above conclusions need to be evaluated in light of the limitations of the data, such as the relatively high percentage of the non-defining-resistance articles; the papers dealing with clinical isolates outnumbering the ones dealing with water samples; and, finally, the large difference in the available information between the tested time periods.


In addition, the foot contact area increased (p = 0.09). Therefore, CCI-induced pain was significantly remitted and spontaneous pain was remitted after repeated administration of NYT. Serum BDNF levels were higher in untreated CCI rats than in normal rats (p = 0.05), but decreased after the repeated administration of NYT , while serum corticosterone levels were lower (p = 0.12) than those in normal rats and increased after the repeated administration of NYT . The blood BDNF level has been suggested to influence pain intensity. The findings demonstrated NYT effectively treats neuropathic pain, suggesting that a NYT-induced decrease in blood BDNF contributed to the mechanism of pain relief. In addition, the variation of corticosterone was observed, suggesting that normalization of responsiveness to stress by NYT contributed to the pain relief.Kampo medicines are frequently used empirically to treat pain in clinical practice. Ninjin'yoeito (NYT), which is associated with few adverse effects, is often used to treat the elderly, but has not yet been examined in detail. We herein investigated the effects of NYT, at 500 and 1,000 mg/kg p.o. (NYT500/NYT1000 group) in single and repeated administrations for 14 days, on pain in rats with peripheral neuropathy induced by loose ligation of the sciatic nerve (chronic constriction injury: CCI). Untreated CCI rats given distilled water were used as a control group. To assess induced pain, the pain threshold was measured using the von Frey test. To evaluate spontaneous pain, the ground-contact area of the paw with neuropathic pain was measured using the Dynamic Weight Bearing test. Serum samples were collected after the test to elucidate the mechanism of action of NYT, and brain-derived neurotrophic factor (BDNF) and corticosterone protein levels, which have been reported to change due to chronic pain, were analyzed. After single administration of NYT, the pain threshold rose in the NYT500 and NYT1000 groups. The pain threshold tended to rise on day 14 of repeated administration in the NYT500 group ( In particular, these drugs must be administered with caution to mental disorder patients and the elderly.Chronic pain has been estimated to affect ~25 million individuals in Japan , and hasin vitro system, identifying that ginsenoside Rg3 is one of the active ingredients . The CCI model was prepared following the method created by Bennet et al. . Rats weThe dried extract powder of NYT (Lot No. 15112017) was used in the present study, and was manufactured by the GMP Pharmaceutical Factory of Kracie Pharma, Ltd. . The von Frey test was performed before NYT administration and animals with CCI which developed pain were selected (pain threshold: 6.0 g or lower). The selected CCI rats were divided into the following 3 groups: Control group, NYT 500 mg/kg treatment group (NYT500), and NYT 1,000 mg/kg treatment group (NYT1000), so as to make no significant difference in the pain threshold among the groups. Then, NYT was administered at 10 mL/kg B.W. once a day for 14 consecutive days starting on the 14th day post-surgery as follows: In the Control group, distilled water was orally administered. In the NYT500 and NYT1000 groups, NYT was suspended with distilled water before use and the specified dose was orally administered. The drug was administered by collaborators and efforts were made to ensure the experimenters were blinded in evaluation of the subsequent von Frey test and dynamic body weight bearing.Mechanical allodynia in rats was evaluated using the von Frey test. In the von Frey test, a series of calibrated von Frey filaments  with a bending force ranging between 1 and 15 g were applied to the midplantar skin of each hind paw at a rate of once per second. Specifically, the paw was