Galectin-1 modulates acute and chronic inflammation, and is associated with glucose homeostasis and chronic renal disease. Whether the serum galectin-1 level can predict short-term and long-term renal outcomes after contrast exposure in patients undergoing coronary angiography (CAG) remains uncertain. This study aimed to evaluate the relationship between the serum galectin-1 level and the incidence of contrast-induced nephropathy (CIN), and to investigate the predictive role of the circulating galectin-1 level for renal function decline in patients undergoing CAG. In total, 798 patients who had undergone CAG were enrolled. Baseline creatinine and serum galectin-1 levels were determined before CAG. CIN was defined as an increase in the serum creatinine level of 0.5 mg/dl or a 25% increase from baseline within 48 h after the procedure, and renal function decline was defined as > 30% reduction of the estimated glomerular filtration rate from baseline. All patients were followed for at least 1 year or until the occurrence of death after CAG. Overall, CIN occurred in 41 (5.1%) patients. During a median follow-up period of 1.4 ± 1.1 years, 80 (10.0%) cases showed subsequent renal function decline. After adjustment for demographic characteristics, kidney function, traditional risk factors, and medications, higher galectin-1 levels were found to be associated independently with a greater risk of renal function decline , but not with CIN, regardless of the presence of diabetes. In conclusion, higher baseline serum galectin-1 levels were associated with a greater risk of renal function decline in patients undergoing CAG, but were not associated independently with CIN. The early detection of individuals at risk of CKD development or progression is especially important, as early-stage CKD is prevalent and contributes greatly to cardiovascular disease. Research clearly indicates that patients with coronary artery disease (CAD) are at increased risk of progressive renal dysfunction2. These patients may be further exposed to the risk of contrast-induced nephropathy (CIN), which has an incidence rate of 4.4–22.1%, if they undergo coronary angiography (CAG)3. CIN has traditionally been considered to be a benign and reversible disease, but it may prolong hospital stays and increase in-hospital mortality4. Accumulating evidence suggests that CIN is related to long-term renal function decline, in addition to short-term outcomes6. Efforts to predict the occurrence of CIN include the development of risk scoring systems composed of baseline risk factors7, renal Doppler evaluation8, 24-h serum creatinine elevation9, and renal injury biomarkers14. However, no predictive marker for long-term renal outcomes in patients undergoing CAG has been established.Chronic kidney disease (CKD) is a serious public health problem, and its incidence and prevalence are increasing15. Galectins comprise three categories: monomeric, chimeric, and tandem-repeat types. They are characterized as homodimers of 14-kDa subunits with two β-galactoside–binding sites, and are expressed in many tissues15. Fifteen galectin isoforms, numbered in order of discovery , are recognized15. Galectin-1 is found in cytoplasm and on cell surfaces, and can be secreted to the extracellular matrix15. It modulates cell signaling, proliferation, and survival, and contributes to the control of inflammation and neovascularization16.Lectin–glycan interactions have been reported to be regulators of extensive physiological and pathological processes. Galectins form a group of proteins that can bind to β-galactoside sugars by N- or O-linked glycosylation through their carbohydrate recognition domains7 and CKD17. Recent evidence indicates that galectin-1 levels are increased in the proteome in subcutaneous interstitial fluid18 and plasma19 in patients with type 2 diabetes. The galectin-1 level is also elevated in the glomerular proteome in mice with CKD20. An in vitro study implicated galectin-1 in diabetic nephropathy, as seen in human podocytes in high-glucose culture21. Furthermore, galectin-1 was reported to be a new fibrosis protein and potential treatment target in the context of diabetic nephropathy22. However, whether the serum galectin-1 level can predict CIN and kidney function decline in patients undergoing CAG remains unclear. This study aimed to evaluate the relationship between the serum galectin-1 level and the incidence of CIN, and to clarify the predictive role of galectin-1 in renal function decline, in patients with suspected CAD undergoing CAG at a single hospital. We hypothesized that elevated galectin-1 levels, which have been reported to be associated with diabetes and diabetic nephropathy, would also be predictive markers of CIN and renal function decline in these patients. We also sought to clarify whether the possible predictive role of galectin-1 would be independent of diabetes.Diabetes is a traditional risk factor for CINIn total, 798 subjects who underwent elective CAG and/or percutaneous coronary intervention (PCI) were enrolled in this analysis Fig. . The meap < 0.001). Patients with prior CIN were also at significantly greater risk of renal function decline than were those without prior CIN (p < 0.001).Kaplan–Meier survival analysis was performed to investigate the potential impact of the baseline galectin-1 level on adverse event–free survival Fig. . PatientOf the 798 study subjects, 275 (34.5%) were diabetic. Table p = 0.04]. After adjustment for age, sex, and all factors that were significant in the univariate analysis, the serum galectin-1 level was not associated significantly with CIN, but associations persisted with age , and the hemoglobin level , 2.64; 95% confidence interval (CI), 1.03–6.76; p = 0.025) and proteinuria  remained associated significantly with renal function decline (Table p = 0.063). The serum galectin-1 level was an independent predictor of renal function decline, rather than CIN, in patients undergoing CAG.In a univariate Cox regression analysis, older age, histories of hypertension and diabetes, proteinuria, higher serum galectin-1 level, lower hemoglobin level, reduced eGFR, and lower LVEF, as well as prior CIN, were associated significantly with a higher incidence of renal function decline. In a multivariable Cox regression analysis, the circulating galectin-1 level [tertile 2: hazard ratio (HR) 5.56, 95% CI 1.79–17.22; tertile 3: HR 5.67, 95% CI 1.97–16.32), hemoglobin level  to up-regulate the galectin-1 protein under hyperglycemic conditions in diabetic mice22. AP4 was identified as a protein binding to the galectin-1 promoter that regulates various functions under high glucose stimulation22.The mechanisms linking galectin-1 elevation to CKD progression are not fully clear. Several groups have postulated the existence of mechanisms relating galectin-1 to CKD31. Cardiac and renal diseases have several common pathways, including those of enhanced systemic inflammation and stress-mediated neurohormonal responses, the development of anemia and bone and mineral disorders, and acid–base and fluid imbalances31. Chronic myocardial dysfunction is also a well-known mediator of progressive renal function decline. The pathophysiology of this so-called type 2 cardiorenal syndrome remains under investigation30. Interestingly, a recent review suggested that galectin-1 is an emergent mediator of cardiovascular inflammation32. In this study, we also found that galectin-1 was associated with the LVEF and CAD in univariate analysis. We speculate that the parallel process affecting both heart and kidney functions also plays an essential role in the non-diabetic association between galectin-1 and CKD. Our findings, in agreement with previous reports, illustrate the association of galectin-1 with renal function decline irrespective of diabetes. Galectin-1 thus may play a critical role in renal function decline in diabetic and non-diabetic patients.Less is known about the association between galectin-1 and CKD in non-diabetic contexts. Emerging evidence suggests that the progression of cardiac and renal functions is often interconnected34. Contrast media may also have cytotoxic effects via the upregulation of reactive oxygen species35 or direct induction of osmotic tubular nephrosis36. Another possible reason is the lower incidence of CIN than renal function decline in our study sample. Furthermore, galectin-1 and diabetes were correlated in this study and in previous reports18. Before adjustment for diabetes, tertile 3 of the galectin-1 level correlated with CIN. After adjustment for all related risk factors, including diabetes, this association was insignificant.Interestingly, we found no significant association between galectin-1 and CIN in patients undergoing CAG or PCI procedures. One possible explanation is that the mechanisms of CIN differ from those of chronic renal decline. Contrast media may alter nitric oxide, endothelin, and adenosine levels, inducing renal vasoconstriction and leading to renal medulla ischemia and acute tubular necrosisThis study has some limitations that should be considered. First, the study population was relatively small, and consisted of Asian patients treated at a single hospital. Further studies larger numbers of different participants are required to confirm our findings. Second, patients enrolled in our study were elders . Caution should be taken when applying our findings to younger populations. Finally, the eGFR was used as the sole renal function endpoint, without consideration of other clinical endpoints affecting renal outcomes, including newly diagnosed diabetes mellitus and the progression of proteinuria. Nevertheless, our study demonstrated that the serum galectin-1 level is a novel risk marker for renal outcomes in patients with suspected CAD undergoing CAG or PCI procedures.Although not a predictor of CIN, the circulating serum galectin-1 level is an independent prognostic marker for subsequent renal function decline in patients undergoing CAG, irrespective of diabetes. These findings provide novel evidence of galectin-1’s involvement in the pathogenesis of renal dysfunction in patients with suspected CAD. Further research exploring the underlying mechanism is needed.37. Patients with stage 5 CKD, defined as creatinine clearance < 15 ml/min/1.73 m2 (n = 15), and those undergoing hemodialysis or peritoneal dialysis (n = 42) were excluded from the analysis. Thus, a total of 798 patients was enrolled in the study and included in the final analysis  collected data for this study consecutively from June 2011 to August 2018. The sample size was not pre-calculated. In total, 855 subjects with stable coronary CAD admitted to Taipei Veterans General Hospital for elective CAG and/or PCI were screened. Each patient’s serum creatinine concentration was measured before CAG, and the eGFR was calculated using the CKD Epidemiology Collaboration equationEach patient’s chart was reviewed in detail to collect data on medications, smoking status, and risk factors for CIN, such as age, existing renal dysfunction, type 2 diabetes mellitus, and volume depletion. Patients’ blood pressure was measured using electronic sphygmomanometers at least four times a day during hospitalization. Hypertension was defined as systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or the use of antihypertensive medications. Type 2 diabetes mellitus was defined as fasting plasma glucose level ≥ 126 mg/dl or the use of hypoglycemic agents. The body mass index was calculated by dividing the weight of the patient (in kilograms) by the square of the height (in meters). A nonionic low-osmolality contrast medium (iopromide) was used for all patients’ examinations. It was administered intra-arterially, mainly through transradial catheters. Metformin and nephrotoxic medications, such as non-steroidal anti-inflammatory drugs, were discontinued 48 h before contrast medium administration. Before and after contrast medium exposure, physiological (0.9%) saline was given intravenously at a rate of 1 ml/kg/h for 12 h. In patients with left ventricular dysfunction (ejection fraction < 40%) or apparent heart failure, the hydration rate was reduced to 0.5 ml/kg/h. This research was conducted according to the principles expressed in the Declaration of Helsinki. This study was approved by the research ethics committee of Taipei Veterans General Hospital, and all participants provided written informed consent.Blood samples were collected after an ≥ 8-h fast. The blood cell count; serum glucose, creatinine, and uric acid levels; and lipid profiles were determined using a Hitachi 7600 autoanalyzer . The serum creatinine concentration was assessed at the time of admission and daily for the following 3 days after contrast medium exposure. Urine dipstick analysis was performed using commercial test strips, and proteinuria was defined as a urine protein concentration ≥ 30 mg/100 ml. Plasma concentrations of galectin-1 were determined using the commercially available Human Galectin-1 Quantikine ELISA Kit DGAL10 ; the sensitivity was 0.129 ng/ml and the assay range was 0.3–20 ng/ml. The intra- and interassay coefficients were 5.7–8.8% and 7.5–9.5%, respectively. Two experienced interventional cardiologists interpreted the coronary angiograms. Coronary lesions causing > 50% diameter narrowing were considered to represent significant stenosis. The LVEF was estimated by left ventriculography. The contrast consumption of each patient was also recorded.38. Patients were advised to visit outpatient clinics regularly after discharge from the hospital. The cohort was followed until January 2019 or the occurrence of death. Patients’ clinical data, including serum creatinine levels, were obtained every 3–6 months during the follow-up period. Renal function decline was defined as a > 30% decrease in the eGFR after discharge40. When it was identified, the serum creatinine level was re-measured 1 month later to ensure the accuracy of the diagnosis.All patients were evaluated for the occurrence of CIN, which was defined as an increase in the serum creatinine concentration of ≥0.5 mg/dl or a 25% increase from baseline within 48 h after CAGData were expressed as medians (interquartile ranges) for numeric variables and as numbers (percentages) for categorical variables. Clinical and laboratory data were compared using the Mann–Whitney U test for continuous variables and Fisher’s exact test for categorical variables. The incidences of CIN and renal function decline were calculated. Survival curves were generated using the Kaplan–Meier method, and survival was compared among groups using the log-rank test. Logistic regression analysis was performed to examine the relationships of various risk factors to CIN, and Cox proportional-hazard regression analysis was performed to identify risk factors for progressive renal function decline. Factors that were significant in univariate regression analysis were entered into the multivariable regression analysis. To investigate modification of the effect of galectin-1 on renal function decline by different comorbidities, we performed subgroup analyses with the study cohort stratified according to the presence of diabetes, presence of proteinuria, and PCI status. Interactions between the galectin-1 concentration and comorbid conditions were examined by adding a product term of galectin-1 and comorbidities to the Cox regression analysis. Data were analyzed using SPSS version 18.0 . P values < 0.05 were regarded as significant.The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.


Patients with advanced chronic kidney disease (CKD) exhibit higher prevalence of coronary artery calcification (CaC) than general population. CaC has been proposed as a risk factor for mortality in end-stage CKD, but most studies in the field are based on short-term follow-up.We conducted a cohort, 10-year prospective longitudinal study of consecutive cases referred to the renal unit. A non-enhanced multislice coronary computed tomography was performed at baseline. CaC was assessed by Agatston method. Patients were stratified according to their CaC score: severe calcification group (CaCs< 400 HU) and mild-moderate calcification group (CaCs≥400 HU). The overall and cardiovascular (CV) mortality, CV events, and factors potentially associated with CaC development were recorded.p < 0.001). The severe calcification group was older, had higher prevalence of type 2 diabetes mellitus, former cardiologic events, and lower albumin serum levels than the mild-moderate calcification group. In a multivariate Cox model, severe CaC was a significant predictor of CV mortality .137 patients with advanced CKD were enrolled and provided consent. Overall mortality rate was 58%; 40% due to CV events. The rate of overall mortality in the severe calcification group was 75%, and 30% in the low calcification group, whereas the rate of CV mortality was 35% vs. 6%, respectively  contains supplementary material, which is available to authorized users. Cardiovascular (CV) disease is one of the leading causes of death and premature mortality among chronic kidney disease (CKD) patients . In addicalcifications affect most of CKD patients (60–90%) and these calcifications develop early in the course of the disease ) was used. Each unit difference in the log-transformed CaCs represents a doubling of the score. Data were analysed using SPSS version 20.0 . The significance level was set at p < 0.05.The influence of each variable on overall and CV mortality was assessed by means of a Cox proportional hazard models. The first stage consisted of a univariate analysis selecting those with a o et al.  to studyThe population-based sample was composed of 137 patients, 85 subjects (62%) undergoing maintenance haemodialysis and 52 patients (38%) with advanced CKD. Subjects were 74 males and 63 females, with a median age of 66 years (IQR 52 to 71). Haemodialysis vintage was 25 months (IQR 10–53). The median follow-up period was 87.5 months (29.5–111). Regarding renal replacement therapy, at baseline, two out five of patients in HD were dialyzed with a membrane of high dialytic efficacy and in four out five it was used a standard dialysis bath. The main baseline subject characteristics are outlined in Table The prevalence of CaC was 87% (range: 0 to 8798 HU). The median of CaCs was 600 HU (IQR 70–1794). Those patients with severe CaC (CaCs ≥400 HU) were older, had a longer haemodialysis vintage, showed higher prevalence of type 2 diabetes, previous cardiologic events, and exhibited higher triglyceride serum concentration and lower serum albumin levels than mild-moderate CaC subjects (CaCs< 400 HU) (Table Variables that showed a significant association with severe CaCs (≥400 HU) were: age , haemodialysis , type 2 diabetes , previous cardiologic events  and low serum albumin values . On the multivariate regression analysis, age , haemodialysis  and type 2 diabetes  kept a statistically significant association with severe CaCs.n = 60) were receiving haemodialysis and 25% (n = 20) had advanced CKD. In terms of CV mortality, 78% (n = 25) of subjects were on maintenance dialysis. Median overall survival, estimated by the inverted KM curve, was 44.6 ± 3.7 months (95% CI 37.4–51.8), while median CV survival was 39.1 ± 5.6 months (95% CI 28.1 to 50.1).During the follow-up period, 58% of subjects died and 40% of deaths were related to CV events. Considering overall mortality, 75% of patients (n = 3) in patients without CaC, 22% (n = 4) in patients with mild CaC, 53% (n = 9) in patients with moderate CaC and 75% (n = 64) in patients with severe CaC. Most of overall mortality events happened in group 4 (80%). By KM analysis, patients with CaCs ≥400 HU had a significantly higher mortality compared to those with CaCs< 400 HU  . By KM analysis, patients with CaCs ≥400 HU showed a significantly higher CV mortality compared to those with CaCs< 400 HU  too .A total of 292 CV events were observed during the follow-up period; 149 of them required admission to hospital. These CV events included: 82 heart failures, 17 atrial fibrillations, 29 ischemic heart diseases and 21 strokes. Figure 2 10.18, p = 0.006)- while, only in haemodialysis patients, severe CaCs seemed to have tendency on overall mortality , although not reaching a statistical significance - still appeared to have an influence on CV mortality  and CV  mortality by 19 and 43% respectively, during a median follow-up period of 87.5 (29.5–111) months.In recent years, mortality of patients in haemodialysis has decreased due to improvements in technique. According to the United States Renal Data System, life expectancy of patients once they start haemodialysis ranges between 8 and 4.5 years. Increasing length of time on dialysis is a related to higher mortality rates .In the present study, we explore the predictive value of CaCs for total and CV mortality in advanced CKD and haemodialysis population. Our results point out that CaCs≥400 HU could be a predictor of higher CV mortality in this population, on a long-term basis. Unfortunately, we were not able to identify a clear relationship between CaCs and overall mortality in the whole sample. A possible limitation of the study might be the use of a relatively small sample size; therefore, further studies should be designed to explore this hypothesis.In addition, we found that a doubling of the CaC score increased the estimated probability of CV mortality by approximately 43% in a 10-year period. In the Detrano study, subjects aged 45 to 84 years with no clinical cardiovascular disease before enrolment were followed for a median of 3.8 years; authors found that a doubling of calcium score resulted in a 20% increase in the risk of a major coronary event  . Our finIn CKD patients, Watanabi et al. investigated a heterogeneous sample of 117 non dialyzed patients with CKD stages 2 to 5. After a follow-up period of 24 months, they found that the presence of CaCs ≥400 HU was associated with a shorter cardiovascular and hospitalization event-free time and a lower survival rate . RecentlShantouf et al. found that haemodialysis patients with CaCs 0 HU had a higher rate of 6-year event-free survival (89%) compared to those with a CaCs> 400 HU (58%). After adjustment for standard CV risk factors and bone-mineral metabolism parameters, they reported a higher total mortality in CaCs> 400 HU group .Some authors have suggested that CaCs could be an independent predictor of all-cause mortality in haemodialysis. After adjusting