stimulated 10 times at a speed of once per second in the order from the filament with the lowest strength and when escape behavior was noted once or more, the rat was judged as positive. Then, stimuli were added using the one-step weaker filament when the animal was positive and using the one-step stronger filament when the animal was negative. Stimulation was repeated until observing positive and negative responses to stimulation with 2 continuous filament types, respectively, and the filament strength to which a positive response was observed was recorded as the pain threshold. Fourteen days after the CCI surgery, the pain threshold of the left foot was measured before administration and on day 1  and day 14  after initiation of administration. The pain threshold of the right foot without surgery before initiation of administration was recorded as the baseline.2) of the foot contacting the floor. The testis and tail were excluded from analysis. The results were presented as the area of the left foot. Fourteen days after the CCI surgery, measurement was performed before administration and on day 14 after initiation of administration (repeated administration) in each rat. The value measured in the right foot without surgery before initiation of administration was recorded as the baseline.The dynamic weight bearing test  was performed referring to the method reported by Quandros et al. . BrieflyBlood samples were collected immediately following the sacrifice of each rat and clotted for 30 min at room temperature before centrifuging for 20 min at 2,000 rpm. The supernatant was then collected for an enzyme-linked immunosorbent assay (ELISA). Serum was stored at −40°C until assayed for brain-derived neurotrophic factor (BDNF) and corticosterone. Serum BDNF  and serum corticosterone  were measured by ELISA following the manufacturer's instructions. Normal rats given distilled water were used as a Normal group.post hoc test followed by Steel test for multiple comparisons. Between normal group and control group were compared by Student's t-test. P < 0.05 were considered to be significant.Data were expressed as the mean ± standard error of means. Significant differences were assessed by a one-way analysis of variance followed by Dunnett's test, or After single administration of NYT, the pain threshold rose in the NYT500 and NYT1000 groups   and it significantly rose at NYT1000 , but decreased after the repeated administration of NYT   .p = 0.12, by Student't-test) than those in normal rats, and increased after the repeated administration of NYT   .We herein evaluated the efficacy of NYT to prevent neuropathic pain in CCI rats. Mechanical allodynia and spontaneous pain were significantly induced by CCI surgery and inhibited by NYT. These results are consistent with clinical findings. In addition, repeated administration of NYT decreased blood BDNF whereas blood corticosterone increased.p > 0.05)  . In addi > 0.05) , and the > 0.05) . These rWhen the serum corticosterone level was measured in rats influenced by chronic pain, it was lower than that in normal rats. It has been reported that in an SD rat continuous stress model, a significant increase was observed on day 1, the level gradually decreased from day 2, and no significant difference from the level in the control was noted on day 5 , suggestMultiple mechanisms resulting from sciatic nerve injury have demonstrated that demyelination, ectopic discharge, and macrophage infiltration are closely associated with the development of neuropathic pain behaviors , 36. MyeAll datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by the Experimental Animal Care Committee of Kracie Pharma, Ltd. Written informed consent was obtained from the owners for the participation of their animals in this study.RiT, SM, L-KH, NF, and RyT performed the experiments and analyzed the data. SM, NF, and RyT initiated and supervised the study. RiT, SM, NF, and L-KH designed experiments. RiT and L-KH wrote the manuscript. All authors contributed to the article and approved the submitted version.All authors are employees of Kracie Pharma, Ltd.