for other CV risk factors, Matsuoka et al. found that the 5-year cumulative survival rate was significantly different between patients with low CaCs (84%) compared to those with high CaCs (68%), although with an adjusted relative risk of death of 1.001 . Block eIn a 7-year follow-up study, reported by Shimoyama , which wIn our study, we did not include CV disease subtypes in Cox analysis. However, it would have been interesting to analyse the subtypes of CV mortality in the haemodialysis group according to their CaCs, since non-atheromatous CV events could predominate in these patients . MatsushAs reported by other authors, we found a high prevalence of CaC, ranging 0–8798 HU. This asymmetric distribution is consistent with other similar studies , 16, 26.Our data showed that patients with high CaCs had lower albumin levels than patients with mild or moderate CaC. Malnutrition-inflammation complex syndrome is a common phenomenon in dialysis patients, and is related to increased morbidity and mortality. In dialysis patients, there seems to be an association between malnutrition-inflammation complex syndrome and vascular calcification , 31. TheWe did not find a significant association between CaC and any biochemical parameter related to mineral metabolism or other traditional CV risk parameters. This lack of association could be explained by the small sample size, the study design or the clinical characteristics of our cohort. It should be mentioned that biochemical parameters were measured only once at baseline, and a single baseline laboratory value may not reflect the time-averaged exposure.Traditionally, hypercalcemia, hyperphosphatemia and secondary hyperparathyroidism have been associated with vascular calcification in patients with CKD. However, it has been described that excessive suppression of PTH, or relative hypoparathyroidism, leads to a decrease in bone remodelling or adynamic bone. It is also related to a greater progression of vascular and coronary calcification. Therefore, calcium and PTH levels do not always are correlated with net calcium balance and vascular calcification severity .Therapeutic changes were not recorded, so might be they have had some influence on endpoints. In relation to this point, it is known that CKD and dialysis patients treated with non-calcium-based phosphate binders have a reduction in all-cause mortality compared with those that use calcium-based phosphate binders , 34. BesMoreover, CaC in our cohort was only measured just once, at baseline conditions. Therefore, we couldn’t report number of patients with CaCs < 400 HU who have change to CaCs > 400 HU during the follow up.We conclude that, within the limits of our relatively small sample size, CaC is highly prevalent in advanced CKD patients. Age, diabetes and haemodialysis therapy may be associated with CaC severity. CaCs seems to be a good long-term predictor of CV mortality in patients with advanced CKD, but larger prospective studies are needed to confirm the clinical utility of performing routine MSCT in this population in order to assess CV risk.Additional file 1:Tables S1 and S2. Description of cardiovascular events and mortality. Multivariate proportional Cox analysis in the haemodialysis group. (DOCX 16 kb)


In China, ginsenoside Rg3 is often used in combination with chemotherapy to treat digestive system cancer. We here performed a meta-analysis and systematic review to provide a much needed high-quality evaluation of the efficacy and safety of ginsenoside Rg3 combined with chemotherapy in these cancers.  The PubMed, EMBASE, Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang, and Weipu (VIP) databases were searched. All randomized controlled trials (RCTs) concerning ginsenoside Rg3 combined with chemotherapy for digestive system cancer were selected. Dichotomous data were expressed as odds ratios (ORs) with 95% confidence intervals (CI). The methodological quality of the included studies was evaluated according to the Cochrane evidence-based medicine system, and the statistical analyses were performed with Review Manager 5.3 and STATA 12.0 software. In addition, the Grades of Recommendation Assessment, Development and Evaluation (GRADE) approach was used to rate the quality of the evidence. Trial sequential analysis (TSA) was used to evaluate information size and treatment benefits.  A total of 18 trials comprising 1531 patients were included in this study. The results revealed that the trials were of sufficient standard to draw reliable conclusions that ginsenoside Rg3 combined with chemotherapy could improve the objective response rate , disease control rate , 1-year survival rate , Karnofsky Performance Scale , gastrointestinal dysfunction , and the decline of leucocyte count .  Ginsenoside Rg3 combined with chemotherapy can improve the clinical efficacy and alleviate treatment-induced side effects for digestive system cancer. Digestive system cancer is the most common and lethal cancer in the world, mainly including gastric cancer (GC), colorectal cancer (CRC), esophageal cancer (EC), liver cancer (LC), and pancreatic cancer (PC). At present, the changes in dietary patterns are considered to be the main causes of digestive system cancers . PatientShenyi capsule , an important Chinese anticancer drug, is composed of the ginseng root extract component, ginsenoside Rg3. Previous studies have found that ginsenoside Rg3 inhibits tumor growth through suppression of pathways linked to oncogenesis, including cell survival, proliferation, invasion, and angiogenesis , 6. For This article was based on the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines (PRISMA guidelines), and the materials were published studies.Studies were retrieved from the PubMed, EMBASE, Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang, and Weipu (VIP) databases. All searches were implemented using MeSH and free words. The search period lasted from the established time to July 28, 2019. All the studies were searched regardless of their publication type and language. Two authors (Linlin Pan and Tingting Zhang) independently searched all the related studies in English and Chinese databases using the following search strategies:For Chinese databases, the following terms were used in combined ways: [Renshen zaodai Rg3 OR Shenyi jiaonang] AND [Hualiao ] AND [Weiai OR Chang ai OR Shidaoai OR Ganai OR Yixianai ]. For English databases, the following terms were used in combined ways: [ Ginsenoside Rg3 OR 20s-ginsenoside rg3 OR Beta-D-glucopyranoside OR 20(R)-ginsenoside Rg(3) OR Ginsenoside 20-rg3 OR Shenyi capsule] AND [Chemotherapy OR Chemotherapies] AND [(Gastric Neoplasms OR Stomach Neoplasms OR Stomach Cancers OR Gastric Cancers OR Stomach tumors OR Gastric tumors OR Gastric Carcinomas OR Stomach Carcinomas) OR  OR  OR (Liver Neoplasms OR Liver Cancers OR Liver tumors OR Liver Carcinomas OR Hepatocellular carcinoma) OR (Pancreatic Neoplasms OR Pancreatic Cancers OR Pancreatic tumors OR Pancreatic Carcinomas)]The inclusion criterias were as follows: (a) interventions: the control group treated with chemotherapy or chemotherapy plus placebo, and the experimental group was treated with ginsenoside Rg3 on the basis of the control group; (b) types of studies: RCTs; (c) participants: patients who were pathologically diagnosed with GC, CRC, EC, LC, or PC; (d) outcomes: objective response rate (ORR), disease control rate (DCR), survival rate (SR), Karnofsky Performance Scale (KPS), and side effects .The exclusion criterias were as follows: (a) nonclinical experimental studies ; (b) patients with other malignancies or serious medical diseases; (c) patients treated with other antitumor Traditional Chinese Medicine (TCM) drugs or therapies .The trials were selected from the relevant literature; two independent authors (Linlin Pan and Tingting Zhang) evaluated each title, abstract, and full-texts and then selected the relevant studies according to the inclusion criteria, and the discrepancies were settled through a consensus discussion. The following information was extracted from the included studies: the name of the first author, year of publication, type of cancer, sample size, sex, age, interventions, follow-up time, outcome measures, and criteria for efficacy.The methodological quality of each RCT was independently assessed by two authors (Linlin Pan and Tingting Zhang) using the Cochrane Risk of Bias tool . DisagreP < 0.05 was considered statistically significant. The Chi2 and I2 tests were used to evaluate the heterogeneity, and the exhibited heterogeneity was P < 0.10 or I2 >50%. The fixed-effect model (FEM) was used for the merging of homogeneity data, and the random effects model (REM) was used for heterogeneous data merging. Sensitivity analysis was assessed by reanalyzing the data using various statistical methods. Publication bias was evaluated by visual assessment of the asymmetry of the funnel plots and Egger's test (STATA 12.0), where P < 0.05 indicated potential bias.The Review Manager (RevMan) Version 5.3 software was used to perform statistical analyses. The risk ratio (OR) with the correspondent 95% confidence intervals (CI) was used to pool the total effectiveness rates of dichotomous data, and The GRADE approach  was usedTSA software (version 0.9.5.10 Beta) was used to calculate the required information size (RIS) for meta-analysis and evaluate treatment benefits based on the sample sizes. The risk of type I error was set at 5% with a power of 80%, the variance was calculated from the data obtained from the included trials, and the relative risk reduction was set at 20% . When cun = 77), cell experiment studies (n = 73), reviews (n = 8), protocols (n = 6), case reports (n = 2), and experimental studies not related to cancer (n = 12). After reading the full-text articles, another 55 studies were excluded due to insufficient outcomes (n = 25), no control group (n = 13), not RCTs (n = 9) or using other complementary and alternative therapies (n = 8). Ultimately, 18 studies were included in the analysis  on the basis of chemotherapy, while the control groups underwent chemotherapy alone or chemotherapy plus placebo. The characteristics of the studies are shown in As shown in P=0.04 in the Z test; for EC, OR = 1.85, 95% CI: 1.17–2.92, and P=0.009 in the Z test; for GC, OR = 2.60, 95% CI: 1.78–3.80, and P < 0.00001 in the Z test; and for LC, OR = 1.77, 95% CI: 1.04–3.03, and P=0.04 in the Z test). The results of EC, GC, and LC did not have heterogeneity , but there was substantial heterogeneity among trials for CRC   .n = 1137) was 52% of RIS (n = 2176), while the cumulative Z-curve crossed the trial sequential monitoring boundary, indicating that current evidence was sufficient to reach a firm conclusion , and none of the three studies showed obvious heterogeneity . However, there was no statistically significant difference in DCR for CRC   .n = 1257) was more than that of RIS (n = 748), and the cumulative Z-curve crossed the trial sequential monitoring boundary, suggesting that the trials sufficiently drew reliable conclusions (TSA showed that the accrued information size (clusions . In addiclusions .P=0.009), while the data had substantial heterogeneity with Chi2 = 10.73, P=0.06, I2 = 53%. Four trials comprising 368 patients focused on 2-year SR between the experimental and control groups; the pooled data showed that ginsenoside Rg3 combined with chemotherapy was significantly better at increasing patients' 2-year SR  without heterogeneity . In addition, two trials comprising 267 patients reporting 3-year SR indicated that ginsenoside Rg3 combined chemotherapy can increase the 3-year SR , and the pooled data did not show heterogeneity   .n = 508) was 44% of RIS (n = 1166) and 2-year SR (n = 368) was 47% of RIS (n = 782). The cumulative Z-curve of 1-year SR crossed the trial sequential monitoring boundary, indicating that current evidence was sufficient to reach a firm conclusion , and the data did not indicate heterogeneity with Chi2 = 3.43, P=0.90, and I2 = 0%  was 90% of RIS (n = 692), and the cumulative Z-curve crossed the trial sequential monitoring boundary, indicating that the evidence will reach a firm conclusion when the sample size is sufficient . Heterogeneity was not observed in GC , but was obvious in LC . As for CRC and EC, there was no statistically significant difference between the control and experimental groups   .n = 1310) was more than that of RIS (n = 936), and the cumulative Z-curve crossed the trial sequential monitoring boundary, indicating that the trials sufficiently drew reliable conclusions (TSA showed that the accrued information size (clusions . In addiclusions .P < 0.00001 in the Z test), and the data among studies did not show heterogeneity   .n = 842) was more than that of RIS (n = 838), and the cumulative Z-curve crossed the trial sequential monitoring boundary, indicating that the evidence will reach a firm conclusion when the sample size is sufficient (TSA showed that the accrued information size (fficient . In addifficient .P=0.35 in the Z test)  .n = 441) was 9% of RIS (n = 4872), and the cumulative Z-curve did not cross the trial sequential monitoring boundary, indicating that the trials were not sufficient to reach a firm conclusion , DCR of CRC , 1-year SR , and gastrointestinal dysfunction of LC . In addition to these four evaluation data, other studies had low or no heterogeneity. As shown in I2 = 0%, P=0.37), but the data was not statistically significant. For the DCR of CRC, after excluding the study Zeng et al., there was no heterogeneity in the result . For the 1-year SR, after excluding the study Zhou et al., the data had no heterogeneity . For gastrointestinal dysfunction of LC, after excluding the study Liu et al., the heterogeneity was significantly reduced . Therefore, the data of this study generally had good consistency.The heterogeneity of the included studies were evaluated by the ChiP value of P > 0.05, so Egger's publication test suggested that there was no publication bias in these three studies, but Egger's publication test suggested that the decline of leucocyte count had a risk of publication bias, with P < 0.05.The funnel plots were nearly symmetrical in ORR , DCR Fi, and gasH. pylori CagA-treated GC cells by regulating SP1 and HSF1 expressions. Ge et al. [Chemotherapy is currently the most common treatment for digestive system cancers, but its many side effects may compromise the therapeutic effect. In TCM, ginseng can reinforce vital energy, which is consistent with enhancement of resistance in modern medicine. Many randomized, prospective clinical trials indicate that ginsenoside Rg3 can effectively treat digestive system cancer and improve its side effects. For example, Tang et al. , 34 repoe et al.  confirmee et al.  confirmeleucocyte count. In addition, the GRADE assessment showed that the quality of evidences were moderate. Therefore, according to the research evidence we had included, ginsenoside Rg3 combined with chemotherapy can significantly control disease progression and reduce the side effects caused by chemotherapy in digestive system cancer.In order to evaluate the efficacy and safety of ginsenoside Rg3 combined with chemotherapy for digestive system cancer accurately, we completed the meta-analysis and systematic review comprehensively for the first time and reported 18 trials with a total of 1,531 patients applied RCTs. In the quality assessment, all the included trials were RCTs with a low risk in attrition bias and reporting bias. About one-third of the trials had a low risk in performance bias and detection bias, two-thirds of the studies had a low risk in other bias. In the publication bias, the funnel plots and Egger's test showed that there was no publication bias in the results of ORR, DCR, and gastrointestinal dysfunction, but the decline of leucocyte count had publication bias. In the sensitivity analysis, after excluding some under- or overestimated trials, the data of this study generally have good consistency. TSA revealed that the trials were of sufficient standard to draw reliable conclusions that ginsenoside Rg3 combined with chemotherapy could improve the ORR, DCR, 1-year SR, KPS, gastrointestinal dysfunction, and the decline of This study has three limitations. First, there is a geographical bias because ginsenoside Rg3 is used for cancer treatment mostly in China. Performing trials in other countries may confirm that ginsenoside Rg3 can be used effectively with chemotherapy for digestive system cancers. Second, some of the included studies were unclear in the allocation concealment and blinding method, although this is unlikely to have a serious impact on the assessment due to the objective criteria (WHO and RECIST). Third, it is not clear which chemotherapy regimens combined with ginsenoside Rg3 is most effective. With the continuous supplementation and improvement of RCTs in this field, we can explore the best chemotherapy regimen combined with ginsenoside Rg3 for digestive system cancer.Despite some limitations, this meta-analysis and systematic review provides assurance that ginsenoside Rg3 combined with chemotherapy can enhance therapeutic effect, improve quality of life, and alleviate side effects of chemotherapy for digestive system cancer patients. In future, we will conduct large and well-designed RCTs to test the above conclusions and explore the best chemotherapy regimen combined with ginsenoside Rg3 for digestive system cancer.


Numerous evidence has suggested that long non-coding RNA (lncRNA) acts an important role in tumor biology. This study focuses on the identification of novel prognostic lncRNA biomarkers predicting tumor recurrence in human colon adenocarcinoma.We obtained the research data from The Cancer Genome Atlas (TCGA) database. The interaction among different expressed lncRNA, miRNA and mRNA markers between colon adenocarcinoma patients with and without tumor recurrence were verified with miRcode, starBase and miRTarBase databases. We established the lncRNA–miRNA–mRNA competing endogenous RNA (ceRNA) network based on the verified association between the selected markers. We performed the functional enrichment analysis to obtain better understanding of the selected lncRNAs. Then we use multivariate logistic regression to identify the prognostic lncRNA markers with covariates. We also generated a nomogram predicting tumor recurrence risk based on the identified lncRNA biomarkers and clinical covariates.We included 12,727 lncRNA, 1881 miRNA and 47,761 mRNA profiling and clinical features for 113 colon adenocarcinoma patients obtained from the TCGA database. After filtration, we used 37 specific lncRNAs, 60 miRNAs and 148 mRNAs in the ceRNA network analysis. We identified five lncRNAs as prognostic lncRNA markers predicting tumor recurrence in colon adenocarcinoma, in which four of them were identified for the first time. Finally, we generated a nomogram illustrating the association between the identified lncRNAs and the tumor recurrence risk in colon adenocarcinoma.The four newly identified lncRNA biomarkers might be potential prognostic biomarkers predicting tumor recurrence in colon adenocarcinoma. We recommend that further clinical and fundamental researches be conducted on the identified lncRNA markers. Colorectal carcinoma (CRC) is among the most common cancers with high morbidity and mortality among all malignancies and the most common CRC is colon adenocarcinoma (CA) . It was HOTAIR has been identified to be related to the overall survival in CA [ATB is associated with poor prognosis of CRC [CCAT1 is reported to be of clinical value in the diagnosis of CA [Long non-coding RNAs (lncRNAs), with lengths of at least 200 nucleotides, modulate gene expression at the post-transcriptional level . With thal in CA . LncRNA s of CRC . LncRNA is of CA . All theThe competing endogenous RNA (ceRNA) hypothesis was presented as a new model demonstrating the association between non-coding and coding RNAs and accepted as one of the most efficient tools in lncRNAs research . It has Few studies have focused on the ceRNA network related to tumor recurrence in CA –22. Thushttps://cancergenome.nih.gov/), an open source of information to identify novel biomarkers in cancer research, using R package “TCGAbiolinks” in June 2018. As a result, 12,727 lncRNA, 1881 miRNA and 47,761 mRNA profiling were obtained.We obtained the RNA sequencing  measurements and clinical characteristics of CA patients from The Cancer Genome Atlas (TCGA) database  with medium follow-up time equal to 1.34 years (488 days). During the follow-up, 6 out of 113 individuals were recorded as deceased.xij is the expression for jth lncRNA or miRNA expression of ith individual, the transformed value of jth lncRNA or miRNA expression of ith individual would equal to ln(xij) (if xij > 0) or 0 (if xij = 0). The transformation can be expressed with:Since the obtained lncRNA and miRNA expression measurements were not normally distributed, we performed a log transformation to normalize and correct their positively skewed distributions. Suppose Then, before incorporating into multi-variate analysis, to obtain a better explanation to the coefficient obtained in regression analysis, we transform the log-transformed lncRNA expressions into binary variables according to whether the log-transformed expression was higher (up-regulated) or lower (down-regulated) than its log-transformed mean.The mRNA profiling obtained from the TCGA database were normally distributed, therefore, no pre-processing was performed on the mRNA expressions.α = 0.05) by Chi-square test , Mann–Whitney test (TNM stage) and t-test (age at diagnosis). We also used t-test to select lncRNAs, miRNAs (log-transformed) and mRNAs with different expression levels between CA patients with and without tumor recurrence (with α = 0.01), the p-value obtained were adjusted with the BH method [All analyses were performed through R . Clinical and demographic characteristics were tested (with H method . Prognoshttp://www.mircode.org/) [http://starbase.sysu.edu.cn/) [http://mirtarbase.mbc.nctu.edu.tw/php/index.php) databases [t-test (with BH correction) to select lncRNAs, miRNAs and mRNAs with different expression levels between cases with and without tumor recurrence. Then, the differentially expressed lncRNAs, miRNAs and mRNAs, which have been verified in the miRcode, starBase and miRTanBase databases, were incorporated into the construction of lncRNA–miRNA–mRNA ceRNA network. The lncRNA–miRNA–mRNA ceRNA network was conducted using the Cytoscape software  [clusterMaker2” [We constructed the lncRNA–miRNA–mRNA ceRNA network to identify miRNAs associated mRNAs based on the interaction among lncRNA, miRNA and mRNA that were verified based on the miRcode (de.org/) , starBasedu.cn/) , and miRatabases . First,  MD, US) . We alsorMaker2”  applicatrMaker2” .clueGO” [http://www.geneontology.org/) [https://www.genome.jp/kegg/pathway.html) [https://www.reactome.org/) databases [We performed the functional annotation analysis using “clueGO”  applicatgy.org/) . Pathwayay.html)  and the atabases .rms”. The prediction performance was evaluated by the ROC analysis and C-index.Finally, we generated a nomogram predicting tumor recurrence risk for asymptotic CA patient based on the lncRNA markers and clinical features identified in the current study using R package “Out of the 113 patients, 50 (44.2%) were males. 