Specifically, a misperception of the SVV was found in patients with migraine  and with TTH . This review shows a misperception of VV in patients with migraine and TTH when assessed with the SVV test, being higher in patients with TTH, although the THH sample size was low.Migraine and tension-type headache (TTH) are the two most prevalent primary headache disorders (PHDs) that may involve visual and vestibular impairments, neck pain, and postural unsteadiness. The perception of visual verticality (VV) has been studied in patients diagnosed with PHD to assess balance disorders showing varying findings. Our study aimed to assess the VV perception in patients diagnosed with PHD in comparison to healthy controls. A systematic review with meta-analysis was carried out in PubMed MEDLINE, Scopus, WOS, CINAHL, and SciELO. The Cohen standardized mean difference (SMD) was used to estimate the differences between exposed and healthy controls. Seven studies with 816 participants were included. The quality of included studies, according to the Newcastle–Ottawa Scale (NOS), was moderate (mean score of 5.2). Patients diagnosed with PHD showed a moderate misperception of VV as assessed with the subjective visual vertical (SVV) test (SMD = 0.530; 95% CI = 0.225, 0.836;  Primary headache disorders (PHDs) are a significant worldwide public health problem . AlthougIt has been possible to observe how certain alterations frequently present in conjunction with headache. In particular, comorbidity between neck pain and headache disorders has been widely assessed in numerous studies ,6,7,8. AThe representation of vertical at the central level makes control of spatial orientation and body stabilization processes possible . Visual The perception of verticality can be assessed through the estimation of one’s own body verticality , the estimation of the verticality of objects that appear in the visual field  or by touch  . The maiDue to the relationship observed among vestibular, visual, and somatosensory alterations in patients with primary headaches, it could be possible to deduce a possible disturbance in vestibular, visual, or somatosensory inputs in patients with headache. Thus, patients with headache could misperceive verticality because of the important role that these three systems of information play in the formation of the central tridimensional pattern of verticality. Different studies have analyzed the visual and vestibular contribution to the sense of verticality in patients with migraine and TTH with varying results. Differences may be able to be explained by methodological differences in the use of the SVV test, or the correct diagnosis of patients with PHD. In light of the differing results of previous studies and the absence of reviews on the subject, our systematic review with meta-analysis aimed to collate the available evidence and analyze the perception of VV in patients with PHD compared to healthy subjects.To perform this meta-analysis, authors have followed the recommendations proposed in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  statemenThe search strategy was performed in PubMed, Scopus, Web of Science, CINAHL, and SciELO by two authors (E.O.-G. and R.L.-V.), independently, between 1 February 2020 and 30 March 2020. The authors also examined reference lists from retrieved full-text studies, guidelines, and reviews. The keywords used in the bibliographic search strategy were “headache disorders, primary,” “migraine disorders,” “tension-type headache,” “subjective visual vertical,” and “perception of visual verticality.” Medical Subject Headings (MeSH), EBSCOhost, and the main keywords found in previous published studies were chosen as keywords. According to the database, a specific keyword combination was employed using the appropriate tags and the Boolean operators “and” or “not”. No publication date and language filters were used. In Studies reported with the search strategy for possible inclusion were assessed in an independent screening for each of the two blinded authors who performed this phase (E.O.-G and R.L.-V). Irrelevant studies were excluded based on the screening of the title and abstract. Each reference was assessed in detail if at least one of the researchers selected it during the title and abstract review. Each reviewer re-evaluated the discrepant studies, and disagreements were resolved by consultation with a third researcher (D.R.-A.) during the full-text review.The inclusion criteria applied were: (1) observational studies ; (2) studies that assessed the ability of patients with migraine or TTH to estimate the perception of VV; (3) studies that used the static SVV test to estimate VV perception; and (4) studies that included a comparison group of healthy controls. The exclusion criteria used were: (1) observational studies with only one group; (2) studies that assessed the perception of VV after any physical or pharmacological intervention; (3) studies that did not report mean or standard deviation of the SVV estimation; and (4) studies that were not susceptible to obtain the absolute error mean and its standard deviation according to validated procedures ,34.Data of the exposed and non-exposed groups of each study were collected by two reviewers (M.M.-N and M.C.O.-P.) independently, employing a standardized data collection form. Discrepancies were resolved with the intervention of a third author (R.L.-V.).Cochrane Handbook for Systematic Reviews of Interventions [The characteristics collected of each included study were the authorship, publication date, research design, sample size, number of participants in the exposed (patients diagnosed with PHD) and non-exposed group , age of participants classified in two age groups (adult: 19–44 years old and middle aged: 45–64 years old), gender sex , type of PHD (migraine or TTH), and phase of progression . The primary outcome measure was the perception of VV obtained in each group using the SVV test. All data included in our analysis according the SVV were obtained without any therapeutical intervention in the control or exposed groups. We obtained the body position, the head position (fixed or non-fixed), and the used SVV test. Following the recommendations of the ventions  and Hozoventions , studiesTo evaluate the quality of the studies included in this meta-analysis, the Newcastle–Ottawa scale (NOS) was applied . The domThis process was performed independently by two authors (D.R.