67 (59.2%) patients were diagnosed with stage I/II CA, 46 (40.8%) were at stage III/IV, 98 (30.9%). 29 (25.7%) patients developed venous invasion, 56 (49.6%) developed lymphatic invasion. The average age at diagnosis for patients with and without tumor recurrence were 71.95 ± 11.93 and 65.33 ± 12.26 years old. The primary diagnosed sites and detailed TNM stage information of included CA patients are shown in Table t-tests with p-value adjusted through the BH method [p-value less than 0.01. The selected lncRNAs, miRNAs and mRNAs are contained in Additional file We selected the lncRNA, miRNA and mRNA measurements with different expression levels between patients with and without tumor recurrence through independent H method . As a reWe used the miRcode, starBase and miRTarBase databases to verify the interaction relationship between the different expressed lncRNA, miRNA and mRNA markers. Based on the verified interaction relationship, we conducted the lncRNA–miRNA–mRNA ceRNA network analysis to reveal the association between selected lncRNAs and miRNAs as shown in Fig. In order to obtain a deep understanding on the selected genes, we performed the functional enrichment analysis to the intersection mRNAs as shown in Fig. The pathways enriched based on the KEGG and Reactome databases are shown in Fig. clusterMaker2” [We used the “rMaker2”  applicatrMaker2” . As a rep < 0.05) in univariate logistic regression and were incorporated into multivariate logistic regression with clinical covariates.We applied the univariate logistic regression model to estimate the ORs for patients with different expression types of the each lncRNA markers in developing tumor recurrence or not separately. Among the 39 lncRNAs identified in ceRNA network, 8 lncRNAs yielded statistical significance  yielded statistical significance (p < 0.05) in multivariate logistic analysis with age at diagnosis and tumor pathology stage as covariates. The result suggests that these lncRNA markers might act as prognostic predictors for the tumor recurrence in CA. The adjusted ORs of the 5 identified prognostic lncRNAs, in particular, indicated that the up-regulation of CASC2 , AL078459.1 , AL390066.1,  STK4-AS1  and HOXA-AS3  may be associated with the development of tumor recurrence of CA.Finally, 5 lncRNAs  of CA patients as shown in Fig. CASC2, AL078459.1, AL390066.1, STK4-AS1 and HOXA-AS3) were identified through the ceRNA network and multivariate logistic regression. We also performed functional enrichment analysis to investigate the molecular role of the identified lncRNA biomarkers.Differential expression of lncRNAs has been widely identified in various cancers. Published studies have revealed that lncRNAs have key roles in vital biological functions of cancers. However, only few studies have described the role of lncRNA profiles in tumor recurrence of CA –22. In tCASC2 has been reported to be associated with colorectal cancer [CASC2 by miRNA hsa-mir-21 and hsa-mir-18a increases the proliferation and migration of cancer cells in colorectal cancer [CASC2 and the prognosis of CA suggested in published literatures were consistent with the results in this study. This also indicated that the results of our research were reasonable. For STK4-AS1, it was associated with protein coding gene STK4 while the down-regulation of STK4 was associated with the invasion and migration of colorectal cancer [STK4-AS1 and the prognosis of CA revealed in this study was reasonable. For HOXA-AS3, though it has not been identified in CA before, the up-regulation of HOXA-AS3 was reported to be associated with tumor progression and poor prognosis in glioma [STK4-AS1 and HOXA-AS3 were also related to tumor recurrence in CA. For lncRNAs AL078459.1 and AL390066.1, though no functional roles have been reported in CA before this work, in our study, have been identified to be related to the tumor recurrence in CA.For the 5 identified lncRNA markers, l cancer –35. Studl cancer –35. The l cancer . This aln glioma . In thisTo obtain a deep understanding of the selected lncRNA markers, the functional enrichment analyses were performed. The enriched in GO terms were related with several human immune process. IL-10 and IL-12, as representative immune factors, play an important role in inflammation and tumorigenesis , and pubFor the subnetwork analysis, we identified 37 distinct subnetworks while only those with at least 10 nodes were selected  and the tumor prognosis of CA patients.Recently, many studies on the identification of prognostic genes in CA based on the TCGA database have been reported and their findings are then verified with functional experiments in succession –60. ThosFirstly, our study was a preliminary work. In this study, we obtained the RNA sequencing and clinical data from public database, but no clinical samples were involved. Thus, the importance of the selected markers still need to be validated in future cohort study. Then, though we tried to incorporated as many clinical factors as possible in our analysis, some important factors were still not available, for example, treatment information , and living habits (such as smoking or drinking habits). These might cause potential bias in analysis.AL078459.1, AL390066.1, STK4-AS1 and HOXA-AS3) were identified for the first time. The hub genes in the network were annotated with functional gens sets associated with colorectal cancer and the mechanism of tumor progression and invasion. Our work also provides a simple-to-use nomogram predicting the tumor recurrence risk for asymptotic CA patients based on the lncRNA markers identified in this study with clinical covariates. Though clinical validation is still needed, it is reasonable to conclude that these miRNAs are worthwhile for further study as novel candidate prognostic biomarkers for the survival of CA.In this study, we identified five prognostic lncRNA markers for the prediction of tumor recurrence in CA based on ceRNA hypothesis and data obtained from the TCGA database, in which, four of the selected lncRNA markers  selected between CA patients with and without tumor recurrence.


It is now well documented that moonlight affects the life cycle of invertebrates, birds, reptiles, and mammals. The lunisolar tide is also well-known to alter plant growth and development. However, although plants are known to be very photosensitive, few studies have been undertaken to explore the effect of moonlight on plant physiology.Coffea arabica genes under full moonlight conditions, particularly at full moon zenith and 3 h later. Among the 3387 deregulated genes found in our study, the main core clock genes were affected.Here for the first time we report a massive transcriptional modification in Moonlight also negatively influenced many genes involved in photosynthesis, chlorophyll biosynthesis and chloroplast machinery at the end of the night, suggesting that the full moon has a negative effect on primary photosynthetic machinery at dawn. Moreover, full moonlight promotes the transcription of major rhythmic redox genes and many heat shock proteins, suggesting that moonlight is perceived as stress. We confirmed this huge impact of weak light (less than 6 lx) on the transcription of circadian clock genes in controlled conditions mimicking full moonlight. Beyond tales and legends, there is no longer a doubt that solar radiation reflected by the moon can be perceived by many organisms on Earth, and the informational role of moonlight as an environmental cue is not questioned . MoonligPeter W. Barlow’s work clearly demonstrated the impact of local gravimetric oscillations on plant growth and development. These gravimetric variations, i.e. the lunisolar gravity cycle or lunisolar tide, occur daily as result of the impact of the sun and moon on the earth’s surface gravity. Lunisolar tide influences plant phenomena such as leaf movement, stem elongation, fluctuations in tree stem diameter, root growth, biophoton emission by seedlings, and chlorophyll fluorescence . RecentlLight is crucial for plant life, and perception of the light environment dictates plant growth, morphology, and developmental changes. Although plants are highly photosensitive, very few studies have explored the effect of moonlight on plant physiology, and most of the results have generally been conflicting. Between 1926 and 1935, Kolisko showed that the particular phase of the moon at sowing time influences the period and percentage of germination as well as the subsequent plant growth . CharlesArabidopsis and genes involved in photosynthesis pathways and regulation are present in the coffee genome. The expression pattern of core clock genes in coffee trees is similar to that in Arabidopsis, suggesting a high level of conservation. While studying the circadian cycle of young Arabica coffee seedlings in an artificial environment , we decided to also check our results by analyzing older plants in the greenhouse. We conducted sampling at three hourly intervals at spring solstice . When we analyzed the key core clock gene LHY using qRT-PCR, we observed a surprising phenomenon. The gene expression profile showed a completely unexpected peak in the middle of the night. By chance, on the night of our study, the moon was full “at the exquisite hour when a vast and tender peacefulness seems to descend from the firmament” . To further investigate this discovery, we analyzed our samples using RNAseq and confirmed our results at spring solstice in plants grown under the same environmental conditions, but also in plants grown in a phytotron.Plants use the circadian clock to synchronize their physiology and development with daily and yearly changes in the environment . The aim− 2 s− 1). The red:far red (R:FR) ratio of sunlight during the day is more than 1.2, while that of moonlight is between 0.18 and 0.22  light is completely unknown. Compared with sunlight, the wavelength of full moonlight is generally centered around 400 nm (580 nm for the sun) with a very low energy level  light Fig. a. Under PHOT1 gene expression was highly correlated with several genes involved in chlorophyll biosynthesis. For example, the correlation with the magnesium chelatase gene was r = 0.91 . Zeaxanthin epoxidase (ZEP), beta-carotene 3-hydroxylase (CRTZ) and phytoene synthase (PSY1) gene expressions were also highly correlated with PHOT1. We observed higher gene expression at ZT15, indicating that the carotenoid biosynthesis pathway was activated by full moonlight.Phytochromes (PHY), cryptochromes (CRY), ZEITLUPE (ZTL) family proteins and phototropins (PHOT) are known to be major red/far-red and blue light photoreceptors. It is likely that several of these photoreceptors could be involved in moonlight perception, but most them are unaffected at the transcription level. Only phototropins were highly expressed at the FM zenith (ZT15) Fig. . We obse.91 Fig. . Not surArabica plantlets and RNA sequencing time-course data, we determined the cycling transcripts by running JTK_CYCLE for two cycles (48 h). Out of the 25,574 genes of the whole transcriptome, we found 4126 (16%) rhythmic genes at their level of transcription, including 83% that were similar to Arabidopsis rhythmic genes , thus showing that the core clock alteration caused by the FM influenced many genes, with most of them being rhythmic genes.The accumulation of coffee putative clock gene transcripts  was affected by full moonlight at ZT3, ZT12, ZT15, ZT18, ZT21, ZT24 , TIMING OF CAB EXPRESSION 1 (TOC1), GIGANTEA (GI), EARLY FLOWERING 3 and 4 , LUX ARHYTHMO (LUX), PSEUDO-RESPONSE REGULATOR , PHYTOCHROME INTEGRATING FACTOR , CONSTANTS-like 2, 4, 9, and 16 (CO)) were affected by full moonlight. Pairwise phase plots . Here we showed . At ZT15, 29 PPR genes were up-regulated and 8 were down-regulated. We also observed high disequilibrium in ribosomal activity at ZT21, where 69 ribosomal genes were up-regulated and only 4 were down-regulated (data not shown).More than 490 putative pentatricopeptides (PPR) have been predicted in the coffee genome , chlorophyll biosynthesis genes (Protochlorophyllide Oxidoreductases a (POR1A)), and starch metabolism gene (Alpha-glucan water dikinase 1 (GWD1)), during the FM and NM. We repeated the experiment during the FM in March 2017, with the same plants in the same greenhouse. In this new experiment, we also placed half of the plants in a phytotron (12/12 h photoperiod) where the plants did not receive any light at night throughout the month of March. We found no difference in gene expression between plants exposed to the NM in 2016 and 2017 and plants placed in the phytotron , so the quantity of energy perceived by the plant was less than 1 photosynthetically active radiation unit (PAR). Technically, we were unable to increase the light intensity to mimic that emitted at moonrise and at the moon zenith. We switched on the light at full intensity at 10:00 pm. Despite the difficulty in reproducing the light of the FM, after 7 days of treatment, plants exposed to this artificial ‘moonlight’ showed atypical transcription at ZT21 of LHY, PHOT1 and PHOT2 genes in the RT-qPCR analysis , cryptochromes (CRY), ZEITLUPE (ZTL) family proteins and phototropins (PHOT) are known to be major red/far-red and blue light photoreceptors , 20. Theed light , CRTZ anArabidopsis and rice [The circadian clock produces rhythmic variations in a suite of biochemical and physiological processes that help to optimize plant growth in daily cycles. Regular environmental changes, especially the sunrise and sunset, coordinate these rhythmic behaviours. Photoreceptors and metabolites produced during photosynthesis, operate to synchronize the internal timing clock with lighting cues. In our study, we hypothesized that massive transcriptional activation would be a good way to demonstrate the effect of moonlight on plants. Moreover, transcript abundance is useful to assess the effects of external clues on circadian oscillations. Light-regulated changes in the morphology of a dicot or monocot seedling are accompanied by an alteration in the expression of up to 20% genes in and rice . The cirand rice . A changand rice , which iand rice . Here weREVEILLE3 (REV3). Several genes showed expression patterns identical to those of core clock genes. REV3 expression was correlated with LHY (r = 0.98), suggesting that these two genes were probably co-regulated  in mitochondria and chloroplasts, where they can affect gene expression in various ways . Here weRegarding photosynthesis-related genes, 50 genes of this pathway were deregulated by moonlight. Many of these genes were found to be down-regulated just before and at dawn, suggesting that full moonlight has a negative effect on the primary photosynthetic machinery at dawn.ROS homeostasis and the transcriptional response [LHY . To determine the effects of moonlight, the plants were cultivated in a glasshouse under natural daylight  at IRD  in 3 L pots containing a GO M2 (Jiffygroup) potting soil mixture with watering as necessary. Leaf samples were collected from 1 year old plants for RNA extraction at Zeitgeber time (ZT) point ZT0 (sunrise), ZT3, ZT6, ZT9, ZT12 (sunset), ZT15, ZT18, ZT21, and ZT24 in March 2016, and from the same plants in March 2017. Sampling was done at the spring solstice FM and the following NM . During the FM, samples were also taken from control plants cultivated in a phytotron under artificial light , which is defined as the time in hours from the start of a normal 12/12 h photoperiod cycle (photoperiod 12 h/12 h). For this purpose, we collected leaf samples at 3 h resolution from ZT0 to ZT24.Rainbow-Light Portable Light Measuring Instrument. This spectrometer uses micro-electromechanical systems (MEMS) and dynamic thermal equilibrium (DTE) technologies, with high accuracy  and high stability (repeated measurement error < 0.04).Solar and lunar light was analyzed in 2016 and 2017 at spring solstice using an MR-16v4 2/s. No background noise was detectable with the light meter or the photometric PAR probe, but the spectrometer showed a background noise spectrum  were taken from 5 plants per condition. The samples were taken from the 3rd and 4th leaves of the coffee plants.In order to simulate the luminous intensity of the moon in a growth chamber, we measured the real luminous intensity emitted by the moon under a FM and NM. We then programmed four types of LED in the growth chamber to emit a light intensity of 6 lx at the main wavelengths: 450 nm (blue), 660 nm (red), 730 nm (red) and in white light. We measured the light intensities to obtain the real intensity value in the growth chamber. We used three devices: a Rainbow-Light Portable Spectrometer (version MR-16 PPF) to generate a light intensity spectrum, a TopSafe light meter to obtain illuminance (lx) and a photometric PAR probe to obtain the photosynthetic photon flux density (PPFD) expressed in μmol/mTotal RNA was extracted from leaves prefrozen in liquid nitrogen that were subsequently ground and processed as described previously . RNA quahttp://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Based on published data, we targeted three key genes of the circadian clock CcLHY (Cc02_g39990), CcGIGANTEA (Cc10_g15270) and CcLUX-ARRYTHMO (Cc06_g20160). The specificity of the PCR products generated for each primer set was checked by analyzing the Tm (dissociation) of the amplified products. PCR efficiency (E) was estimated using absolute fluorescence data captured during the exponential phase of amplification of each reaction with the eq. (1 + E) = 10(− 1/slope)  Ct−ΔΔ, where CΔt,targetC=t,targetgeneC−t,CaGAPDHCand ΔΔtC=Δt,targetC−Δt, reference sample, with the T0 sample used as reference for each construct. Expression levels were normalized with the expression of the CaGAPDH gene (GB accession number GW445811 using primer pair GAPDH-F/R) serving as endogenous control [PCR experiments were performed as previously described . PrimersGANTEA Cc0_g15270  control .Institut de Génomique Fonctionnelle, Montpellier, France; www.mgx.cnrs.fr/). RNAseq libraries were constructed with the TruSeq Stranded mRNA Sample Preparation kit from Illumina. One microgram of total RNA was used for the library construction. SuperScript IV reverse transcriptase and random primers were used to produce first strand cDNA from cleaved RNA fragments. This was followed by second-strand cDNA synthesis. The cDNA fragments were repaired, before the addition of a single ‘A’ base and the subsequent ligature of the adapter. The final cDNA libraries were validated with a Bioanalyzer kit (Standard Sensitivity NGS) and quantified by qPCR (ROCHE Light Cycler 480). Libraries were pooled in equal proportions, before denaturation with NaOH and dilution to 17 pM, and before clustering on two lanes in a flow cell. Clustering and 100 nt single read sequencing were performed with a Hiseq 2500 according to the manufacturer’s instructions. Image analysis and base calling were performed using HiSeq Control Software (HCS) and the Real-Time Analysis component (Illumina). The data quality was assessed using FastQC from the Babraham Institute (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Illumina Sequence Analysis Viewer (SAV) software. We obtained an average of 21 million single end reads per sample.RNA sequencing (RNAseq) was carried out by the MGX platform  algorithm against the Coffea canephora genome (Coffee Genome Hub) (splice junction mapping) and BWA-backtrack 0.7.15 algorithm against the Coffea arabica transcriptome [Before differential expression (DE) analysis, genes whose sum of counts (by summing the counts per repetition (3)) was below 45 were discarded. Reads were then standardized across libraries using the normalization procedure in DESeq2 . FM/NM ccriptome   < 0.05 as rhythmic transcripts. JTK-CYCLE was run with a 21–27 h range of periods. A χ2 test (P < 0.05) was used to determine if the rhythmic genes in the differential expressed gene set were present in greater numbers than expected by chance. Graphs were plotted using Excel, or R. The R codes are available from the corresponding author.Differential expression (DE) analysis was performed using R 3.4.2 software and the DESeq2 1.18.1 package. Rhythmic gene expression, period and phase parameters were measured using JTK_CYCLE implemented in MetaCycle v1.1.0 .. To ideAdditional file 1: Table S0. REPLICATES. Table S1. Genes differentially expressed between Full moon and new Moon at ZT0. Table S2. Genes differentially expressed between Full moon and new Moon at ZT3. Table S3. Genes differentially expressed between Full moon and new Moon at ZT6. Table S4. Genes differentially expressed between Full moon and new Moon at ZT9. Table S5. Genes differentially expressed between Full moon and new Moon at ZT12. Table S6. Genes differentially expressed between Full moon and new Moon at ZT15. Table S7. Genes differentially expressed between Full moon and new Moon at ZT18. Table S8. Genes differentially expressed between Full moon and new Moon at ZT21. Table S9. Genes differentially expressed between Full moon and new Moon at ZT24. Figure S10. Rhythmic genes in common between Coffee and Arabidopsis thaliana. Figure S11. Pairwise phase plots of the core clock genes. Figure S12. Number of genes per phase under new moon and the full moon. Table S13. Correlation matrix between Redox homeostasis genes and LHY. Table S14. Correlation matrix between genes involved in lipids pathways and LHY. Table S14. Primers used for genes studied by qRT-PCR. Figure S16. RT-QPCR of LHY gene during new moon. Figure S17. RT-QPCR time-course of core clock genes, POR1A and GWD1.