-A. and M.Á.L.-O.) who assessed the quality of the included studies. The disagreements that arose were resolved by a third author (R.L.-V.).I2 Statistic of Higgins  and p < 0.1 indicated large heterogeneity [The Comprehensive Meta-Analysis 3.3.070  was used to carry out the meta-analysis . Two autogeneity ,51. We pA sensitivity analysis (using the leave-one-out method) was performed to assess the contribution of each study to the pooled estimate in the meta-analysis .Subgroup analysis was made according to the type of PHD (migraine or TTH), the position of the head in the SVV measure (fixed or non-fixed head position), and the SVV test used .The PRISMA flow chart in The main characteristics of the studies included in the review appears in The methodological quality of the studies included in this review, as evaluated with the Newcastle–Ottawa Scale (NOS), was moderate (NOS mean score of 6.4). Four studies ,54,55,58p < 0.001) , and the number of participants per study was 81.6, showing a low level of precision in our findings. The sensitivity analysis (leave-one-out method) yielded a pooled estimate that varied 22% when compared to the original pooled estimate.Seven studies ,56,57,58< 0.001) . The fun< 0.001) , suggestp = 0.007) , and the precision level was also low (83.62 participants per study). Sensitivity analysis (one study removed) yielded pooled estimates of 21% with respect to the original pooled estimate.Seven studies ,56,57,58= 0.007) . The fun= 0.007) , and thep < 0.001) . The precision level was low because the number of participants per study was 73.5. Sensitivity analysis (one study removed) yielded pooled estimates of 28% with respect to the original pooled estimate.Two studies ,56 with < 0.001) . Risk ofp = 0.026) and non-fixed head position   . Risk of= 0.020) . The prep = 0.019) and in patients that use the rod projected screen test   . Heterog= 0.019) .The present systematic review with meta-analysis attempted to analyze the perception of VV in patients with PHD in comparison to healthy controls. Our results show a misperception of VV in patients with PHD when the SVV test is used to assess the sense of verticality. Both migraine and TTH sufferers present an alteration in verticality perception, but this is more remarkable in patients with TTH, and could be due to the physio-pathological process involved in these disorders.The main reason for these results possibly reflects the important role that cervical, vestibular, and visual structures play in both the perception of verticality and the physio-pathological processes of primary headaches. A recent study revealed the concomitance of neck pain-generating activities, instability-inducing activities, and primary headaches, where high visual attention-demanding activities are essential in this process . VV mispPatients with TTH are known to present with oculomotor disturbances, including a reduction in the speed of smooth pursuit and saccadic eye movements . These oThe relationship between neck pain and headache has been widely documented ,6,7,8. TVestibular disorders or unsteadiness are other pathologies that frequently appear jointly with headache ,67. A liTaking into account the importance of the vestibular, visual, and proprioceptive systems, especially in the structures of the neck, both in the development of migraine and in the perception of verticality, the findings of the present review highlight the importance of considering these three systems in the diagnosis and management of PHD. Our review presents some limitations that must be considered. First, some studies found in the bibliographic search were not included in the meta-analysis due to the absence of statistical data of VV perception in both exposed and healthy control groups. Second, the low number of published studies included in our review may explain the possible publication bias found. In addition, the risk of publication bias could not be studied for TTH comparisons. Third, this low number of studies included and the low sample size must be taken into account in the generalization of our results, especially in TTH group, where fewer studies were included and there was a smaller sample size compared to migraines; therefore, these results should be viewed with caution. Fourth, the moderate quality of the studies included may have contributed to the generalization of our findings. The unequal score obtained in the NOS between exposed and non-exposed groups can produce a possible selection bias that must be considered. Another limitation was that no study analyzed the VV perception using the RFT test. Due to this, this study does not assess the visual and somatosensory contribution to the sense of verticality in patients with PHD. In future studies, it will be interesting to assess VV perception trying to differentiate between the three principal systems involved in the formation of the central pattern of the sense of verticality. In patients with PHD, assessing visual and proprioceptive cues using the RFT is particularly suitable due to the importance of the visual and somatosensory systems in the physiopathology processes of migraine and TTH, allowing deeper knowledge of these processes. Another aspect to be highlighted is the variability of the criteria used to diagnose PHD, which can affect the generalizability of the results. Finally, an analysis that evaluated the perception of VV when the subjects were head tilted could not be performed, due to the fact that only one study that assessed VV in this condition met the proposed inclusion criteria, so no conclusion can be drawn from this. However, it is important to highlight this fact and encourage further research on this condition.Patients with PHD showed a misperception of VV when, mainly, the vestibular contribution to the sense of verticality was assessed with the SVV test. More specifically, a misperception of VV was found separately in patients with migraine and TTH, showing a higher alteration of the SVV in patients with TTH, although the sample size in each subgroup was small. However, a substantial risk of publication and selection bias needs to be kept in mind when interpreting these results. Future studies should analyze the visual and somatosensory contributions to building the sense of verticality using the RFT in patients with PHD.