Secondary osteoporosis may occur in patients with rheumatoid arthritis (RA), causing irreversible joint damage and disability. Bisphosphonates, the recently developed bone resorption inhibitors, have demonstrated significant therapeutic effects on senile and postmenopausal osteoporosis. This study evaluated the efficacy and safety of zoledronic acid (ZOL), with or without methotrexate (MTX), for the prevention and treatment of bone destruction in RA patients.We recruited 66 RA patients with symptoms of secondary osteoporosis. They were randomized into three treatment groups—combined treatment with MTX and ZOL, ZOL monotherapy, or MTX monotherapy—in two consecutive 6-month periods. The participants were followed for 12 months. At the end of each treatment period, improvement in disease activity, bone destruction, and fracture risk were evaluated.P < 0.05). The combination significantly improved the lumbar spine and hip BMD and reduced FRAX scores, suggesting that ZOL combined with MTX reduces bone loss and risk of hip fracture in RA patients with secondary osteoporosis.Combined treatment with ZOL and MTX had significantly better clinical efficacy compared with either ZOL or MTX monotherapy (ZOL has a synergistic effect when combined with MTX, inhibiting RA disease activity, reducing fracture risk, and improving quality of life in RA patients with secondary osteoporosis.http://www.chictr.org.cn/showproj.aspx?proj = 31758Chinese Clinical Trial Registry, ChiCTR1800019290. Registered 3 November 2018–Retrospective registered,  Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease with an estimated global prevalence of 1 to 3% , 5, causThe chronic local and systemic inflammation observed in RA patients is often accompanied by multiorgan dysfunction. Roughly 15 to 36% of RA patients develop osteoporosis  as a comBisphosphonates, a family of drugs that have been widely used as bone resorption inhibitors for osteoporosis for decades , inhibitT value ≤ − 2.5 of L1-L4 and hip bone mineral density (BMD) measurements plus obvious low back pain, night convulsions, and limited life function symptoms; or a T value between − 1.5 and − 2.5 of L1-L4 and hip BMD measurements plus obvious low back pain, night convulsions, limited life function symptoms, and a history of one or more fractures. Exclusion criteria included (a) severe primary diseases in the cardiovascular, cerebrovascular, liver, kidney, or hematopoietic systems, consumptive chronic diseases, or mental illness; (b) hypercalcemia, fresh fractures, liver or kidney dysfunction, thyroid-related diseases, such as hypothyroidism and hyperthyroidism, or malignant tumors; (c) other joint-involving rheumatic diseases, such as systemic lupus erythematosus, dermatomyositis, Sjogren’s syndrome, ankylosing spondylitis, and gout; (d) allergy to ZOL or a history of other allergies; (e) pregnancy, breastfeeding, or planning for pregnancy in the near future; (f) currently participating in other clinical trials; and (g) lack of consent to receive treatment as directed in this protocol.The participants were recruited from patients diagnosed with RA-associated secondary osteoporosis at the outpatient clinic of Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine from June 2013 to December 2015. Eligible participants were defined as those who had been clearly diagnosed with RA and had either a Participants were randomized into three groups: combined treatment with ZOL and MTX, ZOL monotherapy, MTX monotherapy (control) Fig. . A randoParticipants in the ZOL treatment groups received infusions of 0.5 mL of 0.9% NaCl followed by 1.5 mg of sodium zoledronate  intravenously at a controlled rate with instillation time over 30 min and then 100 mL of 0.9% NaCl afterwards. Participants in the MTX treatment groups received 7.5 to 15 mg methotrexate tablets  orally once a week after meals. Additionally, each participant received a 600-mg calcium supplement  and soft capsules containing 0.25 to 0.5 μg alfacalcidol  once daily. Participants completed two consecutive courses of treatment, each lasting 6 months, for a total follow-up of 12 months.For patients taking NSAIDs at enrollment, the NSAID dose was stable for at least 4 weeks before treatment. For those who had been receiving MTX treatment at enrollment, they were left for at least 3 months of treatment and a stable dose for at least 4 weeks before entering the study. A 2-month washout period before treatment was used for those taking oral glucocorticoids at the time of enrollment. A 3-month washout period was used for those who had received other remission medications, including chloroquine, penicillamine, or sulfasalazine.According to the Classification Criteria for Rheumatoid Arthritis revised in 2003 by the American Rheumatology Association , the diaT value from a BMD test according to the WHO standard deviation diagnostic method [T value not less than − 1.0 SD, “reduced bone mass” with a value between − 1.0 and − 2.5 SD, “osteoporosis” with a value less than − 2.5 SD, or “severe osteoporosis” with a value less than − 2.5 SD and one or more fractures.Osteoporosis was diagnosed using the c method . Patient2. FRAX scores were calculated using the FRAX interface (http://www.shef.ac.uk/FRAX/), incorporating collected data on fracture risk factors, to predict the probability of hip fracture in the next 10 years.All participants were assessed before and after treatment for morning stiffness, patient self-evaluation and physician evaluation, C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), BMD scores, and calculated FRAX scores. Disease activity score-28 (DAS28) was calculated with the 28-joint tender joint and swollen joint count, ESR, and health assessment . A visuaSerum hemoglobin, white blood cell count, platelet count, alanine aminotransferase, urea nitrogen, creatinine, routine urine routine, and routine stool were collected before and after each study period. Patients with abnormal results for any of these variables at the end of the study were followed until they returned to normal or clinically stable.χ2) or non-parametric test. Continuous variables were expressed as mean ± SD. An independent T test was used for comparison between two groups with assumed normal distribution and equal variances; a repeated measures data analysis was used for comparison between groups with measurements at multiple time points; and an LSD-T test was performed for further comparison between groups. A rank sum test was used on data that did not fit to the normal distribution or when the variances were not equal. All statistical tests were considered two-sided; P values < 0.05 were considered statistically significant.The data were analyzed on SPSS 19.0 statistical software. Count data were described as frequency or composition ratio, and their changes before and after treatment were analyzed by a chi-square .Sixty-six participants were enrolled in the study and randomized into combined ZOL and MTX treatment, ZOL monotherapy, or MTX monotherapy groups Fig. . During P < 0.05). The combination provided greater improvement in VAS score compared with MTX monotherapy after 6 months of treatment and compared with both ZOL and MTX monotherapy after 12 months (P < 0.05) . Overall, combined treatment was more effective than ZOL or MTX alone.Participants received two consecutive courses of treatment and were evaluated for the main clinical outcomes before and after treatment. An improvement in clinical symptoms, demonstrated with decreased scores in morning stiffness and VAS and DAS28 scores, was observed in all groups after the treatment (data not shown). Improvement in ESR and CRP was observed with combined treatment and MTX monotherapy Table . BetweenP < 0.05), was observed with combination therapy, with gains that were significantly greater compared with those seen with ZOL or MTX monotherapy (P < 0.05). Femoral bone volume significantly improved after 12 months of treatment with ZOL monotherapy (P < 0.05), but there was no difference between ZOL and MTX monotherapy (P > 0.05). In the fracture risk assessment, the FRAX score was significantly reduced with combined treatment after 6 months (P < 0.05), with a further decline observed after 12 months (P < 0.05); at 12 months, the FRAX score was significantly lower in the combination therapy group than in the ZOL and MTX monotherapy groups (P < 0.05)  Table . While t5) Table .Table 2All participants were monitored in the safety analysis. Both ZOL and MTX monotherapy were well tolerated, with most side effects occurring within 48 h after drug administration; they included mild adverse reactions, mainly fever , joint pain (3 last less than 24 h and 3 last between 24 and 48 h), myalgia , or gastrointestinal discomfort (1 lasts less than 24 h). No cases of inflammatory eye disease, jaw osteonecrosis, atrial fibrillation, or serum creatinine or creatinine clearance anomalies were observed. The abovementioned adverse reactions, often been found in patients treated with MTX , are mosIn summary, administration of zoledronic acid, combined with MTX, reduces RA disease activity, risk of fracture, and bone pain in patients with RA-derived secondary osteoporosis.Bisphosphonates, which have a high affinity for bone mineral structures and an ability to chelate calcium ions, inhibit the formation, growth, and dissolution of hydroxyphosphonite crystals and their crystalline materials. The molecular mechanism underlying their action has recently been described . While tRA and bone destruction have been considered two unrelated diseases of the immune system and the bone metabolism system, respectively. Thus, joint inflammation and bone destruction in RA patients have been treated as two major pathological manifestations and as separate therapeutic targets. In the clinic, RA patients typically receive hormones and/or DMARDs to control joint inflammation for symptom relief . On the In recent years, data has accumulated to strongly suggest that the RANKL-RANK system may be involved in RA-mediated bone destruction. It has been known that maturation activation of osteoclasts is induced by RANKL, a critical step in developing both localized bone erosion and systemic osteoporosis . StudiesThese studies all point to the close relationship between RA inflammation and osteoporosis. Thus, a treatment regimen that targets osteoporosis alone may not be sufficient for the optimal therapeutic effect. In this study, we demonstrated that treatment with ZOL alone has little effect on RA-derived secondary osteoporosis, but that when it is combined with MTX it reduces both RA inflammation and bone destruction. Previous studies have shown that for women with postmenopausal RA, whose femoral bone density is correlated with the cross-sectional area of the proximal thigh muscle and the time since menopause, bisphosphonate treatment alone has no significant effect on the bone density of the femoral shaft . On the There is concern whether 12 months of follow-up time in our study is long enough for the evaluation of bone density changes. Several studies –31 reporIn conclusion, inflammation is the most important factor associated with RA bone loss. The combination of methotrexate with bisphosphonate drugs under the premise of complete control of inflammation is the best treatment option for RA-associated secondary osteoporosis.Additional file 1: Table S1. Random number table generated for zoledronate clinical trial


P  =  0.34, I2 = 12%). Severe vitamin D deficiency was significantly associated with an increased risk of DFU . This is the first meta-analysis demonstrating the association between serum vitamin D levels and DFU. Severe vitamin D deficiency is significantly associated with an increased risk of DFU.We aimed to evaluate the association between vitamin D deficiency and diabetic foot ulcer (DFU) in patients with diabetes. Pubmed, EMBASE, BIOSIS, the Cochrane Library, and Web of Knowledge, last updated in July 2018, were searched. We assessed eligible studies for the association between vitamin D deficiency and DFU in diabetic patients. The mean difference (MD) or the odds ratio (OR) was calculated for continuous or dichotomous data respectively. Data were analyzed by using the Cochrane Collaboration’s RevMan 5.0 software. Seven studies that involved 1115 patients were included in this study. There were significantly reduced vitamin D levels in DFU (MD −13.47 nmol/L, 95%CI −16.84 to −10.10;  Patients with DFU have a higher mortality compared with diabetic patients without foot ulcer. Daousi et al.2 reported the mortality rate of DFU is about twice that of nonulcerated diabetic patients. However, there is a paucity of information that explores possible contributory factors on DFU.The diabetic foot ulcer (DFU) is a severe complication in patients with diabetes mellitus (DM)3. Yakob et al.4 found the low levels of vitamin D may be related to the development of diabetic foot infections. Findings from in vitro studies reported that vitamin D could restore the production of antimicrobial peptides in primary cell from DFU and improve the in vitro wound-healing assays5. In rats, the topical application of vitamin D accelerated wound healing in a dose-dependent manner6. Another study found that calcitriol could promote endothelial and keratinocyte cell migration in a DFU model7. Thus, findings from previous studies showed the possible beneficial effects of vitamin D on wound healing in DFU. However, there were no conclusive results available. In this study, we conducted a review and meta-analysis of cohort studies to assess the relationship between serum vitamin D levels with DFU.In recent years, there is an increasing interest in the beneficial role of vitamin D in DM. Some researches have reported its effect on T-cell-mediated immunity, pancreatic insulin secretion and action as well as cell growth and healingPubmed, EMBASE, BIOSIS, the Cochrane Library, and Web of Knowledge were searched for studies published before July 2018. The following combinations of search terms were used:  AND . There were no limitations on language and year of publication. After titles and abstracts review, studies deemed potentially eligible for inclusion were further evaluated by full text reading. Reference lists of the retrieved articles were also reviewed. Two reviewers (J.D. and C.J.) independently conducted the literature search, and disagreements were resolved through discussion with the third authors.All eligible studies must meet the following inclusion criteria: (a) nested case-control study or cohorts study; (b) analyzing the relationship between serum vitamin D levels and DFU; (c) the outcomes were serum vitamin D level or rate of severe vitamin D deficiency; (d) adequate data for extracting or calculating. In addition, the exclusion criteria were as follows: (a) case reports, reviews, editorials and (b) animal studies.We extracted all data with a standardized data-collection protocol. For each study, two investigators independently extracted information on: information on study design, last name of the first author, publication year, country, number of patients, gender, age, serum vitamin D level, rate of severe vitamin D deficiency, and duration of follow-up.8. Serum level less than 20 ng/mL (50 nmol/L) was classified as “vitamin D deficiency” and that less than 10 ng/mL (25 nmol/L) was defined as “severe vitamin D deficiency”.As primary analysis, we assessed mean vitamin D levels in patients with DFU and in control group. As secondary analysis, we evaluated the prevalence of severe vitamin D deficiency in patients with DFU. It is widely accepted that normal 25-hydroxyvitamin D3 supplies a minimal serum value of 20 ng/mL (50 nmol/L)P < 0.05 indicated that there was heterogeneity present between studies. I2 statistic method was also used and I2 > 50% indicated high heterogeneity.Study analyses were performed by the Cochrane Collaboration’s RevMan 5.0 software. Statistical analysis of continuous variables was carried out by using mean difference (MD) with 95% confidence intervals (CIs), and odds ratio (OR) with 95% CIs was used for dichotomous data. Serum vitamin D level is expressed in ng/mL or nmol/L (1 ng/mL = 2.496 nmol/L). Between-study heterogeneity was tested by Q statistics and A sensitivity analysis was performed by repeating the analysis after sequential exclusion of one study at a time.9. The scoring system includes three major areas: participants’ selection, study comparability, and outcomes. Study with 6 or more score indicates higher quality.The Newcastle-Ottawa Scale (NOS) was used to assess the quality of the included studies10, and the Egger’s regression test11. Data were analyzed using STATA 10.0 software. A P value less than 0.05 was considered statistically significant.Potential publication bias was evaluated by funnel plots, the Begg’s rank correlation test18 were included  , and sensitivity analyses showed that the summary results were not significantly influenced by any single study . Based on the forest plot, severe vitamin D deficiency (25-OHD < 10 ng/mL) was significantly associated with the risk of DFU   patients with DFU and 108 (22.78%) in the DM group. No substantial heterogeneity was observed (8.98% patP = 0.217) and Begg’s test (P = 0.764).The funnel plot in the meta-analysis showed no evidence of publication bias Fig. . It was In this study, we conducted a meta-analysis of seven articles with 1115 patients, which provided a comprehensive evaluation of the association between vitamin D and diabetic foot. Meta-analysis reported a significantly lower vitamin D levels in patients with DFU compared with the DM group. Severe vitamin D deficiency was definitely associated with an increased risk of DFU.19. Low serum vitamin D levels are associated with insulin resistance, impaired β-cell function, and the development of DM21. There is also an ongoing interest in the association between lower level of vitamin D and diabetic complications. Zubair et al.22 found that lower 25(OH)D played a significant role in the pathogenesis of foot ulcer. Multiple mechanisms were involved in this process. Hyperglycemia in diabetic patients disturbed the normal process of cytokine production, which in turn impaired wound healing23. Vitamin D supplementation was reported to improve gylcemic control. Sugden et al.24 reported the effect of vitamin D in the improvement of endothelial function. A single high dose of oral vitamin D could substantially improve the brachial artery flow-mediated vasodilatation by 2.3%. Moreover, vitamin D has been suggested as an immune stimulant25. Urashima et al.26 suggested that vitamin D supplementation can reduce the risk of influenza A in schoolchildren. Van et al.27 found vitamin D stimulated the phagocytosis and killed the intestine bacteria by macrophages. Vitamin D also inhibited the secretion of the T helper type 1 cytokines IFN-γ and IL-2 while stimulated the production of Th2 cytokines, which may promote wound healing28. However, the mechanism of the relationship between serum vitamin D levels and DFU is still unclear.Vitamin D has been suggested to play an important role in many chronic diseases, such as diabetes29. More future studies are required to verify the effect of vitamin D supplementation in the prevention or treatment of DFU.This study was the first meta-analysis aiming to estimate the relationship between vitamin D levels and DFU. It showed a significant reduction in vitamin D levels related to DFU with a mean difference of −13.47 nmol/L (95%CI −16.84 to −10.10). Our findings consistently supported the association between vitamin D levels and DFU, and also performed separate analyses for severe vitamin D deficiency. In the independent analyses, we found an increased incidence of DFU for declining levels of vitamin D. Patients with severe vitamin D deficiency had a higher prevalence (48.98 vs. 22.78%) in diabetic foot patients, as compared with those in the DM group. Therefore, the identification of the association of DFU with vitamin D can give us some implications to develop new therapy for DFU. Vitamin D supplementation may be a valid therapeutic option for diabetes with foot ulcer and vitamin D deficiency. It is also supported by the only RCT that evaluated effects of vitamin D supplementation for DFU patientsPotential limitations of this study should be discussed. Firstly, we included only seven studies and the sample size of individual studies was small. It showed that the quantity of researched objects was insufficient. Secondly, two prospective and five retrospective cohort studies were included. Existence of bias was unavoidable in this research. In future, more well-designed trials with large number of participants are urgently required.In conclusion, this is the first meta-analysis demonstrating the association between serum vitamin D levels and DFU. Severe vitamin D deficiency is significantly associated with an increased risk of DFU. More specifically designed studies are needed in the future.