Small intestinal perforation in patients with Burkitt lymphoma is extremely rare. We present the first report of such a case.A 53-year-old woman was admitted with abdominal pain and vomiting. Abdominal examination revealed rigidity and tenderness in the upper abdomen.Computed tomography scan showed thickening of the wall of the jejunum, intra-abdominal free gas, and ascites; the patient was diagnosed with small intestinal perforation, and underwent emergency surgery on the same day. Laparoscopic findings were a 50 mm jejunal perforation and perforation in the transverse mesocolon. A partial jejunal resection of the perforated area, partial transverse colectomy, temporary colostomy, and intra-abdominal drainage were performed. Histological examination showed diffuse infiltration of medium-sized atypical lymphocytes in the perforated area, exhibiting a “starry sky” appearance.Immunostaining results showed that the atypical lymphocytes were CD20 and virtually 100% Ki-67 positive, and CD56, CD30, and EBER negative. The lesion was identified as Burkitt lymphoma (BL). The postoperative course was favorable. On postoperative day 18 the patient began chemotherapy through the hematology department. Currently, the patient is in remission.The majority of the malignant lymphomas occurring in the digestive tract are identified in the stomach; over 90% are B-cell lymphomas and mucosa-associated lymphoid tissue lymphoma Nakamura et al. BL originating from the small intestine accounts for only about 9%.The incidence of BL in the small intestine is low. Pretreatment BL can lead to bowel perforation. Prompt involvement of the hemato-oncologist after definitive diagnosis, and commencing chemotherapy as early as possible after surgery, are thought to improve prognosis. The majWe report a case of acute generalized peritonitis due to BL perforation in the small intestine which required life-saving emergent surgery. The aim of this report is to clarify that Burkitt lymphoma can cause small bowel perforation.The work in this case has been reported in line with the SCARE criteria .2A 53-year-old woman was brought to the hospital in an ambulance with a 1-month history of recurrent abdominal pain and vomiting. The abdominal pain and frequent vomiting usually occurred after eating. She was diagnosed 1 year prior to presentation with left-sided breast cancer. There is no relevant family or medication history. The patient was admitted for medical evaluation at our facility. Physical examination revealed a weight of 46 kg, height 154 cm, blood pressure 110/62 mmHg, temperature 38.5 °C, and a pulse rate of 90/min. She had generalized tenderness with board like rigidity of the abdomen.4/μL, C-reactive protein level of 0.82 mg/dL, and lactate dehydrogenase level of 703 U/L. Abdominal computed tomography scans revealed peripheral wall thickening of the jejunum, small amounts of free gas in the abdominal cavity, and ascites  observed in the jejunum was necrosed. Inflammatory changes were observed in the mesentery of the small intestine and the transverse mesocolon near the perforated area .Fig. 3SuHistological examination of samples from the perforated area of the jejunum, the mesentery of the small intestine, and the transverse mesocolon, revealed a large number of diffuse medium-sized atypical lymphocyte infiltrates and histiocytes with phagocytosed nuclear fragments, and a “starry sky” appearance. Immunostaining showed that the atypical lymphocytes were CD20 positive, and CD56, CD30, and EBER negative. CD3 and CD5 were positive only for a small number of intervening lymphocytes, and CD20 and Ki-67 staining was positive in almost 100% of the cells . A diagnShe had an uneventful postsurgical recovery period and on postoperative day 18 was transferred to the hematology department to commence chemotherapy. To date, the patient has been followed-up for five months and is currently in remission.3BL is a B-cell lymphoma, a form of non-Hodgkin’s lymphoma. It is characterized by aggressive growth and rapid development of clinical features. BL accounts for 1–2% of all malignant lymphomas  and is fBL in the small intestine is often characterized by abdominal pain, nausea, constipation, and an abdominal mass . BL freqA pathognomonic finding is the “starry sky” appearance, characterized by diffuse proliferation of homogeneous cells. These cells are approximately the same size as that of the nuclei of the histiocytes  and exhibit prominent macrophages with phagocytose nuclear fragments. The tendency of these cells to proliferate is extremely high, and Ki-67 staining has been reported to be over 95% positive. Some chromosomal translocations, such as t, t, and t are frequently seen in BL, and most cases exhibit c-MYC gene abnormalities in the eighth chromosome.The diagnostic criteria of malignant lymphomas in the digestive tract have not been established. However, Dawson et al., suggested the following five diagnostic criteria: no superficial lymph node swelling, no distinct swelling of mediastinal lymph nodes, no abnormalities in the peripheral blood results, mainly gastrointestinal lesions with and metastasis limited to the regional lymph nodes, and no metastases in the liver or spleen ,17.AccorMultidrug chemotherapy centered on cyclophosphamide hydrate is reported to have an extremely poor prognosis with an average survival time of 5.6 months; however, recent reports have indicated that, in adults, multidrug treatment has resulted in remission in 75%–90% of patients . There aOur case satisfied all of the five diagnostic criteria for malignant lymphomas of the digestive tract, as proposed by Dawson et al. Histopathological tests revealed the characteristic “starry sky” appearance, and Ki-67 staining was positive for almost 100% of the lymphocytes; therefore a diagnosis of BL was made. Histopathological findings of the excised bowel also showed severe infiltration of diffuse medium-sized atypical lymphocytes in the jejunal wall around the perforated area, and the primary site of the tumor was diagnosed as the jejunum. The postoperative course was favorable, and the patient was transferred to the hematology department, where she continued to receive chemotherapy and remained in remission as of the 5-month follow-up.A search of the terms “Burkitt’s/Burkitt lymphoma”, “perforation”, and “small intestine” in PubMed from 2000 to 2020 showed a number of cases where gastrointestinal perforation occurred during chemotherapy. However, to our knowledge, there are no reports of small intestine perforation due to BL of the digestive tract before treatment, and our report seems to be the first of such a case.In cases of small intestinal perforation in adults, emergency surgery should be conducted as early as possible to avoid life-threatening complications. Remission can be achieved using chemotherapy for a number of gastrointestinal diseases, such as small bowel cancer, gastrointestinal stromal tumor, Crohn's disease, and even aggressive malignancies with an extremely poor natural course, such as BL.4The incidence of BL in the small intestine is low. Prompt involvement of the hemato-oncologist after a definitive diagnosis is made, and commencing chemotherapy as early as possible after surgery, are thought to influence prognosis.All authors declare that there is no conflict of interest.This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sectors.The institutional ethics committee considers that ethical approval is not necessary for a case report.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Yuji Takayama, Masaaki Saito: conceptualization, investigation, data curation, writing and reviewing.Kosuke Ichida, Yuta Muto: conceptualization, investigation, data curation, reviewing.Akira Tanaka, Toshiki Rikiyama: conceptualization, data curation, writing, reviewing.Researchregistry6002.Masaaki Saito, the corresponding author of this paper.Not commissioned, externally peer-reviewed.